The regulation of agarase production by resting cells of Cytophaga flevenis. The regulation of the synthesis of extracellular agarase by Cytophaga flevensis was studied in resting-cell suspensions. Enzyme synthesis was strictly dependent on the presence of a suitable inducer. Enzyme production was maximal at 20 C in phosphate buffer pH 6.9 in the presence of 1.3 mM calcium chloride, 0.03% casamino acids and inducer. Enzyme production was virtually the same at 15 and 20 C, reduced to 50% at 25 C and was not detectable at 30 C. It was highly stimulated by the presence of 0.03% of casamino acids in the incubation mixture and was also favoured by the presence of 1.3 mM calcium ions. Of a variety of compounds tested, only melibiose or neoagaro-oligosaccharides were effective inducers. Among the neoagaro-oligosaccharides, neoagarotetraose was the best inducer. At higher concentrations of inducer compounds catabolite repression of enzyme synthesis was apparent. This was also found when glucose was added to the incubation mixture. This repression was not relieved by the addition of cyclic AMP. Indications were found that the excretion process was limiting the rate of production of extracellular enzyme


Influences of soil acidity on Streptomyces populations inhabiting forest soils. The Streptomyces populations inhabiting five acidic forest soils were examined. It was found that lowering the pH of a medium selective for streptomycetes (starch-casein agar) to the pH of the particular soil horizon being plated influenced both the total numbers and types of streptomycetes that were isolated from the soils examined in this study. On the acidified medium both the numbers of streptomycetes and the percentage of total bacteria on the plates represented by streptomycetes increased (as compared with the same medium with a pH of 7.2). These differences were greatest on the isolations from the most acid soils. The largest concentrations of streptomycetes were found in the surface horizon (0 to 15 cm) and the litter layer immediately over the surface mineral horizon. Acidity tolerance tests demonstrated that random samplings of isolates contained acidophilic, neutrophilic, and acidoduric strains, with the largest numbers of acidophiles being found on the acidified media from the most acid soils. There were no differences between overall utilization of selected carbohydrates among the isolates taken from either the neutral or acidic media, although a larger proportion of the acid media isolates produced acid from the carbohydrates. Evidence is presented which indicates that different types of streptomycetes were isolated on the acid media, and possible reasons for the presence of these acid-tolerant populations are discussed.


Factors affecting production of mold mycelium and protein in synthetic media. The effects of certain cultural conditions on the yield of dry mycelium, protein, and total amino acid content of Rhizopus oligosporus Saito (NRRL 2710), Rhizopus rhizopodiformis (Cohn apud Lichtheim) Zopf (NRRL 6246), and Absidia corymbifera (Cohn) Sacc. et Trotter (NRRL 6247) were studied. The yield of mycelium was found to significantly increase as the spore inoculum was increased from 187,500 to 2,250,000 spores. But the total amino acids (grams/liter) did not change significantly, whereas the percentage of crude protein decreased. An inoculum containing approximately 750,000 spores/ml was used in all of the other experiments. Mycelial production was highest at 37 degrees C for all three molds. However, the best temperature for percentage of crude protein and total amino acids varied with the organism. The mycelial yield and total crude protein of R. oligosporus showed some significant changes as the C/N ratio was increased in 3% glucose medium. In a synthetic medium having a 15:1 C/N ratio, the strains of R. oligosporus, R. rhizopodiformis, and A. corymbifera had better yields from falactose than glucose, not only in dry mycelium but also in total crude protein (grams/liter) and total amino acids (grams/liter). R. oligosporus grew very well on several ammonium salts. but the maximum yield of dry mycelium, total crude protein (grams/liter), and total amino acids (grams/liter) occurred with ammonium sulfate. The optimum pH for both Rhizopus species was 4.0, although R. oligosporus grew equally well at pH 3.0 and slightly less at pH 5.0. The highest yield of mycelium for A. corymbifera was obtained in a medium with an initial pH of 8.0. It was calculated that a fermenter chanrged with an adequate medium and 1,000 lb (about 450 kg) of R. oligosporus or A. corymbifera cells could produce 88 or 90 lb of protein (on a dry-weight basis) per h if the product was removed continuously.


Oxidation of arsenite to arsenate by Alcaligenes faecalis. Alcaligenes faecalis, resistant to the toxic effects of 0.01 M sodium arsenite, was isolated from raw sewage and shown to be capable of oxidizing arsenite to arsenate. When the organisms were grown in chemically defined medium, this conversion was due to the appearance at stationary phase of an intracellular, oxygen-sensitive, inducible enzyme and/or component of the electron transport system; when the organisms were grown in a nutrient broth-yeast extract medium, the enzyme appeared in the late exponential phase of growth. The presence of 0.02 M arsenite in the culture medium affected neither growth rate nor final cell yield.


Defined conditions for synthesis of Bacillus cereus enterotoxin by fermenter-grown cultures. A strain of Bacillus cereus produced high levels of enterotoxin when grown in a semidefined medium in a laboratory scale fermenter. The optimum conditions for enterotoxin synthesis by cultures grown in this medium, which contained Casamino Acids and yeast extract, were found to be: inoculation of vigorously gorwing culture at the 1% level, addition of glucose at a concentration of 1%, control of culture pH at 8.0, incubation at 32 degrees C, use of a moderate stirring rate, and addition of air at low flow rates to minimize foaming. The enterotoxin yield in fermenter-grown cultures was approximately 20 to 50 times higher than the yield obtained in shake flask cultures.


Survival of coliform bacteria in sewage sludge applied to a forest clearcut and potential movement into groundwater. Anaerobically digested dewatered sludge (10 to 15 cm thick) was applied to a forest clearcut as a fertilizer source in northwest Washington on gravelly glacial outwash soil. This sludge is not microbiologically sterile and may contain pathogenic organisms. Fecal coliform bacterial counts in sludge applied in summer (July) fell from 1.08 X 10(5) to 358/g in 204 days and to 0/g in 267 days. Dieoff appeared more rapid in winter (January)-applied sludge, when colnts fell from 1.2 X 10(5) to 20/g in 162 days. Initial death rates were related to sludge temperature, moisture, pH, physical composition, and microbial competition. Aftergrowth of fecal coliforms occurred in warm summer and fall months, but counts were of similar magnitude to background levels in forest soils, where a maximum count of 54/g was recorded. Total coliform counts in fresh sludge ranged from 1.4 X 10(4) to 1.9 X 10(6)/g. Numbers stabilized at 10(3) to 10(4)/g in spring, fall, and summer, with lower numbers in winter. Both total and fecal bacteria moved from the sludge to the soil beneath, but few penetrated past the first 5 cm. The soil acts as an effective biological filter. Few fecal coliform bacteria were recorded in the groundwater, generally being less than 5/100 ml and mostly 0/100 ml. A maximum count of 52/100 ml was recorded. Groundwater contamination from vertical movement of potential pathogens appears unlikely, but hazards from surface runoff and direct handling in the first year may arise.


Dense autotrophic cultures of Alcaligenes eutrophus. Alcaligenes eutrophus was grown autotrophically in 23-liter batch cultures in a controlled H2-O2-CO2 atmosphere. It was demonstrated that the need for periodic supplements of individual nutrients could be anticipated before cell growth depleted these nutrients to the point of becoming growth rate limiting. As a result, exponential growth was extended to optical densities of 44, with doubling times maintained at 2 h. Cultures having an initial optical density of 0.040 to 0.70 reached the final optical density of 60 in about 25 h. The final viable count was 1.2 X 10(11) cells per ml, and the dry weight was 25 g/liter.


Organic flocculation: an efficient second-step concentration method for the detection of viruses in tap water. A method is described for second-step concentration of viruses from water. This method, combined with an adsorption-elution method, yields a mean recovery of about 75%


Reconcentration of poliovirus from sewage. Virus can be adsorbed from effluents of sewage treatment plants on large-surface membranes. Subsequent elution of virus requires large volumes, which in turn requires reconcentration of virus for assay. However, reconcentration of such viral eluates on small adsorbent surfaces is difficult because certain soluble sewage components are adsorbed along with the virus on the initial virus adsorbent and are removed along with the virus by the eluent. Upon acidification of the initial eluate to reconcentrate the virus on smaller membrane surfaces, flocs are formed that interfere with the reconcentration process. To circumvent this problem, the interfering sewage components can be removed by activated carbon and ion-exchange resins. The virus is then readily reconcentrated on small membranes.


Concentration and purification of enteroviruses by membrane chromatography. A simple procedure for the concentration and partial purification of enteroviruses from tissue culture harvests is described. After removal of acid-precipitating components with a cationic detergent, the detergent and most membrane-coating components were removed by treatment with a cationic-exchange resin. The resin effluent was then acidified, and the virus was adsorbed to epoxy-fiberglass membranes. Virus was then eluted with pH 11.5 glycine-NaOH buffer. Since this eluate contains no orgcentrated simply by acidifying the eluate and passing it through a smaller membrane than that used for the first concentration. As high as 500-fold concentrations can be achieved, with a high efficiency of recovery.


Growth and toxin production by Clostridium botulinum in moldy tomato juice. Tomato juice inoculated with Cladosporium sp. or Penicillium sp. developed pH gradients with the upper portions near the mold mats having pH values near neutrality and the lower portions remaining more acid. Clostridium botulinum spores in these moldy tomato juices germinated, grew out, and produced toxin.


Release of immunoreactive serotonin following acid perfusion of the duodenum. Cannulas were placed in the portal vein, hepatic vein, and infrarenal vena cava in eight anesthetized dogs. The duodenum of each dog was irrigated with saline (control) and 0.1 N HCI (50 ml in 10 min). Heparinized blood samples were taken from each cannula 5 min before, and 1, 5, 10, 30 and 60 min after each irrigation for measurement of immunoreactive serotonin concentrations. Serotonin concentrations did not change during saline irrigation of the duodenum. In contrast, serotonin release was consistently observed after duodenal acidification (pH 1.5-2.0). Portal venous serotonin concentrations were increased at 1 min (356 +/- 147 ng/ml) and following a biphasic pattern remained elevated at 30 and 60 min (499 +/- 131 and 489 +/- 187 ng/ml, respectively). Concentrations in the hepatic vein rose more slowly and to a lower peak (357 +/- 123 ng/ml at 10 min). Caval serotonin concentrations were increased at 10 min (342 +/- 121 ng/ml) but promptly returned to baseline levels. In the canine gastrointestinal tract, immunoreactive serotonin concentrations were highest in the duodenal bulb (15.4 mug/gm). This study demonstrated serotonin release from the duodenum following acid perfusion and documented that some of the released serotonin escaped hepatic inactivation. These findings support the possibility that serotonin may be a gastrointestinal hormone involved in the feedback inhibition of gastric acid secretion.


Use of profound hypothermia induced by surface cooling in open-heart surgery. Profound hypothermia with body-surface cooling for use in open-heart surgery is considered a difficult anesthetic technique because of the problems of controlling ether anesthesia. This paper describes our hypothermia technique in detail, with emphasis on its particular usefulness in open-heart procedures in neonates and infants. The results are based on our experience with 281 consecutive patients. Guidelines for safe and effective performance of this technique were established on the basis of continuous monitoring of the electrocardiogram, electroencephalogram, and arterial blood pressure. We conclude from our experience that open-heart operation under profound hypothermia is a safe, effective, and extremely promising method.


Inhibition of adjuvant arthritis by histamine. Histamine was injected subcutaneously to rats at doses of 2--10 mg/kg, twice daily for various periods after an intradermal adjuvant injection into one hind paw. The administration of histamine prevented the appearance of the secondary lesion in the noninjected paw, but did not affect the primary swelling of the injected paw or the established secondary lesion. The histamine effect was dose-dependent with the most effective time of administration being from the 5th to the 10th day after adjuvant injection. Arthritic lesions found in control animals in histological and roentgenographic examinations were also inhibited in histamine-treated animals. Sinomenine, a histamine releaser, likewise showed a suppressive effect on the secondary lesion. Burimamide, a histamine H2-receptor antagonist, blocked these histamine effects, while mepyramine, a H1-receptor antagonist, did not have such a blocking effect. The findings suggest that histamine may inhibit the development of adjuvant arthritis by an immunosuppressive mechanism mediated through activation of H2-receptors on lymphoid cells.


Effects of propranolo, itramin tosylate and dipyridamole on myocardial phosphate metabolism in anoxic perfused rat hearts. Effects of propranolo, itramin tosylate and dipyridamole on coronary flow, cardiac activity and phosphate metabolism were investigated in anoxic perfused rat hearts. During anoxia, heart contractions showed a transient increase followed by gradual declines in amplitude and frequency, while ATP and creatine phosphate (CP) contents strikingly decreased accompanying a pronounced increase in inorganic phosphate (Pi). Propranolol added to the anoxic perfusate attenuated the decrease in CP and increase in Pi, whereas heart contractions wre more markedly depressed than by anoxia alone. Similar effects were also seen in electrically paced anoxic hearts. Itramin decreased ATP contents in non-paced anoxic hearts, but did not in paced ones. Dipyridamole did not produce any significant effect on phosphate metabolism in anoxic myocardium. It was considered that propranolol prevented the anoxia-induced reduction in high-energy phosphates by antagonizing catecholamines released by anoxia and that itramin decreased ATP level by inhibiting ATP generation.


Possible mechanisms involved in the coronary vasodilatory response to dopamine. Dopamine increased coronary blood flow in the dog heart pretreated with alpha-adrenoceptor blocking agents. The coronary vasodilator response was completely inhibited by indomethacin when pretreated with propranolol in addition to alpha blockade. The coronary vasodilation exerted by sodium nitrite was potentiated by indomethacin and adrenergic blockade. In coronary arterial strips, dopamine produced a contraction which was abolished by phenoxybenzamine. Dopamine elicited a dose-dependent relaxation in coronary arterial strips contracted previously by KC1 after pretreatment with phenoxybenzamine. The relaxation was potentiated by indomethacin; this effect was completely blocked by propranolol. Prostaglandin (PG)E1 produced a relaxing response but reduced the dopamine-induced relaxation. From the results it is suggested that beta-adrenoceptor stimulation and release of PG were involved in the coronary vasodilator response to dopamine. PG released by dopamine may increase coronary blood flow on the one hand and reduce beta-adrenoceptor stimulation by inhibiting adenyl cyclase on the other.


Studies concerning dopamine diuresis in the rat. Dopamine is both diuretic and saluretic in the conscious rat. The diuretic effect does not appear to be beta-receptor mediated, but rather may be the result of a combination of alpha- and dopaminergic receptor stimulation. Conclusive proof for the existence of the latter, however, must await the discovery of a more selective peripheral dopamine-receptor stimulant than is dopamine per se.


Selective blocking properties of chloro acetyl catechol (CAC) on the alpha-adrenergic receptor. In anesthetized rats and cats chloro acetyl catechol (CAC, 10 mg/kg i.v. and 10-15 mg/kg i.v., respectively) selectively blocked the arterial hypertensive responses to epinephrine. No adrenergic blockade was produced by CAC in rats pretreated with 20 mg/kg i.p. of the compound 24 hr before the experiment. Norepinephrine-induced contractions of the rat vas deferens and the rat seminal vesicles in vitro were blocked by CAC 10(-4) M. The blockade was prevented by the simultaneous addition of norepinephrine or cysteine. The data are discussed in relation to : (a) the nature of the adrenergic blockade produced by CAC and (b) the possible existence of thiol groups in the vicinity of the active site of the alpha-adrenergic receptor.


The cardiovascular pharmacology and hemodynamic activity of tazolol, a selective myocardial beta-stimulant. The pharmacology and hemodynamics of tazolol (1-iso-propylamino-3-(2-thiazoloxy)-2-propranolol HC1), a selective myocardial beta-stimulant, were studied in pentobarbital anesthetized dogs. Tazolol, i.v., increased myocardial contractile force and heart rate, but induced only minimal changes in arterial pressure. The cardiac effects of tazolol were blocked by pretreatment with the beta-blockers propranolol or practolol and inhibited in animals made tachyphylactic to amphetamine. They were not altered by pretreatment with hexamehtonium, atropine or reserpine. Tazolol increased left circumflex coronary artery flow to a far greater extent than a dose level of isoproterenol which produced a greater increase in cardiac output. Tazolol also increased superior mesenteric artery flow, whereas isoproterenol decreased it. Renal artery flow was not altered. Tazolol was also found to be orally active and to possess some mild general beta-blocking activity.


Influence of adrenaline, dibenamine and dopamine on acidosis, hemoconcentration and lethality in protracted anaphylactic shock of guinea-pigs. Protracted anaphylactic shock of guinea-pigs was accompanied by a marked decrease in blood pH, and an increase in hematocrit. Death ensued in 58.3% of the animals within 3 hr of observation. Infusion of adrenaline (20 mug/kg/min), after eliciting anaphylaxis, intensified the acidosis, and increased the lethality to 100%. Pretreatment with dibenamine (5 mg/kg) reversed the effect of adrenaline. Dopamine, infused in amounts of 200 mug/kg/min, acted similarly to the combination dibenamine/adrenaline. Hemoconcentration was neither prevented nor intensified by adrenaline. Dopamine, however, reduced significantly the anaphylactic increase in hematocrit.


Effects of lithium and rubidium on antinociception and behaviour in mice. I. Studies on narcotic analgesics and antagonists. The effects of acute and chronic LiCl and RbCl treatments on the antinociception caused by morphine, pethidine, methadone, pentazocine, nalorphine or naloxone were studied in mice using the hot plate and phenylquinone writhing tests. In both tests morphine, pethidine, and methadone caused significant antinociception whereas the antagonistic drugs were almost inactive. In the hot plate test both the 21 day administration of LiCl and the 5 and 21 day administration of RbCl decreased the antinociceptive effect of morphine. The 5 day administration of LiCl increased the antinociceptive effect of pethidine whereas the acute and 5 day administrations of RbCl abolished it. There were no significant or consistent interactions of the ions with the antinociceptive effect of methadone or the antagonistic drugs. In the phenylquinone writhing test the effects of the ions were inconsistent. Especially in the acute experiments where LiCl and RbCl had some effects on the behaviour of the mice treated with the analgesics, LiCl mostly impaired motor coordination and motor activity whereas RbCl was inactive or had opposite effects. LiCl enhanced the decrease in rectal temperature whereas RbCl was mostly inactive.


Effects of lithium and rubidium on antinociception and behaviour in mice. II. Studies on three tricyclic antidepressants and pimozide. The effects of LiCl and RbCl on the antinociception caused by three antidepressants and pimozide were studied in mice. On the hot plate LiCl given acutely or chronically did not modify the antinociception of any drug although it augmented hypothermia induced by chlorimipramine and occasionally also that caused by desipramine and doxepine, and also caused definitive changes in motor abilities. RbCl given acutely (2.5 mEq/kg) or for 5 or 21 days in tap water (1 g/liter) abolished the antinociception caused by pimozide and when given acutely or for 5 days that of desipramine. In the phenylquinone writhing test LiCl when given for 21 days enhanced the nearly complete antinociception caused by chlorimipramine, doxepine and pimozide. These effects of ions did not seem to be related to changes in body temperature, motor coordination nor motility.


Utilization of 35S-thiosulphate and an appraisal of the role of ATP-sulphurylase in chemolithotrophic Thiobacillus ferrooxidans. Differentially labelled 35S-thiosulphate was taken up by washed cells of Thiobacillus ferrooxidans which were previously grown on thiosulphate. The uptake was proportional to the biomass over the range 0.5-4.0 mg dry wt. of bacteria and showed typical saturation kinetics with an estimated Km value of 0.5 mM for 35S-thiosulphate. Dithionate and Group VI anions inhibited the uptake, which was under pH control and had a temperature optimum of 50 degrees C. In the absence of thiosulphate, the cells bound 35S-sulphate but the binding did not increase on prolonged incubation and the label could be removed completely by washing with dilute sulphuric acid. Increasing amounts of the label were incorporated from [outer-35S]thiosulphate into cellular materials over a 60-min period, whereas little or no assimilation was observed from either the [inner-35S]thiosulphate or 35S-sulphate. The kinetic properties of the sulphate-activating enzyme ATP-sulphurylase enriched from bacteria grown with either thiosulphate or ferrous-iron were similar although this enzyme has an assimilatory function only when the bacterium is grown with ferrous-iron.


[The activity and properties of adenosine triphosphatase in various swine organs (liver, cerebral and kidney cortex, small intestinal mucosa)]. Studies into the activity of adenosine triphosphatase (ATPase) in homogenates of liver, cerebral cortex, renal cortex, and mucosa of small intestine of swine have shown differentiated activity patterns, with peak activity developing in the liver. This has been related to a particularly high metabolism performance of the liver in fattening pigs. No difference was found to exist between magnesium activation of ATPase of swine tissue homogenates and that in tissue obtained from ruminants. ATPase which could be activated by sodium and potassium ions and inhibited by ouabain was detectable from cerebral and renal cortex. Sodium and potassium ATPases accounts from some 25 per cent of the total activity. ATPase that could be stimulated by calcium ions was recorded only from liver homogenate. The optimum pH values of ATPase were between 7.5 and 8 in the liver, 9 in mucosa of small intestine, and 9.5 in cerebral and renal cortex.


[Circulation load test and quality of meat in fattening pigs of known genetic origin]. 102 fattened pigs with known genetic origin were tested on a conveyor-belt 1--4 days before slaughtering. 64 animals run in the first passage in each case 154 m in a 4 minutes (0.64 m/s), compared to it the 38 animals of the second passage run in each case 770 m in 20 minutes. Measurements of heart frequency and breathing and body temperature were taken before and at intervals until 30 minutes after movement. After slaughtering the values of pH, colour and drip loss of the M. longissimus dorsi were measured. It turned out a difference in the meat quality with a high part of PSE-meat in the first passage (36%), caused by season. The criterion "recovery time of heart action after load" was able to value the stress susceptibility in dependence on meat quality p.m. Animals with a longer recovery time exhibited a lower pH45 value, a lighter colour and a higher drip loss of meat. But only after a certain stress intensity (2. passage) the assertion was given.


[Physico-chemical properties of swine vesicular disease virus]. Titration of SVDV on primary pig kidney cell cultures revealed a plating efficiency of less than or equal to 0,9 X 10(-3). Concentration and purification of the SVD-Virus propagated on pig kidney cell cultures were done by chloroform treatment, adsorption, differential- and density gradient centrifugation. The following physical parameters were found: SVDV is an isometrical RNA-virus having a diameter of 25,1 +/- 1,0 nm. It is resistent to the action of chloroform, ether and pH. The virus has a sedimentation coefficient of 156 +/- 3S and a bouyant density in CsCl of 1,33 +/- 0,01 g/ml. Within the family of picornaviruses the SVDV belongs to the subgroup of enteroviruses and can be distinguished from the foot-and-mouth disease virus by the difference in pH-sensitivity and bouyant density in CsCl.


Feedback of true heart rate during exposure in vivo. Partial replication with methodological improvement. Ten specific phobics improved during a mean of two sessions of experimental treatment by exposure in vivo in a balanced design. Short-term results replicated those of a previous study that self-control of heart rate with the aid of biofeedback significantly reduced heart rate during treatment, but this did not hasten reduction of subjective anxiety, nor of respiratory rate or skin conductance responses. An hour's pretreatment training in self-control of heart rate with the aid of feedback did not enhance the effect. Mere instructions to lower heart rate without feedback had a significant effect during treatment, but the addition of heart rate feedback to instructions significantly augmented the decline in heart rate.


[The influence of partusisten and dilatol on the amino-acid metabolism (author's transl)]. The betamimetica Dilatol and Partusisten have a glycogenolytic and lipolytic effect on the metabolism. In 21 pregnant women significantly higher concentrations were measured in 9 free amino-acids during intravenous tocolysis with Dilatol. After Partusisten infusion 8 amino-acids were increased. After changing to oral medication of the tocolytica the concentration returned to a normal level again. The clinical significance of these findings is being discussed.


The prognostic value of the non-protein nitrogen (NPN) content in the serum and the cerebrospinal fluid. The amounts of total protein and nonprotein nitrogen (NPN), together with the pH were determined in serum and lumbar cerebrospinal fluid (CSF) of controls and comatose patients before and after death. The serum/CSF ratio of NPN was calculated. Under normal conditions the value of this ratio is 1.65. The decrease of this value is a sign of worsening of barrier functions. If the NPN content of the serum and CSF becomes identical, i.e., when the serum/CSF ratio approaches or even equals 1.0, and at the same time the pH decreases in the CSF, then the prognosis is hopeless.


[Kates forefoot arthroplasty (author's transl)]. In a series of 93 forefoot arthroplasties (71 of them followed up), the majority of the cases showed good results of Kates' procedure. The most frequent sources of errors are inadequate treatment of the plantar skin and the formation of steps in the line of resection of the mid-tarsal bones. Rheumatic and degenerative forefoot deformities are equally well suited for this procedure. Forefoot deformities combined with pes equinus appear to be less suitable.


Evaluation of intrinsic sympathomimetic activity of beta-adrenoceptor blocking drugs in the treatment of patients with angina pectoris. Beta-adrenoceptor blocking drugs with intrinsic sympathomimetic activity (ISA) may be less effective in the treatment of patients with angina pectoris than some others that lack this property. A review of 14 trials comparing beta-adrenoceptor blocking drug with ISA and those without ISA in angina pectoris has been made. The overall picture emerges from both acute and chronic studies using subjective and objective endpoints, that there is no striking difference in effectiveness between the two kinds of beta-adrenoceptor blocking drugs. The one exception is pindolol (a drug with ISA) which, at higher doses, has been shown to be consistently worse than propranolol (a drug without ISA). The reasons for the similarity between propranolol and other bets-blocking drugs with ISA in the trials cited are either that the trial design was defective (the trials were mainly fixed dose comparisons) or that the stimulant effects of those drugs with ISA is not of sufficient magnitude to make a difference. It is suggested that further carefully constructed clinical trials should be carried out before the second reason can be accepted.


Characterization of hemoglobin Burke [beta 107 (G9) Gly replaced by Arg]. Hb Burke [beta 107 (G9) Gly replaced by Arg] was discovered in a young woman with hemolytic anemia. A substitution in this position has not been previously reported either in the human beta-chain or in any of the animal beta-chains so far sequenced. The abnormal hemoglobin shows heat instability and a lowered oxygen affinity. The substitution of a large charged arginine residue for the small glycine residue in the G helix next to a heme contact (Leu-106) may be responsible for these effects. Hb Burke is compared with five other hemoglobins having Gly-Arg substitutions in other parts of the molecule.


Genetically defined peptidases of maize. I. Biochemical characterization of allelic and nonallelic forms. A number of biochemical properties have been investigated for both allelic and nonallelic forms of maize peptidases. Four aminopeptidases exist in maize (LAP-A, LAP-B, LAP-C, and LAP-D) and are the products of four diallelic loci. The aminopeptidases fall into two biochemical groups on the basis of these studies. LAP-A and LAP-D have comparatively low apparent Km (Kapp) values for arginine-naphthylamide derivatives and high velocities for arginine-naphthyl-amide and lysine-naphthylamide. LAP-B and LAP-C, on the other hand, have lower Kapp values for leucine-naphthylamide and higher velocities for nonpolar amino acid-naphthylamides than for arginine-naphthylamide. LAP-A and LAP-D are also relatively more heat stable than LAP-B and LAP-C and have somewhat higher molecular weights (71,500) than LAP-B and LAP-C (63,500). In determining molecular weights of the peptidases, use was made of their differential substrate specificities toward amino acid-naphthylamides. Some properties of genetically defined maize endopeptidase are also presented. Maize endopeptidase is inhibited by the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzoate (pCMB), and by tosyl lysine chloromethyl ketone, Maize aminopeptidase activity is inhibited by N-ethylmaleimide, pCMB, and EDTA (ethylenediamine tetraacetic acid).


Aryl hydrocarbon hydroxylase and 16alpha-hydroxylase in cultured human lymphocytes. Aryl hydrocarbon hydroxylase and 16alpha-hydroxylase were examined in intact, cultured human lymphocytes. The two microsomal mixed-function oxygenases had different pH optima and showed competitive inhibition for enzyme induction and activity. Population distributions were lognormal for both enzymes, giving apparent evidence for polygenic control. Induced levels of AHH were slightly higher among first-order relatives of lung or colon cancer patients than in the control group. The correlation coefficient (r) for AHH and SAH coinducibility was -0.08, indicating no correlation and suggesting the absence of association between the two enzymes in man.


Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei. Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5 g of enzyme is obtained from a 400 litre culture. The purification procedure, involving poly-ethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. of approx. 17900 and a turnover number of 4s-1 (50mM-triethanolamine/400mM-KCl, pH 7.2, 25 degrees C) with dihydrofolate and NADPH as substrates. The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.36 muM-dihydrofolate; 0.78 muM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 X 10(6)M-1; NADPH, greater than 10(8)M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.


Determination of the mechanism and kinetic constants for hog kidney gamma-glutamyltransferase. The initial-velocity kinetics of hog kidney gamma-glutamyltransferase were studied. Glutamate gamma-(4-nitroanilide) and its 3-carboxy derivative, glutamate gamma-(3-carboxy-4-nitroanilide), served as gamma-glutamyl donors, and glycylglycine as an acceptor. Reaction products were identified by paper chromatography and amino acid analysis. Inhibited Ping Pong mechanisms and a comprehensive initial- velocity expression were developed which account for the observed simultaneous gamma-glutamyl transfer and autotransfer, competitive inhibition by glycylglycine, and non-competitive inhibition by the carboxy donor. The validity of the proposed Ping Pong mechanisms are supported by enzyme-velocity data obtained with constant ratios of acceptor to donor concentrations. Kinetic constants were determined by a non-linear regression analysis. With glutamate gamma-(4-nitroanilide) as the donor, Michaelis constants for the donor, acceptor and donor-acting-as-acceptor are 1.87, 24.9, and 2.08 mM respectively. With glutamate gamma-(3-carboxy-4-nitroanilide) as the donor, these Michaelis constants are 1.63, 16.6, and 12.3 mM. Glyclyglycine competitive inhibition constants with the parent donor and its carboxy derivative are 275 and 205 mM respectively; the non-competitive inhibition constant of the carboxy donor is 34 mM.


Organ distribution of rat histidine-pyruvate aminotransferase isoenzymes. The organ distribution of rat histidine-pyruvate aminotransferase isoenzymes 1 and 2 was examined by using an isoelectric-focusing technique. Isoenzyme 1 (pI8.0) is present only in the liver and its activity is increased by the injection of glucagon, whereas isoenzyme 2 (pI5.2) is distributed in all tissues (liver, kidney, brain and heart) tested, and is not affected by glucagon injection. Isoenzyme 2 of the liver, kidney, brain and heart was purified by the same procedure and characterized. Isoenzyme 2 preparations from these four tissues were nearly identical in physical and enzymic properties. These properties differed from those previously found for the highly purified isoenzyme 1 preparation of rat liver. Isoenzyme 2 was active with pyruvate but not with 2-oxoglutarate as amino acceptor. Amino donors were effective in the following order of activity: tyrosine greater than histidine greater than phenylalanine greater than kynurenine greater than tryptophan. Very little activity was found with 5-hydroxytryptophan. The apparent Km for histidine was about 0.45 mM. The Km for pyruvate was about 4.5 mM with histidine as amino donor. The amino-transferase activities of isoenzyme 2 towards phenylalanine and tyrosine were inhibited by histidine. The ratio of aminotransferase activities towards these three amino acids was constant through gel filtration, electrophoresis, isoelectric focusing and sucrose-density-gradient centrifugation of the purified isoenzyme 2 preparations. These results suggest that these three activities are properties of the same enzyme protein. Sephadex G-150 gel filtration and sucrose-density-gradient centrifugation yielded mol.wts. of approx. 95000 and 92000 respectively. The pH optimum was between 9.0 and 9.3.


The asymmetric distribution of enzymic activity between the six subunits of bovine liver glutamate dehydrogenase. Use of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid. A method for the preparation of D- and L-glutamyl alpha-chloromethyl ketones (4-amino-6-chloro-5-oxohexanoic acid) is described. These chloromethyl ketones irreversibly inactivated bovine glutamate dehydrogenase, whereas several other related compounds had no adverse effect on the activity of the enzyme. The inactivation process was shown to be due to the modification of lysine-126. The time-courses for the inactivation and the incorporation of radioactivity from tritiated L-glutamyl alpha-chloromethyl ketone into the glutamate dehydrogenase were biphasic. The results were interpreted to suggest the involvement of 'negative co-operative' interactions in the reactivity of lysine-126. From the cumulative evidence it is argued that the first subunit of the enzyme, which takes part in catalysis, makes the largest, and the last the smallest, contribution to the overall catalysis. It is emphasized that three of the six subunits of the enzyme may possess as much as 80% of the total activity of bovine glutamate dehydrogenase.


Guanylate cyclase. Subcellular distribution in cardiac muscle, skeletal muscle, cerebral cortex and liver. 1. Guanylate cyclase of every fraction studied showed an absolute requirement for Mn2+ ions for optimal activity; with Mg2+ or Ca2+ reaction was barely detectable. Triton X-100 stimulated the particulate enzyme much more than the supernatant enzyme and solubilized the particulate-enzyme activity. 2. Substantial amounts of guanylate cyclase were recovered with the washed particulate fractions of cardiac muscle (63-98%), skeletal muscle (77-93%), cerebral cortex (62-88%) and liver (60-75%) of various species. The supernatants of these tissues contained 7-38% of total activities. In frog heart, the bulk of guanylate cyclase was present in the supernatant fluid. 3. Plasma-membrane fractions contained 26, 21, 22 and 40% respectively of the total homogenate guanylate cyclase activities present in skeletal muscle (rabbit), cardiac muscle (guinea pig), liver (rat) and cerebral cortex (rat). In each case, the specific activity of this enzyme in plasma membranes showed a five- to ten-fold enrichment when compared with homogenate specific activity. 4. These results suggest that guanylate cyclase, like adenylate cyclase, and ouabain-sensitive Na+ + K+-dependent ATPase (adenosine triphosphatase), is associated with the surface membranes of cardiac muscle, skeletal muscle, liver and cerebral cortex; however, considerable activities are also present in the supernatant fractions of these tissues which contain very little adenylate cyclase or ouabain-sensitive Na+ + K+-dependent ATPase activities.


Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver. 1. Guanylate cyclase of washed particles and plasma membranes showed S-shaped progress curves when titrated with either GTP or Mn2+ ions; similar results were obtained with Triton X-100-solubilized enzyme preparation from washed particles. Hill plots of these data revealed multiple metal-nucleotide and free-metal binding sites. 2. Guanylate cyclase of supernatant fractions displayed typical Michaelis-Menten properties when enzyme required excess of (free) Mn2+ (over GTP) for maximal activities; Ka (free Mn2+) was about 0.15-0.25 mM at subsaturating concentrations of GTP. 4 MnATP, MnADP, and MnGDP were found to increase the activities of both particulate and superantant enzyme, when MnGTP concentration was below saturation and free Mn2+ ion concentration was low (less than 100 muM); MnATP (50muM-1 mM) inhibited both these activities at high free Mn2+ concentration (1.5 mM) and inhibition of the particulate enzyme was greater than that of supernatant enzyme. 5. Ca2+ ions stimulated supernatant-enzyme activity; the stimulatory concentration of Ca2+ ions depended on the concentration of Mn2+ and GTP. 6. A modest stimulation of particulate guanylate cyclase by pyrophosphate (0.02-1 mM) was observed; the pyrophosphate effect appeared to be competitive with respect to GTP. At a higher concentration (2 mM), pyrophosphate produced a marked inhibition of particulate enzyme; the nature of inhibitory effect appeared complex. 7. Inorganic salts (e.g. NaCl, KCl, LiBr, NaF) produced inhibition of particulate enzyme; the degree of inhibition of Triton X-100-stimulated activity was less than that of unstimulated activity. 9. Treatment of sarcolemmal or microsomal membranes with either phospholipase C or trypsin decreased, whereas phospholipase A increased, the activity of guanylate cyclase.


Changes in conformation of reduced cytochrome c in neutral aqueous solution. In neutral aqueous solution, high concentrations of alcohols perturb cytochrome c in such a way that reduction by means of ionizing radiation results in the formation of abnormal conformers of ferrocytochrome c resembling those previously seen on reduction in alkaline solution. These relax to normal ferrocytochrome c in about 0.1 s.


The effects of diphenyleneiodonium and of 2,4-dichlorodiphenyleneiodonium on mitochondrial reactions. Mechanism of the inhibition of oxygen uptake as a consequence of the catalysis of the chloride/hydroxyl-ion exchange. 1. Increasing the substrate concentration only decreased the inhibition of mitochondrial oxidations by diphenyleneiodonium or by 2,4-dichlorophenyleneiodonium by a small amount. 2. Diphenyleneiodonium and 2,4-dichlorodiphenyleneiodonium lowered the amounts of succinate, citrate and glutamate accumulated in the matrix of mitochondria in the presence of Cl-, but not in its absences. 2,4-Dichlorodiphenyleneiodonium decreased the accumulation of substrates by mitochondria oxidizing glycerol 3-phosphate. 3. Diphenyleneiodonium caused an alkalinization of the medium with an anaerobic suspension of mitochondria, which was only partly reversed by Triton X-100. 4. The rate of proton extrusion by mitochondria oxidizing succinate was not altered by diphenyleneiodonium or by 2,4-dichlorodiphenyleneiodium, although the rate of decay of proton pulses was increased. 5. 2,4-Dichlorodiphenyleneiodonium shifted the pH optimum for succinate oxidation by intact mitochondria from pH 7.2 to 8.0, whereas there was no effect on that of freeze-thawed mitochondria, which was pH 8.0. 6. The concentration of 2,4-dichlorophenyleneiodonium required to inhibit respiration by 50% is less the higher the absolute rate of oxygen uptake. 7. EDTA, but not EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid] increased the inhibition of respiration by diphenyleneiodonium, 2,4-dichlorodiphenyleneiodonium and by tri-n-propyltin. 8. It is concluded that diphenyleneiodonium and 2,4-dichlorodiphenyleneiodonium limit respiration in Cl--containing medium by causing an acidification of the matrix, and that there are pH-sensitive sites in the respiratory chain between NADH and succinate, and between succinate and cytochrome c.


Properties and developmental regulation of an alpha-d-galactosidase from Dictyostelium discoideum. An alpha-D-galactosidase was detected in cells of the cellular slime mould, Dictyostelium discoideum, at all stages of development. Its specific activity was highest during early development (interphase), and this accumulation of enzyme appears to require protein synthesis de novo. Its subcellular distribution differs from that of other D. discoideum glycosidases, since most activity was recovered in the soluble fraction. No evidence was obtained for more than one isoenzymic form after subjection of extracts to electrophoresis and various chromatographic procedures. It is excreted from the cell during development, but no evidence was found for an extracellular function for the enzyme.


Adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterases of guinea-pig cardiac sarcolemma. 1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an 'activated' form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme.


Maternal blood-gases, PAo2--Pao2), hysiological shunt and VD/VT in normal pregnancy. Serial measurements of maternal blood-gases, alveolar-to-arterial oxygen tension difference (PAO2--PaO), calculated pulmonary venous admixture (physiological shunt), deadspace/tidal volume ratio (VD/VT), and respiratory minute volume have been made in a carefully selected group of normal pregnant patients at 12, 24, 32 and 38 weeks of gestation and 5 weeks after delivery. All measurements were made in the semi-recumbent position with a left lateral pelvic tilt. Mean arterial PO2 was consistently greater than 100 mm Hg throughout pregnancy, although the value decreased from 106.4 mm Hg at 12 weeks of gestation to 101.8 mm Hg at the 38th week. Despite this decrease there was no change in (PAO2--PaO2) VD/VT, or percentage shunt with increasing gestation; nor did these values differ significantly from non-pregnant values in the same patients.


Metabolism of lorazepam.

Metabolism of lorazepam. The metabolism of lorazepam by man and four other species is reviewed. Lorazepam and its metabolites in blood, urine and faeces were identified by thin-layer and gas chromatography and by mass spectrometry. The principal metabolite in man, dog, pig and cat is the glucuronide, but the rat produces other metabolites after small doses of lorazepam, and significant amounts of the glucuronide only after high doses. Since all metabolites, except the glucuronide, occur in small quantities only in man, most studies in man have been confined to an estimation of gree and conjugated lorazepam. Blood concentrations of unconjugated lorazepam peak at 1-4 h, significant concentrations persisting for 24 h and decreasing slowly over the next 24 h. About 95% of a dose of lorazepam was accounted for in urine and faeces over a period of 5 days; 74.5% was excreted in the urine as lorazepam glucuronide and 13.5% as minor metabolites. The excretory half-life was 12 h. The blood concentrations and excretion rates are compatible with the clinical effects of lorazepam.


Comparison of the beta2-adrenoceptor selectivity of rimiterol, salbutamol and isoprenaline by the intravenous route in man. 1 The bronchodilating efficacy and the degree of beta2-adrenoceptor selectivity of rimiterol, salbutamol and isoprenaline were determined in seven subjects who exhibited histamine-induced bronchoconstriction. 2 Rimiterol, 0.5 (high dose) and 0.05 (low dose) mug kg-1 min-1, salbutamol, 0.3 and 0.03 mug kg-1 min-1, isoprenaline, 0.05 and 0.005 mug kg-1 min-1 and placebo were administered by a single intravenous injection over 6 min, and the protection against histamine-induced bronchoconstriction, changes in heart rate, pulse pressure and skeletal muscle tremor were measured. 3 Rimiterol (98%), salbutamol (96%) and isoprenaline (69%) protected against histamine-induced bronchoconstriction. For these ventilatory responses, there was a heart rate increase of 31.9, 24.7 and 44.3 beats/min for rimiterol, salbutamol and isoprenaline respectively. The three drugs produced similar increases in pulse pressure and tremor. 4 Significant dose-responses were obtained for all the parameters with each drug. 5 Isoprenaline was approximately 7 and 5 times as potent as rimiterol and salbutamol respectively in bronchodilator action when equimolar doses were compared. Similarly, isoprenaline was approximately 14 and 10 times as potent in increasing the heart rate as rimiterol and salbutamol respectively. 6 Rimiterol, a new beta-adrenoceptor stimulating drug, is an effective bronchodilator and has similar beta2-adrenoceptor selectivity to salbutamol when administered intravenously. The relative potencies and degrees of beta2-adrenoceptor selectivity of these drugs depend partly on their route of administration.


Comparison of the residual effects of two benzodiazepines (nitrazepam and flurazepam hydrochloride) and pentobarbitone sodium on human performance. 1 The residual effects of two benzodiazepines, nitrazepam (10 mg) and flurazepam hydrochloride (30 mg), and pentobarbitone sodium (200 mg) were studied by adaptive tracking and by reaction time. Performance was measured at 10 h, 13 h, 16 h, 19 h and 34 h after ingestion of each drug. Impaired performance on adaptive tracking was observed at 10 h, 13 h, 16 h and 19 h after nitrazepam and pentobarbitone sodium and at 10 h, 13 h and 16 h after flurazepam hydrochloride. Enhanced performance was observed at 34 h after nitrazepam and pentobarbitone sodium. 2 Increased reaction time persisted to 16 h after nitrazepam, flurazepam hydrochloride and pentobarbitone sodium and reaction time was also increased at 34 h after nitrazepam and pentobarbitone sodium. 3 During the morning immediately after ingestion, the subjects as a group were able to differentiate correctly between placebo and drugs, but they were not able to assess accurately the persistence of the residual effects of nitrazepam and pentobarbitone sodium. 4 Flurazepam hydrochloride would appear to be a more promising benzodiazepine than nitrazepam for use as a hypnotic by persons involved in skilled activity. There was a rapid recovery of performance during the afternoon and, unlike pentobarbitone sodium and nitrazepam, subjects retained the ability to recognize impaired skill.


Residual effects of flunitrazepam.

Residual effects of flunitrazepam. 1 Twelve normal subjects were tested on a large battery of tests after a hypnotic dose of flunitrazepam(1 or 2 mg) and a placebo. Psychological tests were given 12 h after the drug and physiological tests 12, 15 and 18 h after the drug. 2 The tests included self-ratings of hypnotic effects and mood, the electroencephalogram, the auditory electroencephalographic evoked response, skin conductance, tapping, card-sorting and the symbol copying test. 3 Both doses of flunitrazepam were effective hypnotics according to the ratings, with an anxiolytic effect the following day. The EEG was significantly altered up to 18 h after the drug and the behavioural tests showed a motor impairment 12 h after drug administration. 4 Nearly all changes displayed linear dose-related trends.


A clinical and psychometric evaluation of flurazepam. 1 The efficacy of flurazepam (15 mg or 30 mg) as a hypnotic, and the residual effects of each dose were compared with placebo in a double-blind cross-over trial involving thirty patients in a general practice setting. Patients received each medication for one week. Daily self-ratings of onset, duration and quality of sleep, together with reports of any untoward effects were made. At the end of each period of medication psychomotor tests (reaction time, pursuit rotor, tapping speed) were administered at 09.00 hours. 2 Both doses of flurazepam were significantly more effective than placebo in inducing sleep, improving the quality of sleep and extending its duration. 3 'Hangover' effects were marked following 30 mg, but not after flurazepam (15 mg). Flurazepam (30 mg, but not 15 mg) significantly impaired performance on the pursuit rotor test and tapping speed. Flurazepam thus appears to be an effective hypnotic drug with the optimum dose for use in general practice being 15 mg at night.


Bioavailability and dissolution of different formulations of oxytetracycline preparations. 1 The concentration of oxytetracycline in plasma was studied by microbiological assay after oral administration of five different preparations of the antibiotic. None of these preparations had been studied previously. 2 There was a statistically significant correlation between the time required for 50% dissolution at pH 2 and biological availability, as assessed by the peak plasma level or the area under the plasma concentration-time curve. 3 The mean bioavailability of oxytetracycline was greatest with preparations of the hydrochloride, and with film-coated tablets of the dihydrate. In contrast, sugar-coated tablets of oxytetracycline dihydrate were associated with poorer dissolution characteristics and reduced biological availability.


A review of the animal pharmacology of labetalol, a combined alpha- and beta-adrenoceptor-blocking drug. 1 The animal pharmacology of labetalol, a drug that blocks both alpha- and beta-adrenoreceptors, is reviewed. 2 In isolated tissues the blockade by labetalol of both alpha- and beta-adrenoreceptors satisfied accepted critera for competitive antagonism. In contrast phentolamine was a competitive antagonist at alpha-adrenoreceptors only, and propranolol a competitive antagonist at beta-adrenoreceptors only. Labetalol was 6-10 times less potent than phentolamine in blocking alpha-adrenoreceptors and 1.5-3 times less potent than propranolol in blocking beta-adrenoreceptors. Labetalol itself was 4-8 times more potent at beta- than at alpha-adrenoreceptors. 3 In anaesthetized dogs labetalol given intravenously blocked vasopressor responses to phenylephrine positive chronotropic, vasodepressor and bronchodilator response to isoprenaline. Phentolamine blocked the effect of phenylephrine only, and propranolol the effects of isoprenaline only. Labetalol was about 7 times less potent than phentolamine in blocking alpha-adrenoreceptors, about 4 times less potent than propranolol in blocking cardiac beta1-adrenoreceptors, and 11-17 times less potent than propranolol in blocking vascular and bronchial beta2-adrenoceptors. This difference in the relative potency of labetalol arises because propranolol is a slightly more potent antagonist at beta2- than at beta1-adrenoreceptors. Labetaol itself was about 16 times more potent at cardiac beta1- than at vascular alpha-adrenoreceptors. In conscious dogs labetaol given orally blocked vasopressor responses to phenylephrine and positive chonotropic responses to isoprenaline. 4 In anesthetized dogs and pithed rats labetaol blocked alpha- or beta-adrenoreceptor-mediated responses to sympathetic nerve stimulation and intravenously administered phenylephrine or isoprenaline to approximately the same extent. 5 Labetalol does not possess partial agonist (intrinsic sympathomimetic) activity at cardiac beta1-adrenoreceptors. 6 The blocking action of labetalol both in vivo was shown to be specific for alpha- and beta-adrenoreceptors. 7 The haemodynamic effects of labetalol are attributable to its adrenoreceptor-blocking actions. The observed responses vary from one experiment situation to another depending on the balance of autonomic influences. For example, in barbitone-anaesthetized dogs, in which sympathetic tone predominates, both labetalol and propranolol reduced heart rate, contractility, output and work--effects which are attributable to beta-adrenoreceptor blockade. Labetalol differed from propranolol in decreasing rather than increasing total peripheral resistance and in causing larger falls in blood pressure at equipotent beta-adrenoreceptor-blocking doses. These differences are probably attributable to peripheral vasodilatation resulting from the vascular alpha-adrenoreceptor-blocking action of labetalol...


Pharmacological effects of labetalol in man. 1 The pharmacological effects of labetalol have been studied in normal healthy subjects. The results of these studies are reviewed. 2 In the evaluation of the beta-adrenoreceptor-blocking effects of labetalol various indices of beta-adrenoceptor blockade in man were used. Labetalol administered orally and intravenously competitively antagonized the effects of isoprenaline on heart rate and diastolic blood pressure. The beta-adrenoceptor blockade induced was regarded as 'non-selective'. In addition, labetalol produced dose-related inhibitory effects on exercise-induced increases in heart rate any systolic blood pressure and similar dose-related inhibitory effects on the tachycardia induced by valsalva's manoeuvre. Labetalol had only a modest inhibitory effect on the tachycardia induced by tilting since blood pressure was reduced on a dose-related basis. 3 Labetalol was a specific competitive antagonist of the alpha-adrenoceptor agonist effects of systemically administered phenylephrine and locally infused noradrenaline. In addition, oral and intravenous administration of labetalol reduced systolic and diastolic blood pressure in the supine, standing and sitting positions. 4 The onset and duration of the alpha- and beta-antagonist effects of oral labetalol did not seem to be dissociated in time and there was a close correlation between the change in plasma concentration and pharmacological effects. 5 In comparative studies with propranolol, similar beta-antagonist effects were observed but propranolol was 4-6 times more potent weight for weight. Precise comparison, however, was complicated by the combined alpha- and beta -effects of labetalol, especially as the predominant effect of labetalol in normotensive subjects was to reduce blood pressure; whereas the predominant effect of propranolol was to reduce heart rate. In addition propranolol had inhibitory effects on ventilatory function in normal subjects, whereas labetalol in equivalent beta-adrenoreceptor-blocking doses did not. 6 From the details of the studies reviewed it was concluded that in man labetalol possesses combined alpha- and beta-adrenoreceptor antagonist properties.


Haemodynamic effects of combined alpha- and beta-adrenoreceptor blockade after intravenous labetalol in hypertensive patients at rest and during exercise. 1 The haemodynamic effects of labetalol 50 mg intravenously were studied in 13 hypertensive patients at rest in the supine and upright positions and during exercise using percutaneous right heart and brachial artery catheterization. 2 Labetalol induced immediate significant reductions in systolic and diastolic blood pressures in all conditions. 3 Heart rate, stroke volume and cardiac output were not significantly altered in the supine position at rest but were significantly reduced in the upright position. In addition systemic vascular resistance was significantly reduced. 4 During exercise, heart-rate, cardiac output and vascular resistance were significantly reduced; stroke volume was increased. Left ventriclac filling pressures were unchanged both at rest and during exercise. 5 It was concluded that these changes resulted from blockade to both alpha- and beta-adrenoreceptor.


Combined alpha- and beta-adrenoreceptors blockade with oral labetalol in hypertensive patients with reference to haemodynamic effects at rest and during exercise. Twelve hypertensive patients received oral labetalol treatment (average final dose 1,200 mg/d) over a period of 16 months. 2 Satisfactory blood pressure control was maintained both at rest and after exercise. 3 Plasma renin activity was significantly reduced. 4 Postural hypotension measured in this group of patients after a single dose of labetalol 50 mg intravenously was considerably reduced during long-term oral treatment. 5 No serious side-effects were observed nor were there any significant changes in haematological and biochemical variables during the period. 6 Haemodynamic data obtained in two of the patients after 16 months' treatment suggests that the haemodynamic profile observed after acute intravenous administration is maintained after prolonged oral treatment.


Labetalol, a new alpha- and beta-adrenoreceptor blocking agent, in hypertension. 1Labetalol is a new compound with antagonistic effects at both alpha- and beta-adrenoreceptor sites. 2 When given to 12 hypertensive patients at an average daily dosage of 273 mg for 7 months statistically significant reductions (compared with pretreatment values) in recumbent and standing blood pressure were observed. 3 Treatment has to be withdrawn in one patient because of vivid dreams, and dosage was reduced in one patient because of dizziness. Otherwise no side-effects of importance were noted. 4 It can therefore be concluded that labetalol offers a useful anti-hypertensive effect and that this compound is well tolerated.


Intravenous labetalol in hypertensive patients treated with beta-adrenoreceptor-blocking drugs. 1 The effect of intravenous injections of labetalol at doses of 1 and 2 mg/kg was studied in 15 patients with severe hypertension inadequately controlled by beta-adrenoreceptor-blocking drugs. 2 At a dose of 1 mg/kg there was a slight but statistically insignificant reduction in blood pressure. When 2 mg/kg was given there was a prolonged (6 h) significant reduction in lying blood pressure. At both dose levels there was a small initial increase in pulse rate but forced peak flow was not affected. 3 Side-effects were limited to transient postural hypotension and a feeling of warmth. 4 It is concluded that intravenous labetalol at a dose of 2 mg/kg is safe and effective in rapidly reducing blood pressure in patients already receiving beta-adrenoreceptor-blocking drugs.


Arterialized ear lobe blood samples for blood gas tensions. The accuracy of arterialized blood samples both at rest and during exercise is described in comparison to simultaneous arterial blood samples. The technique was found to be reliable and sufficiently accurate for clinical exercise testing, with no significant differences for Po2 or Pco2 between the two methods.


Preparation and storage of platelet concentrates. II. Storage variables influencing platelet viability and function. Factors affecting the viability and function of stored platelet concentrates have been investigated in a blood component programme. It was found that platelets could be maintained for up to 72 h without bacterial contamination under the following conditions: (1) surgical skin preparation at venipuncture site; (2) blood collection in CPD or ACD anticoagulant in a closed bag system; (3) centrifugation of PRP at 3000 g for 20 min; (4) storage in Fenwal PL-146, Cutter CL-2383, or McGaw plastic bags; (5) resuspension of the platelet pellet in 70 ml residual plasma; (6) storage at 22+/-2 degrees C; and (7) constant gentle mixing throughout storage. Platelet viability as determined by recovery and survival is largely maintained, as is platelet function measured by template bleeding time. Both viability and function of concentrated platelets stored at 4 degrees C are severely compromised.


Solubilization, partial purification and radioassay for the intrinsic factor receptor from the ileal mucosa. A macromolecule which binds intrinsic factor saturated with vitamin B12 has been solubilized from the guinea-pig ileum by homogenization followed by mechanical disruption without organic solvents or detergents. This intrinsic factor 'receptor' was further purified by precipitation with 30% saturated ammonium sulphate, centrifugation at 105000 g, and filtration through Sephadex G-200. Failure to precipitate the receptor following centrifugation at 105000 g for 3 h and filtration of the receptor with the included volumes through Sepharose 4B and 6B was evidence that it was solubilized. The purification of the receptor was monitored by a radiometric assay where the intrinsic factor-[57Co]vitamin-B12 complex coupled to the solubilized receptor precipitated at 15% sodium sulphate while intrinsic factor-[57Co]B12 alone remained soluble at this salt concentration. This radioassay also permitted the in vitro study of the interaction of the solubilized receptor and intrinsic factor saturated with [57Co]B12. The receptor did not bind intrinsic factor-[57Co]B12 below pH 5 while binding was observed to pH 9.0. Binding was equivalent at 37 degrees C and 25 degrees C, but was markedly reduced at 4 degrees C and 56 degrees C and was destroyed at 100 degrees C. The receptor resisted 60 min of digestion by trypsin, chymotrypsin, pronase and subtilisin. After 180 min digestion, pronase and subtilisin inactivated 90% and 41% of the receptor respectively, whereas trypsin and chymotrypsin inactivated only 21% and 23%. Trisodium EDTA inhibited the binding of intrinsic factor-[57Co]B12 to the receptor and this inhibition could be reversed by the addition of excess Ca2+. Mg2+ and Mn2+ were less effective than Ca2+ for the activity of the receptor. Kinetic analysis of the reaction indicated a maximum velocity of 0.083 nmole IF bound B12/min with a Km of 1.36 x 10(-10) M. The solubilized receptor had a greater affinity for intrinsic factor bound to vitamin B12 than for intrinsic factor free of vitamin B12. The solubilization of this intrinsic factor receptor without chemicals suggests that it is not an integral component of the microvillus membranes hydrophobically bonded to the lipid matrix, but rather a peripheral protein weakly associated with the membrane by non-covalent interaction.


Effect of pyridoxal 5'-phosphate on the oxygen affinity of human erythrocytes. Pyridoxal 5'-phosphate (PLP), an allosteric effector for the oxygenation of haemoglobin, was incorporated readily into erythrocytes and disappeared from them by simple passive diffusion. The disappearance of PLP from the cells was accelerated by the generation of 2,3-DPG in a medium of inosine, pyruvate and phosphate. The oxygen dissociation curve measured at an extracellular pH of 7.4 demonstrated that PLP incorporated into the cells also lowered the oxygen affinity and that PLP functionally compensated for a metabolically reduced 2,3-DPG. However, the dependency of the oxygen affinity on the intracellular PLP concentration showed a different pattern from the observed for 2,3-DPG. On the other hand, the lowering of intracellular pH by organic phosphates accumulated in the cells was much larger with PLP than with 2,3-DPG. The peculiar relationship between the oxygen affinity of erythrocytes and the intracellular PLP concentration is discussed in detail. The present study may offer a new prospect for the preservation of blood with a normal function.


Phosphorus-31 Fourier transform nuclear magnetic resonance study of mononucleotides and dinucleotides. 1. Chemical shifts. A phosphorus-31 nuclear magnetic resonance (NMR) study of adenine, uracil, and thymine mononucleotides, their cyclic analogues, and the corresponding dinucleotides is reported. From the pH dependence of phosphate chemical shifts, pKa values of 6.25-6.30 are found for all 5'-mononucleotides secondary phosphate ionization, independently from the nature of the base and the presence of a hydroxyl group at the 2' position. Conversely, substitution of a hydrogen atom for a 2'-OH lowers the pKa of 3'-monoribonucleotides from 6.25 down to 5.71-5.85. This indication of a strong influence of the 2'-hydroxyl group on the 3'-phosphate is confirmed by the existence of a 0.4 to 0.5 ppm downfield shift induced by the 2'-OH on the phosphate resonance of 3'-monoribonucleotides, and 3',5'-cyclic nucleotides and dinucleotides with respect to the deoxyribosyl analogues. Phosphate chemical shifts and titration curves are affected by the ionization and the type of the base. Typically, deviations from the theoretical Henderson-Hasselbalch plots are observed upon base titration. In addition, purine displays a more deshielding influence than pyrimidine on the phosphate groups of most of the mononucleotides (0.10 to 0.25 ppm downfield shift) with a reverse situation for dinucleotides. These effects together with the importance of stereochemical arrangement (furanose ring pucker, furanose-phosphate backbone conformation, O-P-O bond angle) on the phosphate chemical shifts are discussed.


Reaction of yeast carboxypeptidase C1 with group-specific reagents. The reactions between yeast carboxypeptidase C and the group-specific reagents, phenylglyoxal and iodoacetamide, have been studied in detail and the reactions of residue at the active site with N-tosyl-L-phenylalanine chloromethyl ketone and diisopropyl phosphorofluoridate have been confirmed. Modification of the enzyme by either phenylglyoxal or iodoacetamide results in the loss of peptidase activity, while esterase activity remains unchanged. Inactivation by phenylglyoxal appears to be the result of the modification of a single arginine residue, whereas inhibition by iodoacetamide can be correlated with the modification of a single methionine residue. Inactivation of the enzyme by either N-tosyl-L-phenylalanine chloromethyl ketone or diisopropyl phosphorofluoridate is the result of the modification of a single histidine and a single serine residue, respectively. The pattern of inhibition indicates certain analogies in the mechanism of yeast carboxypeptidase C to pancreatic chymotrypsin, on the one hand, and to carboxypeptidase A, on the other.


Identification and properties of the covalently bound flavin of beta-cyclopiazonate oxidocyclase. Beta-Cyclopiazonate oxidocyclase from Penicillium cyclopium has been previously shown to contain flavin dinucleotide in covalent linkage to the protein. In the present study, a pure flavin mononucleotide peptide was isolated from the enzyme by tryptic-chymotryptic digestion, chromatography on Florisil and on diethylaminoethylcellulose, and hydrolysis with nucleotide pyrophosphatase. The flavin peptide contains 9 amino acids, including histidine in linkage to the flavin, and Asx as the N-terminal residue. The fluorescence of the flavin in the FMN peptide is profoundly quenched even at pH 3.2, where protonation of the imidazole prevents queching of the flavin fluorescence by histidine. This quenching appears to be due to interaction of the flavin with a tryptophan residue, as the quenching is abolished by oxidation of the tryptophan with performic acid. Similarly, the fluorescence of the tryptophan in the peptide is quenched, presumably by the flavin. The flavin of beta-cyclopiazonate oxidocylcase is attached, by the way of the 8alpha-methylene group, to the imidazole ring of a histidine. The aminoacylflavin isolated from the enzyme is identical in the pKa of its imidazole group, in reduction by NaBH4, and in other properties with synthetic 8alpha-(N1-histidyl)riboflavin. The pKa of the histidylriboflavin component of the oxidocyclase is 5.2 before and 5.0 after acid modification of the ribityl chain, as is found in the synthetic derivative. It is concluded that the enzyme contains the N1 isomer of histidylriboflavin and that acid hydrolysis of flavin peptides isolated from the oxidocyclase, while liberating histidylriboflavin, also causes acid modification of the ribityl chain of the flavin moiety.


Potentiometric determination of ionizations at the active site of papain. The ionization behavior of groups at the active site of papain was determined from the pH dependence of the difference of proton content of papain and the methylthio derivative of the thiol group at the active site of papain (papain-S-SCH3). This difference in proton content was determined directly by two independent methods. One method involved potentiometric measurements of the protons released and demethylthiolation of papain-S-SCH3 with dithiothreitol, as a function of pH. The other method involved analogous measurements of the protons released on methylthiolation of papain with methyl methanethiosulfonate. The methylthio pH-difference titrations generated by these measurements indicate that ionization of the thiol group at the active site of papain is linked to the ionization of His-159. The pK of the thiol group changes from 3.3 to 7.6 on deprotonation of His-159 at 29 degrees C/20.05. Similarly, the pK of His-159 shifts from 4.3 to 8.5 when the active site thiol group is deprotonated. The microscopic ionization constants determined in this work for Cys-25 and His-159 indicate that equilibrium constant for transfer of the proton from Cys-25 to His-159 is 8--12, and that in the physiological pH range the active site thiol group exists mainly as a thiol anion.


Studies on the noncooperative binding of the Escherichia coli DNA unwinding protein to single-stranded nucleic acids. The noncooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA oligomers has been studied by means of equilibrium dialysis. Dialyses were performed under a number of solution and temperature conditions using oligomers of varying length and base compositions. The results of these studies, which include a Scatchard analysis of the binding, have allowed us to propose a model for the cooperative binding of the protein to single-stranded DNA. The results of experiments dealing with the interaction of the protein with single-stranded RNA are also presented.


A simple, quantitative approach to the coupling of photophosphorylation to electron flow in terms of proton fluxes. A simple relationship between observed phosphorylation efficiencies (P/e ratios) and internal proton concentration in spinach chloroplast thylakoids has been derived. P/e ratios, varked by either changing the light intensity or by adding the energy transfer inhibitor, 4'-deoxyphlorizin, were found to change with internal proton concentration in accordance with this relationship. A quantitative prediction of the effect of uncouplers on the P/e ratio can probably also be made. By extrapolation of plots of observed P/e ratios against internal proton concentration divided by the overall rate of electron flow, a maximum intrinsic P/e of about 0.66 is obtained. Assuming that two protons appear inside thylakoids per electron transferred, a P/e ratio of 0.66 suggests that three internal protons are consumed for each ATP formed. Internal protons may be considered to be substrates for the phosphorylation reaction. Hill plots of phosphorylation rate vs. internal proton concentration also indicate that three protons are consumed for each ATP synthesized. Thus, the H+ concentration gradient behaves quantitatively, as well as qualitatively, as if it is the connecting link between electron flow and phosphorylation in illuminated thylakoids.


Properties and subcellular distribution of guanylate cyclase activity in rat renal medulla: correlation with tissue content of guanosine 3',5'-monophosphate. The properties of the guanylate cyclase systems of outer and inner medulla of rat kidney were examined and compared with those of the renal cortex. A gradation in steady-state cyclic guanosine 3',5'-monophosphate (cGMP) levels was observed in incubated slices of these tissues (inner medula greater than outer medulla greater than cortex). This correlated with the proportion of total guanyl cyclase activity in the 100 000 g particulate fraction of each tissue, but was discordant with the relative activities of guanylate cyclase (highest in cortex) and of cGMP-phosphodiesterase (lowest in cortex) in whole tissue homogenates. Soluble guanylate cyclase of cortex and inner medulla exhibited typical Michaelis-Menten kinetics with an apparent Km for MnGTP of 0.11 mM, while the particulate enzyme from inner medulla exhibited apparent positive cooperative behavior and a decreased dependence on Mn2+. Thus, the particulate enzyme could play a key role in regulating cGMP levels inthe intact cell where Mn2+ concentrations are low. The soluble and particulate enzymes from inner medulla were further distinguished by their responses to several test agents. The soluble enzyme was activated by Ca2+, NaN3, NaNo2 and phenylhydrazine, whereas particulate activity was inhibited by Ca2+ and was unresponsive to the latter agents. In the presence of NaNo2, Mn2+ requirement of the soluble enzyme was reduced and equivalent to that of the particulate preparation. Moreover, relative responsiveness of the sollble enzyme to NaNO2 was potentiated when Mg2+ replaced Mn2+ as the sole divalent cation. These changes in metal requirements may be involved in the action of NaNO2 to increase cGMP in intact kidney. Soluble guanylate cyclase of cortex was clearly more responsive to stimulation by NaN3, Nano2, and phenylhydrazine that was soluble activity from either medullary tissue. The effectiveness of the agonists on soluble activity from outer and inner medulla cound also be distinguished. Accordingly, regulation and properties of soluble guanylate cyclase, as well as subcellular enzyme distribution, and distinct in the three regions of the kidney.


Thermal transitions of myosin and its helical fragments. Regions of structural instability in the myosin molecule. The structural stabilities of all the familiar proteolytic fragments of myosin have been investigated in melting studies over the pH ranges 5.5-7.0 in 0.5 M KCl. All fragments except subfragment 2 undergo a melting transition manifested by the cooperative uptake of protons in the temperature range 34-47 degrees C, and these fragments experience an increase in transition temperature, Tm as the pH is increased. Subfragment 2 undergoes a melting transition in the 43-55 degrees C range, manifested by the dissociation of protons, and it experiences a decrease in Tm as the pH is increased. These results suggest that pH changes can modulate the relative stabilities of the light meromysin, subfragment-1, and subfragment-2 regions of the myosin molecule.


Relative stabilities of the two quaternary conformations of human fetal hemoglobin. The pH dependence of several functional properties of human fetal and adult hemoglobins have been studied to determine the relative stabilities of the high and low affinity (R and T) quaternary conformations of the two proteins under different conditions. Fetal aqumethemoglobin undergoes changes in sulfhydryl reactivity, absorption spectrum, and circular dichroism in the presence of insitol hexaphospahte which are consistent with a transition from the R to T quaternary state, but only at pH values below 6.8. In adult hemoglobin this transition can be induced pH values below 7.2. Even in the absence of phosphates, the ultraviolet (uv) circular dichroism spectrum of fetal aquomethemoglobin at low pH indicates the presence of some T conformation. The initial value for the second-order rate constant for combination of fetal deoxyhemoglobin with carbon monoxide is comparable to that for adult hemoglobin in the absence of organic phosphates and is not reduced by organic phosphates as much as that for the adult protein. The apparent first-order rate constant for dissociation of CO from fully liganded fetal hemoglobin, measured by replacement with NO, increases threefold in the absence of organic phosphates, and fourfold in the presence of organic phosphates, with decreasing pH; the midpoint of the pH dependent transition occurs around 6.8. A similar increase in the apparent first-order rate constant for O2 dissociation as measured by replacement with CO, can also be seen with decreasing pH. NO-hemoglobin F can be converted to the T state even when fully liganded simply by lowering the pH, as judged by uv circular dichroism, visible difference spectrum in the region of the alpha and beta bands, and a dramatic increase in the rate of NO dissociation, measured by replacement with CO in the presence of dithionite. These results are all consistent with a model for fetal hemoglobin in which the organic phosphate site may be functionally weakened by replacement of a residue involved in ionic interactions with the negatively charged phosphate groups, but in which the low affinity T conformation is intrinsically more stable than that of adllt hemoglobin. According to this model,the differences between fetal and adult hemoglobin can be accounted for primarily in terms of the relative stabilities of R and T conformations in each of the proteins with differences in the intrinsic properties of the individual conformations contributing effects of only secondary importance.


Adenine nucleotide metabolism of blood platelets. IX. Time course of secretion and changes in energy metabolism in thrombin-treated platelets. Changes in the energy metabolism of washed human platelets were compared with the kinetics of secretion induced by thrombin (5 units/ml). A 50% decrease in the level of metabolic ATP (3H-labelled), which was essentially complete in 30s, was matched in rate by adenine nucleotide secretion from storage in dense granules. Incubation of platelets with antimycin before thrombin addition increased the rate of fall in metabolic ATP, but did not affect the rate of adenine nucleotide secretion. beta-N-Acetylglucosaminidase secretion, which was slower than adenine nucleotide secretion in control platelets, was noticeably inhibited by antimycin, confirming previous reports that different regulatory mechanisms exist for dense and alpha-granule secretion. The rates of rephosphorylation of metabolic ADP to ATP via glycolysis and oxidative phosphorylation were estimated by measuring lactate production and O2 consumption in resting and thrombin-stimulated platelets and compared to the level of metabolic ATP (9-10 nmol/mg of platelet protein in the resting state). The rate of ATP production was stimulated at least two fold from 12 nmol to 24 nmol/min/mg within seconds of thrombin addition. This increased rate was maintained over the observed period of 5 min although the level of metabolic ATP had decreased to 4-5 nmol/mg within 30 s; the turnover of the remaining metabolic ATP thus increased four fold over the resting state although the actual stimulation of energy production was only two fold.


Ammonia production and pathways of glutamine utilization in rat kidney slices. Ammonia production from glutamine was studied in slices from non-acidotic and acidotic rat kidneys. Slices from non-acidotic kidneys made 53% as much ammonia from D-glutamine as from L-glutamine during the initial 15 min of incubation. Thereafter the production rate from the L-isomer accelerated while that from the D-isomer remained constant. The accelerated rate of ammonia production from L-glutamine was dependent upon tissue swelling since prevention of swelling reduced the production rate. Swelling activates the mitochondrial glutaminase I pathway as evidenced by the rise in ammonia produced per glutamine utilized ratio as well as by the accelerated rate of CO2 production derived from the oxidative disposal of glutamin's carbon skeleton. Cortical slice swelling activates the mitochondrial pathway in a manner not unlike that seen in vivo during chronic acidosis and may reflect increased permeability to glutamine. Acidotic rat kidneys are not swollen in vivo while cortical slices initially produce 4-fold more ammonia than do non-acidotic slices. After 15 min, this 4-fold difference in total ammonia production drops to only a 2-fold difference due to the swelling-induced activation of the mitochondrial pathway. Consequently, slice swelling obliterates the important fact that ammonia production by the mitochondrial pathway is 15-fold greater in acidotic than in non-acidotic kidneys.


Effect of imidazole on renal gluconeogenesis. The metabolic effects of imidazole were tested in rat renal cortex. Imidazole enhanced the activity of renal cortical phosphodiesterase in vitro. Imidazole inhibited glucose production in a dose-dependent fashion from a variety of substrates in the gluconeogenic pathway proximal to the triose phsophates. The stimulation in renal gluconeogenesis resulting from isoproterenol and parathyroid hormone was inhibited by imidazole. These changes correlated with an inhibition of the augmented levels of renal cortical cyclic AMP levels produced by these hormones. These studies indicate that imidazole is an effective activator of phosphodiesterase in intact renal cells and lend further support to the suggestion that the stimulation of renal gluconeogenesis produced by isoproterenol and parathyroid hormone is mediated by a release of cyclic AMP.


Vitamin B-6-catalyzed beta-elimination of serine and O-phosphoserine. Qualitative and quantitative aspects of catalytic influences at the rate-limiting step, a comparison with the rate of enzymatic beta-elimination. The overall reaction rates for the beta-elimination of serine and O-phosphoserine, catalyzed by various vitamin B-6 analogs (pyridoxal 5'-phosphate, 5'-deoxypyridoxal and N-methylpyridoxal 5'-phosphate) in the presence or absence of Cu2+ ions, are determined. The comparison of the pH-dependence of the molar activities of the three vitamin B-6 aldehydes in beta-elimination of serine enables the characterization of the different active Schiff base species and the single catalytic events. The Schiff base which has a positive charge on the pyridine ring nitrogen and a fully ionized phosphate group shows the highest molar activity. The phosphate group acts as an intramolecular general base catalyst, most probably at the alpha-carbon proton of the amino acid. Furthermore general acid catalysis by buffer species occurs at the beta-hydroxy group serine. These facts together provide a kinetically unambiguous description of the mechanism of the reaction: the removal of the proton at the alpha-carbon atom of serine is the rate-limiting step and is followed by the more rapid elimination of the b-hydroxy group of serine. The forward rate constant of the rate-limiting step is calculated for each of the reactions mentioned. The rate constants are compared with respect to the effectiveness of the individual catalytic components in the vitamin B-6-dependent beta-elimination. For optimal conditions the reaction of O-phosphoserine is faster by a factor of 10(4) in the velocity of the beta-elimination than the corresponding acid-catalyzed beta-elimination of serine. For the eliminations at the alpha- and beta-carbon atoms of O-phosphoserine in vitamin B-6-catalysed reactions a common transition state is discussed. From a comparison of the fastest vitamin beta-6-dependent model reaction with the rate of an enzymatic beta-elimination it is suggested that for those beta-elininating enzymes where the rate-limiting step is the same as in the model, the catalytic components mentioned could suffice to explain the velocity of the rate-limiting step.


Properties and subcellular localization of CMP-N-acetylneuraminic acid hydrolase of calf kidney. The properties and subcellular distribution of CMP-N-acetylneuraminic acid (CMP-NAcNeu) hydrolase were studied in the cortex of calf kidney. The pH optimum was 9.0 in both Tris - HCl and glycine/NaOH buffer. The apparent Km was 0.47 mM and the apparent V 15.3 mumol/h/g wet wt of calf kidney cortex. A stimulation by divalent metal ions (Ca2+ and Mg2+) was demonstrated for the hydrolase. In the presence of Triton X-100 an increase in enzyme activity was observed. CMP-NAcNeu hydrolase was inhibited by EDTA, beta-mercaptoethanol, nucleoside phosphates and nucleotide-sugars. The inhibition was more pronounced when a sub-optimal CMP-NAcNeu concentration was used. The enzyme appeared to be localized in the plasma membranes. In the plasma membrane preparation of calf kidney cortex, which was derived mainly from the proximal tubule cells, the yield of CMP-NAcNeu hydrolase (13%) and its increase in specific activity (9-fold) was as high as for the plasma membrane marker enzymes. From subcellular distribution studies it appeared that the enzyme was localized mainly at the bursh border side of the plasma membrane of the proximal tubule cell.


Localization and some properties of lysosomal dipeptidases in rat liver. 1. The rates of hydrolysis of 26 synthetic dipeptides by extracts from highly purified lysosomal fractions from rat liver at pH 5.0 and by whole liver homogenates at pH 7.4 have been determined. Extracts from the lysosomal fractions hydrolysed most peptides at a lower rate per mg protein than the homogenates, and some peptides not at all. 2. Properties of two dipeptidases present in the extracts from the lysosomal fractions, splitting Ile-Glu and Leu-Gly, respectively, were studied in greater detail. The enzyme that hydrolysed Ile-Glu was strongly activated by dithiothreitol, showed optimal activity at pH 4.5 and had a molecular weight of about 120 000. Leu-Gly dipeptidase did apparently not contain an essential thiol group and had a molecular weight of approx. 90 000. It showed maximal activity at pH 6.5. 3. After differential centrifugation of liver homogenates, Ile-Glu and Leu-Gly-splitting activities were determined in the fractions, under the optimal conditions mentioned above. The Ile-Glu-hydrolysing enzyme activity showed about the same distribution as the lysosomal marker enzyme acid phosphatase. Leu-Gly-splitting activity, however, was largely present in the cytosol fraction, with only a small peak in the lysosomal fraction. We obtained evidence that the activities present in the lysosomal fraction and in the cytosol fraction were due to different enzymes, and that one of these enzymes was localized exclusively in lysosomes. 4. It is concluded that some dipeptides originating from intralysosomal proteolysis might be split by lysosomal dipeptidases, whereas others are probably hydrolysed only in the extra-lysosomal compartment of the cell.


Some properties of erythrocuprein treated by organic solvents. The effect of various types of organic solvents on the properties of bovine erythrocuprein was studied. Three organic solvents were found in which the protein is soluble, these were: dimethyl sulfoxide, formamide and N-methylformamide. It was shown that in formamide and dimethyl sulfoxide media the protein possees superoxide dismutase activity, but in N-methylformamide the protein has negligible activity. In organic solvents the substrate (superoxide radical) and solvated electron result in reduction of the protein copper. At high concentrations of superoxide radical or solvated electrons an inactivation of protein and stabilization of superoxide radicals was noted. The stabilization is most pronounced in N-methylformamide. The protein that is reduced by the radical or the solvated electron may be reoxidized by molecular oxygen, the latter being reduced to the superoxide radical.


Oxygen equilibrium characteristics of adult and fetal hemoglobin of Japanese monkey (Macaca fuscata). Adult hemoglobin and fetal hemoglobin were obtained from Japanese monkey (Macaca fuscata) and their oxygen equilibrium characteristics were studied. (1) The oxygen affinity of fetal hemoglobin was higher than that of adult hemoglobin both in the presence and absence of 2,3-diphosphoglycerate. The presence of diphosphoglycerate lowers the oxygen affinity of adult hemoglobin much greater than does that of HbF and the diphosphoglycerate levels of red cells of adult and newborn monkeys are about the same. (2) The intensity of the Bohr effect, as expressed by -deltalogP50/deltapH, at pH 7.4 was in the order of fetal hemoglobin-diphosphoglycerate greater than adult hemoglobin-diphosphoglycerate greater than fetal hemoglobin greater than adult hemoglobin.


Studies on the binding of FMN by apoflavodoxin from Peptostreptococcus elsdenii, pH and NaCl concentration dependence. 1. The pH and ionic strength dependence of the interaction of FMN with apoflavodoxin has been studied by fluorometry in the pH region 2-5, at 22 degrees C. 2. The rate constant of dissociation and the dissociation constant were experimentally determined; the rate constants of association were claculated at a given pH value. These constants depend on the ionic strength. The plots of these constants against the square root of the ionic strength are straight. 3. Our data have been interpreted in terms of the Brönsted theory, which relates chemical reaction rates to ionic strength. The data indicate that the apoenzyme reaches its maximum net positive charge at pH 2.0-2.6. The calculated net charge in this pH region is between 11 and 12 and is in agreement with the theoretical value of 12 as deduced from the primary structure of the protein. The isoelectric point of the holoenzyme is about 4. 4. The rate constant of association extrapolated to zero ionic strength is 3.2-10(5)M-1-s-1 and is pH-independent. 5. The rate constant of dissociation and the dissociation constant extrapolated to zero ionic strength depend on the pH. The results are explained by assuming that there are two protein ionizations with a pK value of 3.4; these ionizing groups are possibly close to the FMN binding site.


Alkalin titrations of human somatotropin, human choriomammotropin and ovine prolactin by circular dichroism and fluorescence. Structural transitions occurring during the alkalin titration of human somatotropin, human choriomammotropin, and ovine prolactin have been investigated by means of circular dichroism and fluorescence emission spectra. Human somatotropin exhibited an isodichroic point at 287 nm, with all spectral changes being reversed upon back titration from pH 12.50 to pH 8.0. Fluorescence quenching as a function of pH produced a simple sigmoidal curve. Human choriomammotropin exhibited an isodichroic point at 288 nm. The fluorescence and circular dichroism spectra of this protein were found to be reversible between pH 8.0 and 11.0. However, on titration above pH 11, the isodichroic point and the reversibility of the circular dichroism spectra were lost. This conformational transition was accompanied by a sharp increase in fluorescence quantum yield. The circular dichroism spectra of ovine prolactin showed essentially no change on titration to pH 11.0. However, between pH 11.0 and 12.0, a sharp conformational transition was observed similar to that seen in human choriomammotropin, but not exhibiting the same increase in fluorescence quantum yield. The fluorescence titration of prolactin was found to be essentially reversible upon back titration from pH 12.5, although the circular dichroism spectra were not reversible from this pH.


A pH-dependent conformational change in the coat protein subunits from potato virus X. Both the circular dichroism and fluorescence spectra of the dissociated coat protein subunits from potato virus X changed substantially over the pH range 8 to 4, irreversible changes resulted below pH 4, with tyrosyl and tryptophanyl residues affected most. The titration curves show a pKa of about 5.6 and do not require cooperative interactions between the coat protein subunits, thus they are in marked contrast to titrations of tobacco mosaic virus A-protein. The spectra of the intact virus were little changed between pH 8 and 4 and suggested that the coat protein was locked into a conformation similar to that of the subunits in solution at pH 7. It is proposed that the pH induced conformational change is responsible for determining the acidic branch of the pH profile for reconstitution of potato virus X from its dissociated coat protein subunits and RNA.


On the allosteric transition between the structures of high and low ligand affinity in carp hemoglobin. The variation of magneto-optical rotatory dispersion with pH for carp deoxyhemoglobin in the presence and absence of inositol hexaphosphate was interpreted as a pH-induced allosteric transition between the structures of high and low ligand affinity (the R and T states in terms of the two state model of cooperativity). Increasing the pH from 6 to 11 causes a decrease in the fraction of molecules in the T state from 1 to 0.65. In the absence of inositol hexaphosphate the pH dependence of this fraction has a midpoint at 7.8, addition of inositol hexaphosphate shifts this midpoint by 1.5 units toward high pH. From the analysis of the data obtained and the pH dependences of functional properties (Tan, A.L., Noble, R.W. and Gibson, Q.H. (1973) J. Biol. Chem. 248, 2880-2888) the parameters of the two state model of cooperativity for carp hemoglobin were estimated.