The effect of denervation and dystrophy on the adaptation of sarcomere number to the functional length of the muscle in young and adult mice. In young animals the elongation of the limb bones increases the functional lengths of the muscles. In adult animals the functional length of a muscle can be increased by immobilizing it in the lengthened position. In both cases the muscle adapts by adding on more sarcomeres in series. The role of the nerve supply in this adaptation has been investigated using denervated muscles and muscles from dystrophic animals where there is thought to be an abnormality of the nerve supply. Postnatal sarcomere addition in denervated muscles falls short of that of controls. Although this might mean that the nerve supply is necessary for normal addition of sarcomeres, it is just as likely that there is a change in gait resulting from denervation, which affects the sarcomere number. Sarcomere number in fully grown mice is not affected by denervation, nor is the ability of the muscle to adapt to immobilization in the lengthened position. This is true for fast-twitch as well as slow-twitch muscles. In dystrophic muscles postnatal sarcomere addition is normal, although the presence of a few short fibres in the muscle may mean that some muscle fibres cannot adapt to an increase in the functional length of the muscle accompanying bone growth. Adult dystrophic muscle is capable of adapting to immobilization in the lengthened position. However, although the total number of additional sarcomeres is the same as in normal immobilized muscle, they are added on at a slower rate. The experiments show that although denervated and dystrophic muscle fibres are in a state of atrophy they are still capable of adding on sarcomeres in series when the functional length of the muscle is increased. It would appear that the mechanism which enables the muscle to respond in this way to an increased functional length does not involve the nerve supply. This work was supported by a grant from the National Fund for Research into Crippling Diseases.


The development and structure of the chimpanzee mandible. The sites of growth and remodeling, and the associated changes in cortical bone structure, have been studied in the chimpanzee mandible and compared with those previously reported in the human and macaque mandibles. The location of the principal sites of growth, and the distribution of the areas of deposition and resorption in the ramus, were found to be similar in all three species. In the chimpanzee, unlike Man, the bone being deposited at the condyle, posterior border of the ramus and coronoid process was plexiform in nature, indicating very rapid growth. The pattern of remodeling in the mandibular body, on the other hand, showed marked species differences at the chin and on the submandibular lingual surface, which account for the contrasts seen in the adult morphology of these regions. Although the pattern of distribution of cortical densities differed from that of surface remodeling, the information they give is complementary in analysing bone growth. The densest regions were found to coincide with sites of consistent lamellar deposition, while the least dense regions were those where plexiform bone was formed. Areas where remodeling led to the greatest reorientation of bone tissue within the cortex showed the greatest disparity between the two patterns.


Descriptive analysis of eating regulation in obese and nonobese children. Bite rate, sip rate, and concurrent activities of six 7-yr-old children, three obese and three nonobese, were observed at lunchtime over a six-month period. A procedure for decreasing bite rate, putting eating utensils down between bites, was implemented in a multiple-baseline across-subjects design. Sip rates and concurrent activities were observed to assess behavioral covariations. In addition, bite rate and amount of food completed were computed over six food categories to analyze food preferences. Results indicated the control of bite rate acorss all subjects, with a significant reduction in amount of food consumed. Correlations between the response classes indicated they were at least partially independent. Differences in eating behavior of obese and nonobese subjects were observed for breadstuffs and milk drinking.


Teaching pedestrian skills to retarded persons: generalization from the classroom to the natural environment. Little attention has been given to teaching adaptive community skills to retarded persons. In this study, five retarded male students were taught basic pedestrian skills in a classroom- Training was conducted on a model built to simulate city traffic conditions. Each subject was taught five specific skills involved in street crossing in sequence, viz. intersection recognition, pedestrian-light skills, traffic-light skills, and skills for two different stop-sign conditions. Before, during, and after training, subjects were tested on generalization probes on model and under actual city traffic conditions. Results of a multiple-baseline design acorss both subjects and behaviors indicated that after receiving classroom training on the skills, each subject exhibited appropriate pedestrian skills under city traffic conditions. In addition, training in some skills appeared to facilitate performance in skills not yet trained.


Improving social interaction in chronic psychotic using discriminated avoidance ("nagging"): experimental analysis and generalization. Three social-interaction behaviors of a withdrawn chronic schizophrenic were increased using a discriminated avoidance ("nagging") procedure. The three behaviors were: (a) voice volume loud enough so that two-thirds of his speech was intellibible at a distance of 3m; (b) duration of speech of at least 15 sec; (c) placement of hands and elbows on the armrests of the chair in which he was sitting. "Nagging" consisted of verbal prompts to improve performance when the behaviors did not meet their criteria. A combined withdrawal and multiple-baseline design was used to evaluate the effectiveness of the procedure, and the contingency was sequentially applied to each of the three behaviors in each of four different interactions to determine the degree of stimulus and response generalization. Results indicated that the contingency was the effective element in increasing the patient's appropriate performance, and that there was a high degree of stimulus generalization and a moderate degree of response generalization. After the patient's discharge from the hospital, the durability of improvement across time and setting was determined in followup sessions conducted at a day treatment center and at a residential care home. Volume and duration generalized well to the new settings, while arm placement extinguished immediately.


Aerobic power as a factor in women's response to work in hot environments. Twelve young women, athletes (n = 6) and nonathletes (n = 6), walked on a treadmill at loads equivalent to approximately 30% Vo2 max for two 50-min periods in three environments: 1) 28 degrees C, 45% rh, 2) 35 degrees C, 65% rh, and 3) 48 degrees C, 10% rh. There were no differences between groups in rectal temperature, heart rate, evaporative heat loss, or mean skin temperature at 28 or 35 degrees C or during the first work period in the 48 degrees C environment. However, a significantly lower cardiac output (Q) and stroke volume (SV) observed for nonathletes by the 46th min of work at 48 degrees C may explain why no nonathletes were able to complete a 2nd h of work while four of six athletes successfully finished the period. It appears that in conditions of severe heat stress (48 degrees C) athletes were able to maintain a cardiac output sufficient to meet the metabolic requirements and the large increase in peripheral blood flow for a longer period of time than nonathletes.


An analysis of multiple misplaced parental social contingencies. This study analyzed the training of a mother to modify five subclasses of her attention to her young child's noncompliance with instructions, and also displayed the changes in her child's behavior correlated with these events. Training in four subclasses consisted of teaching the mother to withhold various forms of social attention to her daughter's undesired behavior; training in the fifth subclass involved introduction of a brief room-timeout procedure for noncompliance. The effectiveness of the parent-training procedure, consisting of initial instructions and daily feedback, was demonstrated through a multiple-baseline design across the five subclasses of parent behavior. Sequential decreased in the first three subclasses of the mother's social attention to undesired child behavior resulted in incomplete improvements in some child responses; however, a decrease in the fourth subclass resulted in a significant increase in undesired child behavior. Complete remediation of all child behaviors was achieved following the training of a timeout procedure for noncompliance. Postchecks conducted up to 16 weeks later showed that these effects were durable.


Cardiorespiratory responses of sedentary college women as a function of training intensity. Twenty-seven sedentary college women trained on a treadmill 3 times weekly over a 9-wk experimental period. Subjects exercised at a heart rate (HR) of either 50 or 65% of the HR reserve added to the resting HR with the duration of each session limited to the time required to elicit 1,000 beats above the resting value. Treadmill speed was adjusted automatically to maintain the prescribed exercise heart rate (EHR) within +/- 5 beats-min(-1). A comparison of the pretraining and posttraining results revealed that both training intensities caused significant increases in VO2max (1-min(-1) and ml-kg(-1)-min(-1)), V at VO2max, and O2 pulse at VO2max, and a significant decrease in VEO2 at VO2max. There was no alteration in EHR at VO2max for either intensity. For every dependent variable in which training effects were noted, the absolute gain made by the subjects training at the 65% intensity was greater than for those exercising at 50%. In no instance, however, was the difference between groups statistically significant. It was concluded that training at an EHR of either 50 or 65% of the HR reserve plus resting HR is sufficient to elicit a training response.


A performance-based lottery to improve residential care and training by institutional staff. Two experiments were conducted on four units of a residential facility for the multiply-handicapped retarded in an attempt to improve daily care and training services. Experiment I compared the effects of two procedures in maintaining the work performance of attendants, using an A-B design on two units. One procedure consisted of implementing specific staff-resident assignments, the other consisted of allowing attendants who had met performance criteria to be eligible for a weekly lottery in which they could win the opportunity to rearrange their days off for the following week. Results showed that the lottery was a more effective procedure as measured by the per cent of time attendants engaged in predefined target behaviors, and by their frequency of task completion in several areas of resident care. Experiment II replicated and extended these results to the area of work quality on two additional units, using a multiple-baseline design. The performance lottery was found to be an effective econimical procedure that could be implemented by supervisory staff on a large scale.


Interactions between local and reflex influences on human forearm skin blood flow. A three-part experiment was designed to examine interactions between local and reflex influences on forearm skin blood flow (SkBF). In part I locally increasing arm skin temperature (Tsk) to 42.5 degrees C was not associated with increases in underlying forearm muscle blood flow, esophageal temperature (Tes), or forearm blood flow in the contralateral cool arm. In part II whole-body Tsk was held at 38 or 40 degrees C and the surface temperature of one arm held at 38 or 42 degrees C for prolonged periods. SkBF in the heated arm rose rapidly with the elevation in body Tsk and arm Tsk continued to rise as Tes rose. SkBF in the arm kept at 32 degrees C paralleled rising Tes. In six studies, SkBF in the cool arm ultimately converged with SkBF in the heated arm. In eight other studies, heated arm SkBF maintained an offset above cool arm SkBF throughout the period of whole-body heating. In part III, local arm Tsk of 42.5 degrees C did not abolish skin vasoconstrictor response to lower body negative pressure. We conclude that local and reflex influences to skin interact so as to modify the degree but not the pattern of skin vasomotor response.


The effects of social punishment on noncompliance: a comparison with timeout and positive practice. The effects of social punishment, positive practice, and timeout on the noncompliant behavior of four mentally retarded children were assessed in a multitreatment withdrawal design. When programmed, the experimental procedure occurred contigent on non-compliance to experimenter-issued commands. Commands were given at 55-sec intervals throughout each experimental session. The results showed (1) lower levels of noncompliance with social punishment than with the positive-practice or timeout conditions, and (2) that relatively few applications of social punishment were required to obtain this effect. The advantages of social punishment over other punishment procedures, considerations to be made before using it, and the various aspects of the procedure that contribute to its effectiveness were discussed.


Effect of altitude exposure on thermoregulatory response of man to cold. The thermoregulatory responses to 10 degrees C (for 3 h) were investigated in 1) 12 natives from sea level (lowlanders) at 150 m, and on arrival at 3,350 and 4,340 m; 2) 6 of these during a 6-wk sojourn at 4,360 m, and on return to sea level; and 3) 5 natives from each of the two altitudes (highlanders) in their respective habitat, and after descent to 150 m. The cold-induced increase in the rate of O2 consumption (Vo2) of the lowlanders was significantly smaller at both altitudes than at sea level. It did not recover substantially during the 6 wk at altitude, but was restored to its initial rate on return to sea level. By contrast, visible shivering activity was augmented on arrival at altitude. It persisted throughout the 6 wk there, but was greatly depressed on return to sea level, despite the increased Vo2. Mean skin temperatures (Tsk) stabilized in the cold at significantly higher values at altitude. Rectal temperature (Tre) decreased similarly at all altitudes. Vo2 of the highlanders in the cold was significantly greater at sea level than at their resident altitudes, although shivering activity was less intense; Tsk stabilized at significantly lower levels at 150 m than at either altitude. These results indicate that altitude exposure reduces the calorigenic response of man to cold, and that this effect is not moderated by acclimatization to altitude, yet is reversible immediately on descent to sea level. The component of cold thermogenesis which appeared to be reduced by altitude exposure was nonshivering thermogenesis rather than visible shivering.


Teaching coin summation to the mentally retarded. A procedure to teach four mild and moderately retarded persons to sum the value of coin combinations was tested. Subjects were first taught to count a single target coin, and then to sum that coin in combination with coins previously trained. Five American coins and various combinations were trained. Modelling, modelling with subject participation, and independent counting by the subject constituted the training sequence. The subjects improved from a mean pretest score of 29% to 92% correct at posttest. A four-week followup score showed a mean of 79% correct. A multiple-baseline design suggested that improvement in coin-counting performance occurred only after the coin was trained. The results indicate that this procedure has potential for teaching the retarded to sum combinations of coinds in 5 to 6 hr of instruction.


Pulmonary function changes in ozone-interaction of concentration and ventilation. A total of 28 healthy young subjects have been exposed for 2 h to ozone (0.37-0.75 ppm) under conditions of either rest or intermittent light exercise (sufficient to increase the respiratory minute volume by a factor of 2.5). All pulmonary function tests (vital capacity, forced expiratory volume, maximum expiratory flow-volume curve, slope of phase III of alveolar nitrogen plateau) showed a significant deterioration relative to parallel control experiments. Responses were related to the dose of ozone as calculated from the product of concentration, exposure time, and respiratory minute volume during exposure, changes at 1 h averaging approximately one-half those seen at 2 h.


The changing criterion design.

The changing criterion design. This article describes and illustrates with two case studies a relatively novel form of the multiple-baseline design called the changing criterion design. It also presents the design's formal requirements, and suggests target behaviors and circumstances for which the design might be useful.


Ventilatory control in peripheral chemoreceptor-denervated ponies during chronic hypoxemia. The present study was designed to provide further insight into the role of the carotid and aortic chemoreceptors in ventilatory (VE) acclimatization during sojourn at altitude. Measurements were made: 1) on 10 ponies near sea level (SL, 740 Torr) under normal conditions, 2) on 6 of these at SL following chemoreceptor denervation (CD), and 3) subsequently on all 10 during 4 days of hypobaric hypoxia (PaO2 = 40-47 Torr). CD resulteo in hypoventilation at SL (deltaPaCO2 = d8 Torr, P less than 0.05), and it prevented hyperventilation normally observed with injection of NaCN and acute exposure to hypoxia (less than 1 h). In contrast, hyperventilation was evident in normal ponies during acute hypoxia (deltaPaCO2 = -6.7 Torr). Ventilation increased in both groups between the 2nd and 8th h of hypoxia (deltaPaCO2 from 1 h = -4 Torr, P less than 0.05). This change, a common characteristic of acclimatization, persisted throughout 4 days of hypoxia in the normal ponies. However, in the CD ponies this change was evident consistently only through the 12th h and after the 44 h hyperventilation was no longer evident. We conclude that the peripheral chemoreceptors are essential in ponies for normal VE acclimatization to this degree of hypoxemia. Two additional findings in CD ponies suggest the presence of a CNS inhibitory influence on the VE control center during chronic hypoxemia. First, acute hyperoxygenation on the 4th day of hypoxemia induced hyperventilation (deltaPaCO2 = -5 Torr, P less than 0.05). Second, again on the 4th day and during hyperoxygenation, VE responsiveness to CO2 and doxapram HCl was greater than at sea level.


Effect of beta-adrenergic blockade during exercise on ventilation and gas exchange. The ventilatory effects of beta-adrenergic blockade during steady-state exercise were studied in eight normal subjects using intravenous propranolol hydrochloride (0.2 mg/kg). Heart rate decreased in all subjects by an average of 17%. Coincident with the phase of decreasing heart rate was a significant decrease in both minute ventilation (VE) and CO2 output (VCO2), averaging 9.6 and 9.2%, respectively. Both functions returned to prepropranolol levels after heart rate had reached its reduced steady-state value. The change in VE was significantly correlated with the change in VCO2 (r = 0.85, P less than 0.005), and was associated with negligible changes in endtidal CO2 tensions and ventilatory equivalents for CO2. We interpret these studies as showing that the transient isocapnic hypopnea concomitant with an acute reduction in cardiac output was secondary to a transient decrease in CO2 flux (cardiac output x mixed venous CO2 content). This decrease in VE appears to be induced by the acute decrease in cardiac output ("cardiodynamic hypopnea"), in fashion similar to the previously described cardiodynamic hyperpnea.


Sodium o-iodobenzoate and hemoglobin-oxygen affinity: in vivo effects. Sodium o-iodobenzoate (OISB) was given intravenously to 15 dogs to test the in vivo effect of this drug on the oxyhemoglobin dissociation curve. Administration of a single dose of 500 mg/kg was followed by an average increase in P50 (PO2 at 50% oxyhemoglobin saturation) of 3.6 mmHg from 26.8 +/- 0.5 to 30.4 +/- 1.8 mmHg (corrected to pH 7.4). This elevation was sustained for 7 days. During intravenous infusions of 200 mg/kg every other day for 3 wk, there was a sustained increase in P50 of 2.6 mmHg from 27.8 +/- 1.1 to 30.4 +/- 0.9 mmHg. All dogs survived the experiment and no ill effects of the drug were noted. An increase in serum lactate and pyruvate occurred in all animals following acute or chronic exposure to the drug. There was no significant change in whole blood pH, 2,3-diphosphoglycerate concentrations, intracellular pH, or serum total phosphate. Multiple infusions of sodium cyanate (50 mg/kg per day) reduced P50 by an average of 12.2 +/- 0.3 mmHg. A subsequent single infusion of OISB (500 mg/kg) failed to increase P50. Our preliminary data indicate that pharmacological manipulation of hemoglobin O2 affinity is possible with organic compounds unrelated to erythrocyte metabolism.


Cardiorespiratory deconditioning with static and dynamic leg exercise during bed rest. Bed rest deconditioning was assessed in seven healthy men (19-22 yr) following three 14-day periods of controlled activity during recumbency by measuring submaximal and maximal oxygen uptake (VO2), ventilation (VE), heart rate, and plasma volume. Exercise regimens were performed in the supine position and included a) two 30-min periods daily of intermittent static exercise at 21% of maximal leg extension force, and b) two 30-min periods of dynamic bicycle ergometer exercise daily at 68% of VO2max. No prescribed exercise was performed during the third bed rest period. Compared with their respective pre-bed rest control values, VO2max decreased (P less than 0.05) under all exercise conditions; -12.3% with no exercise, -9.2% with dynamic exercise, but only -4.8% with static exercise. Maximal heart rate was increased by 3.3% to 4.9% (P less than 0.05) under the three exercise conditions, while plasma volume decreased (P less than 0.05) -15.1% with no exercise and -10.1% with static, but only -7.8% (NS) with dynamic exercise. Since neither the static nor dynamic exercise training regimes minimized the changes in all the variables studied, some combination of these two types of exercise may be necessary for maximum protection from the effects of the bed deconditioning.


Flow field and mass transport analysis in arteries with longitudinal ridges. Recent observations have indicated that the earliest lesions of atherosclerosis frequently take the form of longitudinal and helical ridges in arteries of man. Since longitudinal vortices are expected to be present in the troughs between the longitudinal ridges, an analysis was carried out to investigate the three-dimensional flow field and the trasport of lipoproteins and oxygen molecules to arterial walls in the presence of such vortices. The calculations revealed that local hypoxia and lipoprotein accumulation may occur at the ridges, leading to subsequent intimal thickening and ridge growth. Higher shear stresses, calculated in the troughs between ridges, may also partially damage the endothelium and lead to intimal thickening and subsequent merging of the ridges. Meaningful measurements are needed in vivo to determine the strength of the vortices, their time-varying behavior, and the actual transverse variations in shear stress, oxygen transport, and lipoprotein accumulation from trough to ridge regions, in order to appraise the present findings and to learn more about the observed progressive thickening and widening of ridges with increasing degrees of intimal thickening and atherosclerosis.


Effect of pretest temperature on aerosol penetration and clearance in donkeys. Pretest temperature and humidity were correlated with tracheobronchial particle penetration and clearance data from donkeys housed in unheated outdoor facilities and tested after spending 1-2 h in a temperature- and humidity-controlled laboratory. The animals inhaled an inert insoluble radioisotope-labeled monodisperse aerosol for several minutes. Its retention was monitored continuously for 3 h by external gamma detection. Aerosol deposition pattern and bronchial clearance were linearly correlated with pretest outdoor temperature which ranged from -10 to 30 degrees C. The fraction depositing in the unciliated regions of the lung decreased 0.6% per degrees C drop in outdoor temperature. Overall bronchial transport decreased at least 1.5% per degrees C decrease. Multiple linear regression analysis and correction for the positive correlation between temperature and humidity left no significant residual humidity dependence. Acclimatization of the animals in the laboratory for 6 h before testing significantly reduced these effects.


Left ventricular effects on right ventricular developed pressure. The possibility that left ventricular (LV) performance might affect right ventricular (RV) function through the myocardium was examined by using isolated, flow-perfused, paced rabbit hearts beating isovolumically. Reducing LV volume from its optimal volume to zero caused a 5.7% decrease (N = 10, P less than 0.001) in right ventricular developed pressure (RVDP). Ligating the anterior ventricular branches of the left coronary artery which in the rabbit supply the LV free wall resulted in an additional 9.3% decrease in RVDP (N = 5, P = 0.05) within 3 min of ligation. Finally, cutting the LV free wall from the atrioventricular orifice to the apex (thereby preventing any developed LV free wall force during systole) caused a 45% further decrease in RVDP (N = 2, P less than 0.02). Cineradiographic study showed that the alterations in RVDP resulting from changes in LV volume and coronary occlusion correlated significantly (N = 5, P less than 0.01) with the magnitude of septal bulging into the RV cavity during systole. The results indicate that alteration in LV free wall function and changes in LV volume can directly effect RVDP through the myocardium.


New respiratory gas valve support for graded exercise studies. We describe a new support structure for respiratory gas valves for use in exercise studies. Use of this structure largely avoids problems of large inertial reactions and feelings of confinement on the part of the subject which have characterized previous systems. The support mechanism consists of a spring-reacted, pivoted boom carried on the end of a counterbalanced swinging beam. This device was designed for use on an adjustable slope motor-driven treadmill, but may be adapted to a variety of other experimental arrangements.


Computer simulation of brainstem respiratory activity. A mathematical model of the medullary respiratory oscillator, composed of two mutually inhibiting populations (inspiratory and expiratory) of computer-simulated neurons, is presented. Each population consists of randomly interconnected subpopulations of excitatory and inhibitory neurons, is presented. Each population consists of randomly interconnected subpopulations of excitatory and inhibitory neurons. Neuronal coupling is such that either the inspiratory or expiratory population alone is capable of cyclic activity. Weak inhibitory connections between inspiratory and expiratory populations provide satisfactory reciprocating activity independent of the natural frequency of either population alone. Initiation and persistence of rhythmic activity is dependent on a diffused noncyclic excitatory input. Vagal discharge, simulated by phasic inhibition of inspiratory neurons, results in increased respiratory frequency with decreased inspiratory activity. In the absence of simulated vagal discharge, uniform facilitation of synaptic connections increases averaged activities of inspiratory and expiratory populations, with minor effect on frequency. In the presence of simulated vagal discharge, facilitation of synaptic connections increases both frequency and amplitude. The simulated effects of synaptic facilitation, with and without vagal discharge, mimic the physiological response to CO2 in the intact and vagotimized animal.


Transducer for recording electrical and mechanical chronic intestinal activity. An extraluminal displacement transducer has been developed for simultaneously recording the mechanical activity in two perpendicular directions and the electrical activity of the intestinal serosa. The length variations in two perpendicular directions were measured by means of strain gauges bounded on two pairs of lamellae embedded in a rigid stand. The electrical activity was recorded by means of four electrodes situated at the extremity of these lamellae. The electrical gauges of each pair of lamellae are connected to form a Wheatstone bridge. This device allows establishment of a correlation between the mechanical displacement of the intestinal wall serosa and electrical potentials by means of studies of long duration.


Venous ultrasound catheter-tip technique for evaluation of arterial hemodynamics. A variety of devices has been used for measuring flow properties of deep-lying arteries, but many have limitations. This paper describes a relatively nontraumatic intravenous approach which uses a catheter in connection with a pulsed ultrasonic Doppler velocity meter (PUDVM) and an ultrasound echo track. The venous ultrasound catheter (VUC) has permitted measurements of local instantaneols blood velocity, flow, and wall motion in the abdominal aorta and iliac arteries of beagle dogs; evaluation studies have been conducted to compare the VUC recordings with an independent method for measuring blood flow and wall motion. Coupling of this catheter-tip device with the PUDVM and echo track provides chronic measurements of hemodynamic parameters in these deep vessels which were virtually impossible to obtain previously. This technique may prove useful in monitoring vessel pathology longitudinally as well as in basic experimental situations requiring flow and arterial wall mechanical properties.


Ventricular dimensions measured noninvasively by echocardiography in the awake dog. A surface echocardiographic technique was employed in 37 mongrel dogs for measurement of left ventricular dimensions. Intracardiac bolus injections of dextran were used as an echo contrast material to visualize cardiac chambers, and the identification of intracardiac structures was further confirmed by postmortem examination. The left ventricle could be satisfactorily visualized from both the right and left chest, providing two transverse left ventricular diameters nearly perpendicular to each other. End-diastolic and end-systolic diameter measurements in the two separate transverse planes differed by an average of only 0.07 and 0.1 cm, respectively, and dimensional measurements were reproducible from day to day. Diastolic diameter ranged from 3.0 to 4.7 cm (mean 3.7 cm) and systolic diameter 1.9 to 3.3 cm (mean 2.6 cm). Diameter was directly related to animal weight. Stroke volume calculated from the dimension measurements correlated with stroke volume calculated from indicator-dilution curves in 16 dogs. Ejection fraction averaged 0.67 and 0.54, depending on the formula used to calculate volumes. These results indicate that surface echocardiography can be employed to evaluate left ventricular dimensions in the awake dog.


Method to optimize high-pressure, multicomponent gas mixing. By use of the equations derived herein, a method is outlined to determine the optimum filing sequence and to obtain the maximum possible pressure when two or more pure high-pressure gases are to be transferred to a receiver cylinder in order to prepare a multicomponent gas mixture. The method is valid for any number of gas components, originating from high-pressure storage cyclinders of arbitrary size and pressure and for a receiver cylinder to contain initially one or more of the component gases. Percentage concentrations within 1% of desired are easily obtained with this method.


Distribution of chondroitin sulfate in cartilage proteoglycans under associative conditions. Proteoglycan aggregates and proteoglycan subunits were extracted from bovine articular cartilage with guanidine-HC1 folowed by fractionation by equilibrium centrifugation in cesium chloride density gradients. The distribution of chondroitin sulfates (CS) in the cartilage proteoglycans was studied at the disaccharide level by digestion with chondroitinases. In the proteoglycan aggregate fraction, it was observed that the proportion of 4-sulfated disaccharide units to total CS increased from the bottom to the top fractions, whereas that of 6-sulfated disaccharide units was in the reverse order. Thus, the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units increased significantly with decreasing density. The proportion of non-sulfated disaccharide units to total CS tended to increase with increasing density. These data indicate a polydisperse distribution of CS chains, under the conditions used here, in proteoglycan aggregates from bovine articular cartilage.


Partial purification and properties of porcine thymus lactosylceramide beta-galactosidase. Porcine thymus lactosylceramide beta-galactosidase was purified by a simple procedure. In the final step of isoelectric focusing the enzyme was separated into two peaks of pI 6.3 (peak I) and 7.0 (peak II), which showed 3,600- and 4,000-fold enhancement of lactosylceramide-hydrolysing activity, respectively. The two peaks had identical mobility on polyacrylamide gel electrophoresis. The apparent molecular weight was 34,000. Neither monosialoganglioside (GM1) nor galactosylceramide was hydrolysed by the purified enzyme fractions. The optimal pH was at 4.6, and sodium taurocholate was essential for the reaction. The apparent Km was 2.3 x 10-5 M. The reaction was stimulated by sodium chloride and linoleic acid, while it was strongly inhibited by Triton X-100 and bovine serum albumin. Galactosylceramide, p-nitrophenyl beta-galactoside, and p-nitrophenol were weak inhibitors. No effects of GM1 and galactose were observed on the hydrolysis of lactosylceramide.


Dietary control of triglyceride and phospholipid synthesis in rat liver slices. 1. The effect of dietary manipulation on the synthesis of triglycerides and phospholipids was investigated by determining the incorporation of labeled long-chain fatty acid or glycerol into these lipids in liver slices derived from normally fed, fasted, and fat-free refed rats. 2. Triglyceride synthesis was affected markedly by the dietary regime of the animal; the lowest rates were measured with fasted rats, and the highest ones with fat-free refed rats. 3. In contrast to triglyceride synthesis, phospholipid synthesis occured at virtually constant rates regardless of the dietary conditions. 4. Addition of large amounts of fatty acid to the incubation mixture resulted in a marked stimulation of triglyceride synthesis, whereas phospholipid synthesis was affected to a much smaller extent. 5. These results indicate that the synthesis of triglycerides and that of phospholipids are controlled independently, and that the availability of fatty acid in the cell contributes to the control of triglyceride synthesis.


Kinetic study of alpha-chymotrypsin catalysis with regard to the interaction between the specificity-determining site and the aromatic side chain of substrates. In order to investigate how changes in the structures of side-chain aromatic groups of specific substrates influence binding and kinetic specificity in alpha chymotrypsin [EC 3.4.21.1]-catalyzed reactions, a number of nucleus-substituted derivatives of the specific ester substrates were prepared and steady-state kinetic studies were carried out at pH 6.5 and 7.8. Ac-Trp(NCps)-OMe was hydrolyzed more readily at low substrate concentration than Ac-Trp-OMe due to its smaller Km(app) value, suggesting that the bulky 2-nitro-4-carboxyphenylsulfenyl moiety interacts with outer residues rather than with those in the hydrophobic pocket and that this interaction increases the binding specificity. Inhibition experiments using the corresponding carboxylate and analogous inhibitors, however, showed that the carboxy group at the para position of the phenyl nucleus of the substituent sterically hinders association with the active site of alpha-chymotrypsin at pH 7.8 but not at pH 6.5. The kcat values of Ac-Trp(CHO)-0Me, Ac-Tyr(3-NO2)-OMe, and Ac-m-Tyr-OMe were much higher than those of the corresponding specific substrates, indicating that derivatives with a substitute as large as a formyl, nitro or hydroxyl group at the xi-position are stereochemically favorable to the catalytic process. Remarkable increases in Km(app) were also observed. The individual parameters for Ac-Dopa-OMe, however, were comparable to those for Ac-Tyr-OMe.


Properties of non-polymerizable tropomyosin obtained by carboxypeptidase A digestion. Tropomyosin digested with carboxypeptidase A [EC 3.4.12.2] (CTM) shows a lower viscosity than the undigested protein in solution. From the relation between the viscosity decrease and the amount of amino acids liberated from the carboxyl terminus during this digestion, it is inferred that loss of the tri-peptide-Thr-Ser-Ile from the C-terminus is responsible for the decrease in viscosity. The secondary structure of -TM was not affected by the digestion according to circular dichroic measurements. The viscosity of CTM did not increase in methanol-water mixtures, whereas that of tropomyosin increased markedly. These results indicate that polymerizability was lost upon the removal of a small peptide from the C-terminus without change in the secondary structure. A decrease in the viscosity of tropomyosin solutions was observed on the addition of CTM, indicating that CTM interacts with intact tropomyosin. The dependence of the viscosity decrease on the amount of CTM showed that CTM binds tropomyosin in a one-to-one ratio as a result of end-to-end interaction. Since paracrystals having a 400 A repeated band structure could be grown in the presence of Mg ions at neutral pH, side-by-side interactions in CTM molecules remain intact, even though polymerizability is lost. The disc gel electrophoretic pattern showed that troponin could bind to CTM, but no increase in viscosity due to the complex was observed in solution. That is, the C-terminal part of tropomyosin is not required for the formation of the complex. The amount of CTM bound to F-actin was less than half of that bound to undigested tropomyosin, and could be reduced to one-tenth by a washing procedure. In the presence of troponin, however, the amount recovered to the level of tropomyosin normally bound to F-actin. Therefore, it is concluded that troponin is bound in the middle of the tropomyosin molecule and strengthens the binding of tropomyosin to F-actin.


Regulation of AMP deaminase from chicken erythrocytes. A kinetic study of the allosteric interactions. The allosteric properties of AMP deaminase [EC 3.5.4.6] from chicken erythrocytes have been qualitatively and quantitatively accounted for by the concerted transition theory of Monod et al., on the assumption that this enzyme has different numbers of binding sites for each ligand. Theoretical curves yield a satisfactory fit for all experimental saturation functions with respect to activation by alkali metals and inhibition by Pi, assuming that the numbers of binding sites for AMP, alkali metals, and Pi are 4, 2, and 4, respectively. The enzyme was inhibited by concentrations of ATP and GTP below 0.1 and 0.25 mM, respectively, whereas activation of the enzyme was observed at ATP and GTP concentrations above 0.4 and 1.5 mM, respectively. These unusual kinetics with respect to ATP and GTP could be also accounted for by assuming 2 inhibitory and 4 activating sites for each ligand.


Regulation of chicken erythrocyte AMP deaminase by phytic acid. AMP deaminase [EC 3.5.6.4] purified from chicken erythrocytes was inhibited by phytic acid (inositol hexaphosphate), which is the principal organic phosphate in chicken red cells. Kinetic analysis has indicated that this inhibition is of an allosteric type. The estimated Ki value was within the normal range of phytic acid concentration, suggesting that this compound acts as a physiological effector. Divalent cations such as Ca2+ and Mg2+ were shown to affect AMP deaminase by potentiating inhibition by lower concentrations of phytic acid, and by relieving the inhibition at higher concentrations of phytic acid. These results suggests that Ca2+ and Mg2+ can modify the inhibition of AMP deaminase by phytic acid in chicken red cells.


Gamma-Actinin, a new regulatory protein from rabbit skeletal muscle. I. Purification and characterization. A new regulatory protein which we have designated as gamma-actinin has been isolated from native thin filaments of rabbit skeletal muscle. Depolymerized native thin filaments were fractionated by salting out with ammonium sulfate, and the precipitates obtained at 40--60% ammonium sulfate saturation were further subjected to DEAE-Sephadex and Sephadex G-200 column chromatography. The purified gamma-actinin was shown to have a chain weight of 35,000 daltons and had a strong inhibitory action on the polymerization of G-actin. The results of amino acid analysis indicated a unique amino acid composition of gamma-actinin as compared with other structural proteins of muscle. Non-polar and neutral amino acid residues were abundant. One cysteine residue was contained per one molecule of gamma-actinin and played a critical role in the maintenance of the inhibitory activity. Pelleting of gamma-actinin with F-actin showed that gamma-actinin binds to F-action.


Effective method for activity assay of lipase from Chromobacterium viscosum. A method was devised for activity assay of the lipase [triacylglycerol acyl-hydrolase, EC 3.1.1.3] excreted from Chromobacterium viscosum into the culture medium; olive oil emulsified with the aid of Adekatol 45-S-8 (a non-ionic detergent, the ethoxylate of linear sec-alcohols having chain lengths of 10--16 carbon atoms) was used as the substrate. This method was specifically effective for Chromobacterium lipase acitvity assay, and was approximately twice as sensitive as the conventional method, in which polyvinyl alcohol is used for the emulsification of the substrate.


A new bile acid conjugate, ciliatocholic acid, from bovine gall bladder bile. This study was carried out to investigate the occurrence of ciliatocholic acid in bovine gall bladder bile. Ciliatocholic acid was synthesized according to the method described by Bergstrôm and Norman for the synthesis of taurocholic acid. Elemental analysis, melting point, and the infrared spectrum of this substance were determined. An isolation procedure for ciliatocholic acid was established by stepwise elution with an HCl-ethanol solvent system using a Dowex-1 anion exchange resin column chromatographic technique. Ciliatocholic acid amounting to 158 mug (as ciliatine) per 100 ml of gall bladder bile was found in the fraction eluted with 0.01 N HCl in 50% ethanol. This coumpound was purified by preparative thin-layer chromatography and confirmed to be ciliatocholic acid from the hydrolytic stability, phosphorus determination, and chromatographic behavior. Thus, bovine gall bladder bile contains a small amount of ciliatocholic acid.


The structure of a glycopeptide (GP-II) isolated from Rhizopus saccharogenic amylase. Mild alkaline treatment of glycopeptide (GP-II) resulted in the loss of 1 mole of serine and 5 moles of threonine per mole of GP-II, suggesting the presence of O-glycosyl bonds between 1 serine and 5 threonine residues and carbohydrate chains. Treatment of GP-II with alkaline borohydride released only disaccharide. Methylation studies of the carbohydrate moiety gave 2,3,4,6-tetra-O-methyl and 2,4,6-tri-O-methyl derivatives of mannose in a ratio of approximately 1:1. In addition, one step of Smith degradation resulted in the loss of about 6 residues of mannose per mole of GP-II. Moreover, alpha-mannosidase [EC 3.2.1.24] liberated about 6 residles of mannose per mole of GP-II. On the basis of these data, the structure of the carbohydrate moiety of GP-II was confirmed to be 3-O-alpha-mannosylmannose. The amino- and carboxyl-terminal amino acids of GP-II were determined to be threonine and serine, respectively. On reductive cleavage of N-proline bonds with metallic sodium in liquid ammonia,  2 moles of alanine per mole of GP-II were lost. From the compositions of three fragments isolated from the reductive cleavage products, the amino acid sequence of the peptide portion of GP-II was determined. Based on these data, a probable structure was proposed for GP-II.


Connectin, an elastic protein from myofibrils. The elastic protein isolated from myofibrils of chicken skeletal muscle was compared with extracellular non-collagenous reticulin prepared from chicken liver and skeletal muscle. The amino acid compositions of these proteins were similar except that their contents of Phe, Leu, Cys/2, and Hyp were different. The impregnations of the elastic protein and reticulin with silver were also different. The reticulin was not at all elastic. It also differed from reticulin in solubility and antigenicity. It is proposed to call the intracellular elastic protein connectin.


Enrichment of enzyme activity on deformylation of 1-NFK-lysozyme. The formamide linkage of an inactive lysozyme derivative (1-NFK-lysozyme), formed by selective ozonization of tryptophan 62 in hen egg-white lysozyme [EC 3.2.1.17] was hydrolyzed with dilute acid faster in the frozen state at about --10 degrees than at 20 degrees. On hydrolysis of 1-NFK-lysozyme the low lytic activity increased to approximately 80% of that of native lysozyme. It is suggested that the binding ability associated with kynurenine 62 in the lysozyme derivative formed by this hydrolysis may be responsible for increase in enzymatic activity.


Isolation and characterization of nerve growth factor from the venom of Naja naja atra. Nerve growth factor was isolated from the venom of Naja naja atra by ion exchange and gel permeation chromatography and was found to be homogeneous by disc gel electrophoresis. The molecular weight was estimated to be approximately 20,000 by gel filtration and 22,000 by ultracentrifugation. This protein, which showed an isoelectric point of pH 7.02, probably consists of two subunits of equal molecular weight which are held together or interact with each other noncovalently. The biological activity survives treatment by a number of proteolytic enzymes, such as trypsin [EC 3.4.21.4], chymotrypsin [EC 3.4.21.1], and pepsin [EC 3.4.23.1].


The biosynthesis of transfer RNA in insects. II. Isolation of transfer RNA precursors from the posterior silk gland of Bombyx mori. The occurrence of precursors to tRNA in the post-polysomal fraction of the posterior silk gland of Bombyx mori was demonstrated by pulse-chase labeling and DNA-RNA hybridization competition experiments. These precursors had molecular sizes ranging from 4S to 5S on polyacrylamide gel electrophoresis. Analysis of the incorporation of the methyl group from [methyl-14C]methionine revealed that a radioactive peak on polyacrylamide gel appeared in the 4.5S region during brief labeling. This suggested that some methylation occurred at the 4.5S precursor step.


delta-Aminolevulinate synthetases in the liver cytosol fraction and mitochondria of mice treated with allylisopropylacetamide and 3,5-dicarbethoxyl-1,4-dihydrocollidine. Hepatic delta-aminolevulinate (ALA) synthetase was induced in mice by the administration of allylisopropylacetamide (AIA) and 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC). In both cases, a significant amount of ALA synthetase accumulated in the liver cytosol fraction as well as in the mitochondria. The apparent molecular weight of the cytosol ALA synthetase was estimated to be 320,000 by gel filtration, but when the cytosol ALA synthetase was subjected to sucrose density gradient centrifugation, it showed a molecular weight of 110,000. In the mitochondria, there were two different sizes of ALA synthetase with molecular weights of 150,000 and 110,000, respectively; the larger enzyme was predominant in DDC-treated mice, whereas in AIA-treated mice and normal mice the enzyme existed mostly in the smaller form. When hemin was injected into mice pretreated with DDC, the molecular size of the mitochondrial ALA synthetase changed from 150,000 to 110,000. The half-life of ALA synthetase in the liver cytosol fraction was about 30 min in both the AIA-treated and DDC-treated mice. The half-life of the mitochondrial ALA synthetase in AIA-treated mice and normal mice was about 60 min, but in DDC-treated mice the half-life was as long as 150 min. The data suggest that the cytosol ALA synthetase of mouse liver is a protein complex with properties very similar to those of the cytosol ALA synthetase of rat liver, which has been shown to be composed of the enzyme active protein and two catalytically inactive binding proteins, and that ALA synthetase may be transferred from the liver cytosol fraction to the mitochondria with a size of about 150,000 daltons, followed by its conversion to enzyme with a molecular weight of 110,000 within the mitochondria. The process of intramitochondrial enzyme degradation seems to be affected in DDC-treated animals.


Ca-releasing action of beta, gamma-methylene adenosine triphosphate on fragmented sarcoplasmic reticulum. beta,gamma-Methylene adenosine triphosphate (AMPOPCP) has two effects on fragmented sarcoplasmic reticulum (FSR), i.e., inhibition of the rate of Ca uptake and the induction of Ca release from FSR filled with Ca. The Ca release brought about by AMPOPCP has many features in common with the mechanism of Ca-induced Ca release: i) it is inhibited by 10 mM procaine; ii) the amount of Ca release increases with increase in the extent of saturation of FSR with Ca; iii) increase of the Ca concentration in the extent of saturation of FSR with Ca; iii) increase of the Ca concentration in the medium facilitates the release of Ca. However, no facilitation of Ca release upon decrease of Mg concentration in the medium is observable. AMPOPCP and caffeine potentiate each other remarkably in their Ca-releasing action, irrespective of the kind of substrate. From the mode of action of AMPOPCP on the rate of Ca uptake, the amount of phosphorylated intermediate (EP), and the effect on Sr release, it is suggested that the state of the FSR-ATP complex is crucial for Ca-induced Ca release.


Control of ketogenesis from amino acids. IV. Tissue specificity in oxidation of leucine, tyrosine, and lysine. In vitro and in vivo studies were made on the tissue specificity of oxidation of the ketogenic amino acids, leucine, tyrosine, and lysine. In in vitro studies the abilities of slices of various tissues of rats to form 14CO2 from 14C-amino acids were examined. With liver, but not kidney slices, addition of alpha-ketoglutarate was required for the maximum activities with these amino acids. Among the various tissues tested, kidney had the highest activity for lysine oxidation, followed by liver; other tissues showed very low activity. Kidney also had the highest activity for leucine oxidation, followed by diaphragm; liver and adipose tissue had lower activities. Liver had the highest activity for tyrosine oxidation, but kidney also showed considerable activity; other tissues had negligible activity. In in vivo studies the blood flow through the liver or kidney was stopped by ligation of the blood vessels. Then labeled amino acids were injected and recovery of radioactivity in respiratory 14CO2 was measured. In contrast to results with slices, no difference was found in the respiratory 14CO2 when the renal blood vessels were or were not ligated. On the contrary ligation of the hepatic vessels suppressed the oxidations of lysine and tyrosine completely and that of leucine partially. Thus in vivo, lysine and tyrosine seem to be metabolized mainly in the liver, whereas leucine is metabolized mostly in extrahepatic tissues and partly in liver. Use of tissue slices seems to be of only limited value in elucidating the metabolisms of these amino acids.


A correlation between changes in gamma-aminobutyric acid metabolism and seizures induced by antivitamin B6. The effects of DL-penicillamine (DL-PeA), hydrazine and toxopyrimidine (TXP, 2-methyl-6-amino-5-hydroxymethylpyrimidine) on gamma-aminobutyric acid (GABA) metabolism in mouse brain were studied. All these compounds inhibited the activity of glutamate decarboxylase [EC 4.1.1.15] (GAD) and slightly inhibited that of 4-aminobutyrate: 2-oxoglutarate aminotransferase [EC 2.6.1.19] (GABA-T). In contrast, very different effects were observed on GABA levels; hydrazine caused a marked increase, DL-PeA had no effect, and TXP caused a slight decrease in the content of the amino acid. These results could be described by an equation which related the excitable state to changes in the flux of the GABA bypass. Since the values obtained from the equation clearly reflect the seizure activity, it is suggested that the decreased GABA flux might be a cause of convulsions induced by these drugs.


Magnesium ion-induced changes in the binding mode of adenylates to chloroplast coupling factor 1. The effect of Mg2+ on the binding of adenylates to isolated chloroplast coupling factor 1 (CF1) was studied using CD spectrometry and ultrafiltration. At adenylate concentrations smaller than 100 muM, one mole of CF1 binds three moles of ATP (or ADP) regardless of the presence of Mg2+. In the presence of Mg2+, the first two ATP's bind to CF1 independently with the same binding constant of 2.5 X 10(-1) muM-1, then the third ATP binds with a much higher affinity of 10 muM-1. In the absence of Mg2+, the first ATP binds to CF1 with a binding constant of 2.5 X 10(-1) muM-1 then the other two ATP's bind less easily with the same binding constant of 4.0 X 10(-2) muM-1. The binding mode of ADP to CF1 is quite similar to that of ATP. In the presence of Mg2+, the binding constants of the first two ADP's are both 7.6 X 10(-2) muM-1, that of the third ADP being 4.0 muM-1. In the absence of Mg2+, the binding constant of the first ADP is 7.6 X 10(-2) muM-1, the constants of the other two ADP's both being 4.0 X 10(-2) muM-1. AMP caused a negligible change in CD.


Separation of molecular species of glucosylceramide by high performance liquid chromatography of their benzoyl derivatives. The method of separation of glucosylceramide by HPLC was reported. Glucosylceramide was perbenzoylated and separated on a packed muBondapack C18 column, using methanol as eluting solvent. The pattern obtained by HPLC closely resembled that obtained by GLC of the TMS-glucosylceramide, and reflected the molecular species of fatty acid components. This method is reproducible, and sensitive as GLC. This method also can be used for analysis of higher glycolipids.


Further studies on the mechanism of adrenaline-induced lipolysis in lipid micelles. Lipase [EC 3.1.1.3] depleted lipid micelles, in which lipolysis was not elicited by adrenaline, were prepared from lipid micelles. When these lipase-depleted lipid micelles incubated with adipose tissue extract containing lipase activity, adrenaline-induced lipolysis was restored to almost the same level as that of native lipid micelles. Adrenaline-induced lipolysis was not restored when the lipase-depleted lipid micelles were homogenized or sonicated. Various tissue extracts from kidney, lung, liver, and pancreas, and post-heparin plasma, which contained lipase activity, restored adrenaline-induced lipolysis in lipase-depleted lipid micelles.


Determination of the anomeric configurations of Corbicula ceramide di- and trihexoside by chromium trioxide oxidation. The anomeric configurations of Corbicula ceramide dihexoside and ceramide trihexoside were determined by chromium trioxide oxidation and the structures of these lipids were shown to be Man-beta(1 leads to 4)-Glc-beta(1 leads to 1)-ceramide and Man-alpha(1 leads to 4)-Man-beta(1 leads to 4)-Glc-beta(1 leads to 1)-ceramide. These results are compatible with those obtained by enzymic hydrolysis reported previously.


A method for analyzing enzyme kinetics with substrate activation and inhibition and its application to the alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetates. A general kinetic method was developed to analyze enzyme-catalyzed systems complicated by the presence of activation or inhibition by substrate. The method was applied to the alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis of p-chlorophenyl and p-methoxyphenyl acetates. Deacylation rate constants which were not complicated by substrate activation were obtained. The analysis shows that the abnormal substituent dependence of kcat in the steady state hydrolysis is due not to substrate activation but to inappropriateness of the two-step mechanism or the existence of more than one acetyl-enzyme intermediate.


Formation of lipid-linked sugar compounds in Halobacterium salinarium. Presumed intermediates in glycoprotein synthesis. The ability of bacitracin to inhibit the growth of Halobacterium salinarium suggested that glycosylation of the major envelope component, a high molecular weight glycoprotein, might occur via a pathway involving lipid intermediates. This report demonstrates that the cells have enzymatic activities for formation of lipid-linked sugar compounds having the expected properties of such intermediates. Whole cell homogenate catalyzed the transfer of sugar from UDP-glucose, GDP-mannose, and UDP-N-acetyglucosamine to endogenous lipid acceptors. Two lipid products were formed from UDP-glucose, two from GDP-mannose, and one from UDP-N-acetylglucosamine. Characterization of the partially purified lipids by ion exchange chromatography, thin layer chromatography, and mild acid and base hydrolysis showed the major product in each case to have the properties expected for polyisoprenyl phosphoglucose, polyisoprenyl phosphomannose, and polyisoprenyl pyrophospho-N-acetylglucosamine. Estimates of chain length by thin layer chromatography indicate that the lipid has 11 to 12 isoprene identity as a C55-60-polyisoprenyl pyrophospho-N-acetylglucosamine. The N-acetylglucosamine transferase, present in cell envelope preparations, was partially characterized. The enzyme was found to be extremely halophilic, specifically requiring a high concentration of KCl. Optimum activity was obtained at 4 m KCl and partial substitution of K+ by Na+ resulted in a decrease in activity.


mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase from vaccinia virions. Donor and acceptor substrate specificites. Characterization of the donor and acceptor specificities of mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase isolated from vaccinia virus cores has enabled us to discriminate between alternative reaction sequences leading to the formation of the 5'-terminal m7G(5')pppN-structure. The mRNA guanylyltransferase catalyzes the transfer of a residue of GMP from GTP to acceptors which possess a 5'-terminal diphosphate. A diphosphate-terminated polyribonucleotide is preferred to a mononucleoside diphosphate as an acceptor suggesting that the guanylyltransferase reaction occurs after initiation of RNA synthesis. Although all of the homopolyribonucleotides tested (pp(A)n, pp(G)n, pp(I)n, pp(U)n, and pp(C)n) are acceptors for the mRNA guanylyltransferase indicating lack of strict sequence specificity, those containing purines are preferred. Only GTP and dGTP are donors in the reaction; 7-methylguanosine (m7G) triphosphate specifically is not a donor indicating that guanylylation must precede guanine-7-methylation. The preferred acceptor of the mRNA (guanine-7-)-methyltransferase is the product of the guanylyltransferase reaction, a polyribonucleotide with the 5'-terminal sequence G(5')pppN-. The enzyme can also catalyze, but less efficiently methylation of the following: dinucleoside triphosphates with the structure G(5')pppN, GTP, dGTP, ITP, GDP, GMP, and guanosine. The enzyme will not catalyze the transfer of methyl groups to ATP, XTP, CTP, UTP, or to guanosine-containing compounds with phosphate groups in either positions 2' or 3' or in 3'-5' phosphodiester linkages. The latter specificity provides an explanation for the absence of internal 7-methylguanosine in mRNA. In the presence of PPi, the mRNA guanylyltransferase catalyzes the pyrophosphorolysis of the dinucleoside triphosphate G(5')pppA, but not of m7G(5')pppA. Since PPi is generated in the process of RNA chain elongation, stabilization of the 5'-terminal sequences of mRNA is afforded by guanine-7-methylation.


Identification of 17-methyl-18-norandrosta-5,13(17-dien-3beta-ol, the C19 fragment formed by adrenal side chain cleavage of a 20-aryl analog of (20S)-20-hydroxycholesterol. Incubation of (20R)-20-phenyl-5-pregnene-3beta,20-diol, an aromatic analog of (23S)-20-hydroxycholesterol, with an adrenal mitochondrial preparation leads to the formation of four compounds: pregnenolone, phenol, a C8 ketone, acetophenone, and a nonpolar C19 compound. This latter compound has now been identified by reverse isotope dilution analysis and by gas chromatography/mass spectrometry as 17-methyl-18-norandrosta-5,13(17)-dien-3beta-ol. From these results it is evident that enzymatic fission of the C-17,20 bond of this synthetic derivative occurs. On the other hand, when (20S)-20-hydroxy[21-14C]cholesterol was used as substrate, the analogous cleavage did not take place. Thus, substitution of an aromatic group on C-20 facilitates side chain cleavage between that carbon atom and the nucleus whereas neither of the naturally occuring precursors, cholesterol or its 20-hydroxylated counterpart, are metabolized to a C8 fragment.


Isolation and amino acid sequence of a neurotoxic phospholipase A from the venom of the Australian tiger snake Notechis scutatus scutatus. The complete amino acid sequence of notechis 5, a neurotoxic phospholipase A from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The main fragmentation of the 119-residue peptide chain was accomplished by digesting the reduced and S-carboxymethylated derivative of the protein with a staphylococcal protease specific for glutamoyl bonds. Tryptic peptides were used to align and complete the sequence of the four staphylococcal protease peptides. The sequence was determined by Edman degradation by means of the direct phenylthiohydantoin method. Notechis 5 differs in seven positions from the recently elucidated sequence of the presynaptic neurotoxin notexin from the same venom. Notechis 5 has a 50% higher specific prospholipase A activity than notexin when assayed against egg yolk but is only one-third as toxic.


Regulation of de novo purine biosynthesis in human lymphoblasts. Coordinate control of proximal (rate-determining) steps and the inosinic acid branch point. Purine nucleotide synthesis de novo has been studied in a permanent tissue culture line of human splenic lymphoblasts with particular attention to coordination of control of the proximal (rate-determining) steps with the distal branch point of the pathway. An assay was used which permits simultaneous determination of the overall rate of labeling of all intracellular purines with sodium [14C]formate, as well as the distribution of isotope into all intracellular guanine- and adenine-containing compounds. The guanine to adenine labeling ratio was used as an index of IMP branch point regulation. It was found that exogenous adenine and guanine produce feedback-controlling effects not only on the first step in the de novo pathway, but also on the IMP branch point. Concentrations of adenine which produce less than 40% inhibition of the overall rate of de novo purine synthesis do so by selectively inhibiting adenine nucleotide synthesis de novo by 50 to 70% while stimulating guanine nucleotide synthesis de novo by up to 20%. A reciprocal effect is seen with exogenous guanine. The adenosine analog 6-methylmercaptopurine ribonucleoside selectivity inhibits adenine nucleotide synthesis via the de novo pathway but not from exogenous hypoxanthine. Thus, the reactions of purine nucleotide interconversion, in particular adenylosuccinate synthetase, may be regulated differently in cells deriving their purine nucleotides solely from de novo synthesis than when deriving them via "salvage" of preformed hypoxanthine.


H5 Histone and DNA-relaxing enzyme of chicken erythrocytes. Interaction with superhelical DNA. The interaction of closed circular duplex DNA with the lysine-rich H5 histone fraction of avian erythrocytes has been studied. H5, like H1 histone, interacts preferentially with superhelical DNA. The extent of interaction increases with increasing negative or positive superhelicity. Salt-extracted lysine-rich histones show the same specificity for interaction with superhelices as do acid-extracted preparations. Chicken erythrocyte nuclei contain DNA-relaxing enzyme. This enzyme is extracted from the nuclei at lower salt concentrations than those required to extract H1 and H5 histones and is, therefore, probably a function of a protein distinct from H1 and H5 histones.


Isolation of rat liver albumin messenger RNA. Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.


Polymerization of Acanthamoeba actin. Kinetics, thermodynamics, and co-polymerization with muscle actin. The kinetics and thermodynamics for the polymerization of purified Acanthamoeba actin were studied and compared to muscle actin. Polymerization was qualitatively similar for the two actins with a rate-limiting nucleation step followed by rapid polymer extension. Polymerization occurred only above a threshold critical concentration which varied with polymerization conditions for each actin. In the presence of 2 mM MgCl2, nucleation of both actins was rapid and their critical concentrations were similarly low and not detectably dependent on temperature. In 0.1 M KCl, the rates of nucleation of both actins were much slower than when Mg2+ was present and were significantly different from each other. Also, under these conditions, the critical concentrations of Acanthamoeba and muscle actin were significantly different and both varied markedly with temperature. These quantitative differences between the two actins could be attributed to differences in both their enthalpies and entropies of polymerization, Acanthamoeba actin having the more positive deltaH and delta S. Co-polymerization of the two actins was also demonstrated. Overall, however, there were no qualitative differences between Acanthamoeba and muscle actin that would suggest a unique role for the monomer-polymer equilibrium of cytoplasmic actin in cell motility.


Stimulation of phosphorylase kinase autophosphorylation by peptide analogs of phosphorylase. Autoactivation of phosphorylase kinase in the presence of substrates has been studied to determine the cause of the hysteresis, or lag, in the phosphorylase kinase reaction. Peptide analogs corresponding to the convertible serine region of phosphorylase have been used as low molecular weight alternative substrates. Autophosphorylation of the kinase molecule was measured under conditions that favored autoactivation. Phosphorylase b and a tetradecapeptide, which was found to be a good model of phosphorylase, stimulated autoactivation by 86- and 37-fold, respectively. The tetradecapeptide also stimulated autophosphorylation of subunits A and B of the kinase molecule. This increased autophosphorylation coincided with an increased ability to convert phosphorylase. This finding supports the hypothesis that autophosphorylation is responsible for the lag in the phosphorylase kinase reaction. No evidence was obtained to suggest that the lag could be due to dissociation of the kinase. The stoichiometry of phosphate incorporation into phosphorylase kinase subunits by autophosphorylation was much greater than that reported to occur by protein kinase phosphorylation. Multiple phosphorylation sites in subunit A accounted for most of the phosphate incorporation during autophosphorylation. Saturating levels of hexa- and octapeptide analogs also caused stimulation of autophosphorylation. Possible mechanisms and experimental implications of substrate-stimulated autophosphorylation are discussed. Consideration also is given to the possible role of effectors in autophosphorylation in vivo.


Gangliosides of human kidney.

Gangliosides of human kidney. Five gangliosides isolated from human kidney have been characterized. The two main fractions were shown to be typical extraneural gangliosides in having lactose as their neutral carbohydrate moiety. Their structures were identified as: AcNeu(alpha2-3)Gal(beta1-4)Glc(beta1-1)Cer and AcNeu(alpha2-8)AcNeu(alpha2-3)Gal(beta1-4)Glc(beta1-1)Cer. The two main hexosamine-containing gangliosides are structurally related to human blood group substances of glycosphingolipid nature. The following structures are postulated: AcNeu(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc(beta1-1)Cer and AcNeu(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc(beta1-1)Cer. The third hexosamine-containing ganglioside belongs to a different series of glycolipids and was shown to have the structure of a major ganglioside of human brain: AcNeu(alpha2-3)Gal(beta1-3)GalNAc(beta1-4)[AcNeu(alpha2-3)]Gal(beta1-4)Glc(beta1-1)Cer. The fatty acid structure of different gangliosides was shown to resemble that of neutral glycolipids of human kidney with the nonhydroxy acids C16:0, C22:0, and C24:0 as major components.


Hemoglobin Providence. A human hemoglobin variant occurring in two forms in vivo. Hemoglobin Providence Asn and Hemoglobin Providence Asp are two abnormal hemoglobins which apparently arise from a single genetic change that substitutes asparagine for lysine at position 82 (EF6) in the beta chain of human hemoglobin. The second form appears to be thr result of a partial in vivo deamidation of the asparagine situated at position beta 82. Cellulose acetate and citrate agar electrophoresis of hemolysates from patients with this abnormality shows three bands. Globin chain electrophoresis at acid and alkaline pH shows three beta chains. These three chains correspond to the normal beta A chain and two abnormal beta chains. Sequence analysis indicates that the two abnormal chains differ from beta A at only position beta 82. In the two abnormal chains, the residue which is normally lysine is substituted either by asparagine or by aspartic acid. These substitutions are notable because beta 82 lysine is one of the residues involved in 2,3-diphosphoglycerate binding. Additionally, beta 82 lysine is typically invariant in hemoglobin beta chain sequences. Sequence data on the two forms of Hemoglobin Providence are given in this paper. The functional properties of these two forms are described in the next paper.


Studies on a nonpolysomal ribonucleoprotein coding for myosin heavy chains from chick embryonic muscles. A messenger ribonucleoprotein (mRNP) particle containing the mRNA coding for the myosin heavy chain (MHC mRNA) has been isolated from the postpolysomal fraction of homogenates of 14-day-old chick embryonic muscles. The mRNP sediments in sucrose gradient as 120 S and has a characteristic buoyant density of 1.415 g/cm3, which corresponds to an RNA:protein ratio of 1:3.8. The RNA isolated from the 120 S particle behaved like authentic MHC mRNA purified from chick embryonic muscles with respect to electrophoretic mobility and ability to program the synthesis of myosin heavy chain in a rabbit reticulocyte lysate system as judged by multi-step co-purification of the in vitro products with chick embryonic leg muscle myosin added as carrier. The RNA obtained from the 120 S particle was as effective as purified MHC mRNA in stimulating the synthesis of the complete myosin heavy chains in rabbit reticulocyte lysate under conditions where non-muscle mRNAs had no such effect. Analysis of the protein moieties of the 120 S particle by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows the presence of seven distinct polypeptides with apparent molecular weights of 44,000, 49,000, 53,000, 81,000, 83,000, and 98,000, whereas typical ribosomal proteins are absent. These results indicate that the 120 S particles are distinct cellular entities unrelated to ribosomes or initiation complexes. The presence of muscle-specific mRNAs as cytoplasmic mRNPs suggests that these particles may be involved in translational control during myogenesis in embryonic muscles.


Lipolytic action of cholera toxin on fat cells. Re-examination of the concept implicating GM1 ganglioside as the native membrane receptor. The possible role of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM1) ganglioside in the lipolytic activity of cholera toxin on isolated fat cells has been examined. Analyses of the ganglioside content and composition of intact fat cells, their membranous ghosts, and the total particulate fraction of these cells indicate that N-acetylneuraminylgalactosylglucosylceramide (GM3) represents the major ganglioside, with substantial amounts of N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM2) and smaller amounts of other higher homologues also present. Native GM1 was not detected in any of these preparations. Examination of the relative capacities of various exogenously added radiolabeled sphingolipids to bind to the cells indicated that GM2 and glucosylsphingosine were accumulated by the cells to extents comparable to GM1. Galactosylsphingosine and sulfatide also exhibited significant, although lesser, binding affinities for the cells. The adipocytes appeared to nonspecifically bind exogenously added GM1; saturation of binding sites for GM1 could not be observed up to the highest concentration tested (2 X 10(-4) M), wherein about 7 X 10(9) molecules were associated with the cells. Essentially all of this exogenously added GM1 was found bound to the plasma membrane "ghost" fraction. Investigation of the biological responses of the cells confirmed their sensitivities to both cholera toxin and epinephrine-stimulated lipolysis, as well as the lag period displayed during the toxin's action. While we could confirm that the toxin's lipolytic activity can be enhanced by prior treatment of the fat cells with GM1, several of the observed characteristics of this phenomenon differ from earlier reported findings. Accordingly, added GM1 was able to enhance only the subsequent rate, but not the extent, of toxin-stimulated glycerol release (lipolysis) from the cells. We also were unable to confirm the ability of GM1 to enhance the toxin's activity at either saturating or at low toxin concentrations. The limited ability of added GM1 to enhance the toxin's activity appeared in a unique bell-shaped dose-response manner. The inability of high levels of GM1 to stimulate a dose of toxin that was ineffective on native cells suggests that the earlier reported ability of crude brain gangliosides to accomplish this was due to some component other than GM1 in the crude extract. While several glycosphingolipids and some other carbohydrate-containing substances that were tested lacked the ability to mimic the enhancing effect of GM1, 4-methylumbelliferyl-beta-D-galactoside exhibited an effect similar to, although less pronounced than, that of GM1. The findings in these studies are unable to lend support to the earlier hypothesis that (a) GM1 is cholera toxin's naturally occurring membrane receptor on native fat cells, and (b) the ability of exogenously added GM1 to enhance the toxin's lipolytic activity represents the specific creation of additional natural receptors on adipocytes...


Hydrophobic interaction of human, mouse, and rabbit interferons with immobilized hydrocarbons. Interferons of human, mouse, and rabbit origin bind to straight chain hydrocarbons immobilized on agarose. The hydrophobic nature of binding is established by the following observations: (a) a positive correlation between the length of hydrocarbon ligand and the strength of interaction; (b) a stronger interaction with hydrocarbon ligands terminated with apolar rather than polar head groups; (c) a lack of dependence of binding on ionic strength and pH of the solvent; (d) a reversal of binding by ethylene glycol, a hydrophobic solute; (e) an increasing eluting efficacy of tetraalkylammonium ions with the length of their alkyl substituents. The hydrophobic interactions of human interferon underlie the efficiency of two-step chromatographic procedures. For example, human embryo kidney interferon can be purified about 3,600-fold by sequential chromatography on (a) concanavalin A-agarose, (b) octyl-agarose. Another two-step procedure: (a) concanavalin A-agarose, (b) L-tryptophan-agarose, gives about 10,000-fold purification. The overall recovery of interferon in both cases in close to 90%.


Subunit dissociation in fish hemoglobins. The tetramer-dimer dissociation equilibria (K 4,2) of several fish hemoglobins have been examined by sedimentation velocity measurements with a scanner-computer system for the ultracentrifuge and by flash photolysis measurements using rapid kinetic methods. Samples studied in detail included hemoglobins from a marine teleost, Brevoortia tyrannus (common name, menhaden); a fresh water teleost, Cyprinus carpio, (common name, carp); and an elasmobranch Prionace glauca (common name, blue shark). For all three species in the CO form at pH 7, in 0.1 M phosphate buffer, sedimentation coefficients of 4.3 S (typical of tetrameric hemoglobin) are observed in the micromolar concentration range. In contrast, mammalian hemoglobins dissociate appreciably to dimers under these conditions. The inability to detect dissociation in three fish hemoglobins at the lowest concentrations examined indicates that K 4,2 must have a value of 10(-8) M or less. In flash photolysis experiments on very dilute solutions in long path length cells, two kinetic components were detected with their proportions varying as expected for an equilibrium between tetramers (the slower component) and dimers (the faster component); values of K 4,2 for the three fish hemoglobins in the range 10(-9) to 10(-8) M were calculated from these data. Thus, the values of K 4,2 for liganded forms of the fish hemoglobins appear to be midway between the value for liganded human hemoglobin (K 4,2 approximately 10(-6) M) and unliganded human hemoglobin (K 4,2 approximately 10(-12) M). This conclusion is supported by measurements on solutions containing guanidine hydrochloride to enhance the degree of dissociation. All three fish hemoglobins are appreciably dissociated at guanidine concentrations of about 0.8 M, which is roughly midway between the guanidine concentrations needed to cause comparable dissociation of liganded human hemoglobin (about 0.4 M) and unliganded human hemoglobin (about 1.6 M). Kinetic measurements on solutions containing guanidine hydrochloride indicated that there are changes in both the absolute rates and the proportions of the fast and slow components, which along with other factors complicated the analysis of the data in terms of dissociation constants. Measurements were also made in solutions containing urea to promote dissociation, but with this agent very high concentrations (about 6 M) were required to give measureable dissociation and the fish hemoglobins were unstable under these conditions, with appreciable loss of absorbance spectra in both the sedimentation and kinetic experiments.


Reactivity of cyanate with valine-1 (alpha) of hemoglobin. A probe of conformational change and anion binding. The 3-fold increase in the carbamylation rate of Val-1 (alpha) of hemoglobin upon deoxygenation described earlier is now shown to be a sensitive probe of conformational change. Thus, whereas this residue in methemoglobin A is carbamylated at the same rate as in liganded hemoglobin, upon addition of inositol hexaphosphate its carbamylation rate is enhanced 30% as much as the total change in the rate between the CO and deoxy states. For CO-hemoglobin Kansas in the presence of the organic phosphate, the relative increase in the carbamylation rate of this residue is about 50%. These results indicate that methemoglobin A and hemoglobin Kansas in the presence of inositol hexaphosphate do not assume a conformation identical with deoxyhemoglobin but rather form either a mixture of R and T states or an intermediate conformation in the region around Val-1 (alpha). Studies on the mechanism for the rate enhancement in deoxyhemoglobin suggest that the cyanate anion binds to groups in the vicinity of Val-1 (alpha) prior to proton transfer and carbamylation of this NH2-terminal residue. Thus, specific removal with carboxypeptidase B of Arg-141 (alpha), which is close to Val-1 (alpha) in deoxyhemoglobin, abolishes the enhancement in carbamylation. Chloride, which has the same valency as cyanate, is a better competitive inhibitor of the carbamylation of deoxyhemoglobin (Ki = 50 mM) compared with liganded hemoglobin. Nitrate and iodide are also effective inhibitors of the carbamylation of Val-1 (alpha) of deoxyhemoglobin (Ki = 35 mM); inorganic phosphate, sulfate, and fluoride are poor competitive inhibitors. The change in pKa of Val-1 (alpha) upon deoxygenation may be due to its differential interaction with chloride.


Hemocyanin of the horseshoe crab, Limulus polyphemus. Structural differentiation of the isolated components. The high molecular weight hemocyanin found in the hemolymph of the horseshoe crab, Limulus polyphemus, is composed of at least eight different kinds of subunits. Ion exchange chromatography at high pH in the presence of EDTA yields five major zones, hemocyanins I to V, three of which are electrophoretically heterogeneous. The subunits have similar molecular weights, 65,000 to 70,000, and their amino acid compositions are remarkably similar to each other and to other arthropod and molluscan hemocyanins. Digestion of the native subunits of Limulus hemocyanin by formic acid or trypsin shows considerable structural diversity which is supported by cyanogen bromide cleavage patterns and by peptide mapping of the tryptic peptides prepared from denatured hemocyanin subunits. The structural differentiation of the subunits is accompanied by functional differentiation, as shown in previous investigations of their O2 and CO affinities (Sullivan, B., Bonaventura, J., and Bonaventura, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2558-2562; Bonaventura, C., Bonaventura, J., Sullivan, B., and Bourne, S. (1975) Biochemistry 13, 4784-4789). The subunit diversity of Limulus hemocyanin suggests that other electrophoretically heterogeneous hemocyanins may be composed of structurally distinct subunits.


Aggregation of deoxyhemoglobin S at low concentrations. The self-association of deoxyhemoglobin S was measured in dilute solutions (0 to 5 g/dl) by Rayleigh light scattering at 630 nm and osmometry in 0.05 M potassium phosphate buffer (pH 7.35). Weight and number average molecular weights (Mw and Mn, respectively) and the second or higher virial coefficients, B' were determined. No experimentally significant differences were observed between oxy- and deoxy-Hb S up to the concentration of 2 g/dl; their apparent average molecular weights were within experimental error. Above that concentration, both Mn and Mw of deoxy-Hb S were significantly different from that of oxy-Hb S. The negative second viral coefficent of deoxy-Hb S, observed by both techniques, is consistent with the self-association of this protein. The lack of effect of 0.4 M propylurea on the state of aggregation and the significant influence of 0.1 M NaCl suggests that polar interactions are involved in formation of these aggregates.


Carbon 13 NMR study of nonenzymatic reactions of pyridoxal 5'-phosphate with selected amino acids and of related reactions. Carbon 13 nuclear magnetic resonance spectroscopy has been used to monitor the nonenzymatic reactions of pyridoxal 5'-phosphate with glycine, alanine, valine, serine, and with several other model compounds. Isotopically enriched amino acids were employed so that low concentrations could be utilized while still allowing relatively rapid acquisition of spectral data. The results for alanine and serine are particularly noteworthy in that alanine is deaminated to pyruvate and pyruvate is aminated to alanine, but contrary to the enzymatic reactions of various serine dehydratases wherein serine is converted to pyruvate, the nonenzymatic reaction utilizing serine results in hydroxypruvate rather than pyruvate formation. In the reverse reaction, hydroxypyruvate is aminated to serine but very inefficiently relative to the amination of pyruvate to alanine. The experimental results have been formulated into a proposed reaction mechanism for deamination of amino acids by pyridoxal-P.


Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes. Initiation factor IF-E2 was isolated from rabbit reticulocytes and purified 120-fold to near homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and phosphocellulose, and, when suitable, by sucrose density gradient centrifugation. The factor is a complex protein containing three nonidentical polypeptides of molecular weight 57,000, 52,000, and 36,000. It behaves as a complex throughout its purification and during polyacrylamide gel electrophoresis in nondenaturing buffer but its thress components are readily separated by electrophoresis in denaturing buffers. None of its components corresponds to any of the polypeptides of the other initiation factors or to any proteins of ribosomes washed in buffers containing a high salf concentration. A stoichiometric ratio of 1:1:1 was determined for the three polypeptides; based on the assumption of one copy each per complex, the calculated factor molecular weight is 145,000, a value in agreement with the measured value of 160,000. Initiation factor IF-E2 was radioactively labeled in vitro by reductive alkylation or by phosphorylation with a protein kinase also isolated from rabbit reticulocytes. Neither procedure causes a measurable change in the ability of the factor to form a ternary complex with GTP and the initiator methionyl-tRNA. 5'-Guanylyl-methylenediphosphonate may substitute for GTP, but only at relatively high concentrations. The binding of labeled initiation factor IF-E2 and methionyl-tRNA to the 40 S ribosomal subunit was studied by sucrose density gradient centrifugation. Appreciable binding of the factor is seen only when all three components of the ternary complex are included in the reaction mixture. The binding of either the factor or methionyl-tRNA was not stimulated by the addition of globin messenger RNA and initiation factor IF-E3. It was shown that all three polypeptide components of initiation factor IF-E2 are bound to these nascent initiation complexes.


Isolation and characterization of major intrinsic microsomal membrane proteins. Treatment of the membrane matrix derived from hepatic microsomes with buffered 1 M urea resulted in the selective extraction of a group of proteins together with a portion of the membrane lipid. Thorough chemical characterization of this fraction has been performed, and the proteins have been fractionated by two different procedures. The first of these, preparative polyacrylamide gel electrophoresis, has produced five highly homogeneous membrane proteins which have been characterized with regard to molecular weight, electrophoretic behavior in five different polyacrylamide systems, NH2 terminus, relative carbohydrate content, isoelectric point, and amino acid composition. The five proteins of this group fell in the molecular weight range of 54,000 to 96,000 and had isoelectric points ranging from pH 4.9 to pH 6.7. Further fractionation of the urea-soluble proteins by gel filtration in a sodium dodecyl sulfate-containing medium resulted in the isolation of four homogeneous molecular weight classes of proteins which have been characterized with respect to various physicochemical parameters. The major membrane glycoprotein (apparent molecular weight, 171,000) was isolated by this procedure and found to contain approximately equal amounts of NH2-terminal glycine and serine. suggesting the presence of at least two polypeptide chains in this molecular weight region. From the urea-insoluble fraction of the membrane comprising approximately 80% of the total protein, five intrinsic polypeptides designated S-5 through S-9 were isolated. S-5 (54,000) and S-6 (49,000) represent the most prominent components in the microsomal membrane, accounting for close to 30% of the total protein. Also isolated and characterized is the smallest membrane protein (S-9), a hydrophobic polypeptide of molecular weight 16,000. All of the urea-insoluble proteins are glycoproteins, and S-7 (35,000) gives the second most intense stain for carbohydrate of all proteins in the microsomal membrane.


Negligible amount of copper in hepatic L-tryptophan 2,3-dioxygenase. During the purification of L-tryptophan 2,3-dioxygenase, a protohemoprotein from rat liver, both copper and heme contents of the preparations were found to be progressively increased as purification proceeded. However, the greater part of copper was removed in the late stages of the purification giving a copper to heme ratio less than 0.4. The small amounts of copper could further be reduced by one-half, by a mild treatment of enzyme with chelators such as ethylenedi aminetetraacetate, without any accompanying decrease in enzymatic activity. Since the turnover number of these enzyme preparations expressed per mol of enzyme-bound heme, 200 to 277 min-1 at 25 degrees, were either comparable to or slightly higher than those reported with homogeneous enzyme preparations, the heme in the preparation was considered to be of fully active L-tryptophan 2,3-dioxygenase and, therefore, such a small ratio of copper to heme, 0.1 to 0.3, indicated that copper is not a constituent of L-tryptophan 2,3-dioxygenase of rat liver. The findings were thus inconsistent with the results of Brady et al. (Brady, F. O., Monaco, M. E. Forman, H. J. Schutz, G., and Feigelson, P. (1972) J. Biol. Chem. 247, 7915-7922), who found that L-tryptophan 2,3-dioxygenase contained 2 g atoms of copper and 2 mol of heme/mol of enzyme. Possible reasons for this discrepancy have been discussed.


Isolation and characterization of two mouse L cell lines resistant to the toxic lectin ricin. Two variant mouse L cell lines (termed CL 3 and CL 6) have been selected for resistant to ricin, a galactose-binding lectin with potent cytotoxic activity. The resistant lines exhibit a 50 to 70% decrease in ricin binding and a 300- to 500-fold increase in resistance to the toxic effects of ricin. Crude membrane preparations of CL 3 cells have increased sialic acid content (200% of control), while the galactose, mannose, and hexosamine content is within normal limits. Both the glycoproteins and glycolipids of CL 3 cells have increased sialic acid, with the GM3:lactosylceramide ratios for parent L and CL 3 cells being 0.29 and 1.5, respectively. In contrast, the membranes of CL 6 cells have a decrease in sialic acid, galactose, and hexosamine content with mannose being normal. Both cell lines have specific alterations in glycosyltransferase activities which can account for the observed membrane sugar changes. CL 3 cells have increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, while CL 6 cells have decrease UDP-GlcNAc:glycoproteinN-acetylglucosaminyltransferase and DPU-galactose:glycoprotein galactosyltransferase activities. The increased sialic acid content of CL 3 cells serves to mask ricin binding sites, since neuraminidase treatment of this cell line restores ricin binding to essentially normal levels. However, the fact that neuraminidase-treated CL 3 cells are still 45-fold resistant to ricin indicates that either a special class of productive ricin binding sites is not being exposed or that the cell line has a second mechanism for ricin resistance.


Role of glucagon on the control of hepatic protein synthesis and degradation in the rat in vivo. The effect of glucagon on hepatic protein systhesis and proteolysis has been investigated. The intraperitoneal administration of 200 mug of glucagon produced an increase of the polypeptide chains completion time which was maximal 5 min after its administration and approached control values at 20 min. The increase of the polypeptides chains completion time observed at 5 min after the hormone administration represents a 38% inhibition of the hepatic protein synthetic rate. When glucagon was continuously supplied by intravascular infusion, maximal inhibition was attained throughout the experiment. This inhibition of protein synthesis brought about by glucagon was accompanied by an increase in the polyribosomal state of aggregation, indicating that the hormone acts mainly if not exclusively, on the elongation or termination step, or both. The administration of glucagon produced also a progressive increase in the hepatic valine concentration. This increase could not be accounted for the the decrease in plasma valine levels, suggesting that the rise in haptic valine concentration is an expression of hepatic proteolysis rather than the result of an accelerated transport of amino acids across the hepatocyte plasma membrane. The different time sequence in the glucagon-induced effects of protein synthesis and proteolysis suggests that both effects are independent and probably mediated by different mechanisms.


Participation of a trisaccharide-lipid in glycosylation of oviduct membrane glycoproteins. Preincubation of a hen oviduct membrane preparation with UDP-Nactyl[14C]glucosamine and bacitracin, followed by incubation with GDP-mannose,  leads to formation of a chloroform/methanol (2/1)-extractable glycolipid.  Treatment of the lipid with mild acid results in the release of a trisaccharide shown to have the structure beta-mannosyl-N-acetylglucosamineyl-N-acetylglucosamine.  Incubation of purified trisaccharide-lipid with oviduct membranes in the presence of sodium deoxycholate, Mn2+, and GDP-mannose leads to formation of a labeled glycoprotein with an apparent molecular weight of 25,000...


Nuclear location of mammalian DNA polymerase activities. Nuclei were isolated from monolayer cultures of mouse and human cells using a nonaqueous procedure of cell fractionation in which lyophilized cells were homogenized and centrifuged in 100% glycerol. In previous work we have shown that the nuclear pellet and cytoplasmic supernatant fraction contained 10% or less of the nucleic acids characteristic of the other cell fraction. Aqueous extracts made from fresh cultures and from nonaqueous material at each step of the fractionation procedure were assayed fro DNA polymerase activity. Activities were normalized to DNA contents of extracted material. Specific activity was preserved quantitatively through freezing and drying the cells, but was found to be unstable in glycerol suspensions with approximate half-lives and 1 h at 23 degrees and 4 h at 0-4 degrees. Activities were relatively stable at -25 degrees, however, so that by homogenizing only 15 min at 4 degrees and centrifuging at -25 degrees we preserved approximately 85% of the specific activity of fresh cultures in the nonaqueous nuclear fraction. Sedimentation analyses showed that the nuclear fraction contained both DNA polymerase-alpha and-beta in approximately the proportions expected if all polymerase activities were confined to the nucleus in living cells. DNA polymerase-alpha was found to be more unstable in glycerol suspensions than DNA polymerase-beta. Nuclear location of both activities was found in exponential cultures and in 3T3 mouse cultures synchronized in the G1 and S phases of the cell division cycle. We found no evidence for cytoplasmic factors affecting nuclear polymerase activities. We have concluded that the two major DNA polymerases are nuclear although one, DNA polymerase-alpha, frequently is present as a weakly bound nuclear protein.


Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 60 S ribosomal subunit proteins L4, L5, L7, L9, L11, L12, L13, L21, L22, L23, L26, L27, L30, L33, L35', L37, and L39. The proteins of the large subunit of rat liver ribosomes were separated into seven groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Seventeen proteins (L4, L5, L7, L9, L11, L12, L13, L21, L22, L23, L26, L27, L30, L33, L35', L37, and L39) were isolated from three of the groups (B60, D60, G60) by ion exchange chromatography on carboxymethylcellulose and by filtration through Sephadex. The amount of protein obtained varied from 0.5 to 15 mg. Eight of the proteins (L9, L11, L13, L21, L22, L35', L37 and L39) had no detectable contamination; the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.