Immunoglobulin deposits in lepromatous leprosy skin. Presence of deposits in apparently uninvolved skin and occurrence of serum antiepithelial antibodies. Immunoglobulin deposits were detected in ten of 13 biopsy specimens from apparently uninvolved skin of patients with lepromatous leprosy. There were deposits of IgM at the dermoepidermal junction in the skin of five patients, and deposits of IgM along the dermal collagen and elastic fibers in the skin of the other five. The deposits were eluted with acid buffers and high molarity salt solution. Circulating IgG antibodies to intercellular substance of epithelial cells, similar to those present in pemphigus vulgaris, were found in 25% of patients with lepromatous leprosy who were studied. These antibodies appeared to be different from the skin-bound immunoglobulin deposits.


Subclinical neuropathy at "safe" levels of lead exposure. Electrophysiological methods revealed subclinical neuropathy in 26 workers, exposed from 1 to 17 years to lead and whose blood lead (PbB) values had never exceeded 70mu/100ml, as ascertained by checking the monitor reports of the factory and by careful exposure history. The PbB determinations had been tested repeatedly and had been found valid. The main findings were slowing of the maximal motor conduction velocities of the median and ulnar nerves and particularly the conduction velocity of the slower fibers of the ulnar nerve. Electromyographical abnormalities comprised fibrillations, diminution of the number of motor units on maximal contraction, and an abnormally long duration of the units. Earlier similar measurements from heavily exposed workers had been even more abnormal. Thus, a dose-response relationship exists on a group basis. Since the regular monitoring of PbBs in most workers during their entire period of exposure excludes the possibility of a body burden out of proportion to the PbB slight neurological damage is produced at exposures hitherto regarded as quite safe.


Synovial sarcoma.

Synovial sarcoma. Of 34 cases with synovial sarcoma, the five-year survival rate was 36%. A high local recurrence rate results when local excision is performed. Wide excision which may necessitate amputation is the treatment of choice. Prophylactic and even therapeutic node dissections are ineffective in increasing survival because of the disease. Evaluation of radiation therapy was impossible, although some patients obtained significant paliation. Adriamycin appears to have a tumoricidal effect and provided clinically significant responses in several patients.


The effect of puromycin and cycloheximide on vacuole formation and exocytosis in Tetrahymena pyriformis GL-9. It has been shown that both puromycin and cycloheximide, at concentrations of 434 and 100 mug/ml respectively, produce a marked inhibition of vacuole formation and exocytosis in Tetrahymena pyriformis GL-9. These effects were analysed in a quantitative manner. At the same time as these inhibitions occurred the incorporation of 1-C14 leucine into trichloroacetic acid precipitable material was inhibited by 90% and 100% respectively over a 40 min period. This inhibition of protein synthesis by cycloheximide occurred almost immediately, whereas the inhibition of vacuole formation and egestion was delayed. The results suggested that the latter processes were dependent upon a continuing supply of proteinaceous material, of which there was only a small store within the cell. Cycloheximide inhibited exocytosis completely -nder the conditions employed (with 100% inhibition of protein synthesis) whereas puromycin (with a 90% inhibition of protein synthesis) only inhibited it by about 50%. This suggested that the amount of newly synthesized protein required for the exocytic egestion process was very small in relation to the total cell requirement for protein synthesis. The entry of both inhibitors into the cell was by means other than vacuole formation. Puromycin appeared to have some effect on vacuole formation which was unconnected with protein synthesis. Microscopic observations of living cells indicated that oral apparatus function and endocytic vacuole formation were probably both affected by the inhibitors. Chloramphenicol, at 200 mug/ml, had little effect on vacuole formation by starved cells with an exposure of an hour. The uptake of 1-C14 leucine from the growth medium was found to be a selective process, giving a concentration of about 2000 times into the cells over a 1 hr period. The results are discussed


Stable polypeptides associated with the 250S mengovirus-induced RNA polymerase structure. One host polypeptide (40,000 daltons) synthesized prior to infection is associated with the 250S RNA polymerase structure partially purified by a combination of velocity sedimentation and isopycnic separation. A series of pulse-chase experiments have shown that a 56,000 dalton polypeptide made during the eclipse phase of infection is inserted into the 250S viral RNA polymerase structure. This 56,000 dalton polypeptide is bound in a stable manner since labeled 56,000 dalton polypeptide is not removed from the 250S polymerase structure by a 2-hour chase (3 to 5 hours after infection) and it is the major labeled polypeptide species remaining. However, the 56,000 dalton polypeptide (viral-specific polypeptide E) made at 4 hours after infection is not present in the 250S polymerase structure following a 50 minue chase. Levels of cycloheximide which inhibit protein synthesis 95 per cent in the infected cell have no effect on the amount of viral-specific RNA polymerase activity (in vitro) when the inhibitor is added for 30 minutes at the time of maximum rate of viral RNA synthesis in whole cells. These inhibitor studies support the hypothesis that the viral-specific RNA polymerase polypeptide may be a stable polypeptide that is not rapidly turning over in the infected cell. In view of these results the stable 56,000 dalton polypeptide (polypeptide E) made early in infection may be a candidate for the viral-specific polymerase polypeptide.


Some investigations on the adjuvant mechanism of DEAE dextran. In vitro it was shown that adsorption of inactivated FMDV onto DEAE-D kieselgur columns did not occur in the presence of 0.1--0.15M NaCl. These NaCl concentrations are present in DEAE-D/FMDV vaccines and in the tissues of animals. Therefore, adsorption of virus antigen does not appear to be responsible for the adjuvant effect of DEAE-D. In pigs it was demonstrated that DEAE-D exerts its optimal adjuvant effect, as measured by the formation of neutralizing antibodies and protection against challenge infection, when injected together with inactivated FMDV as vaccine. Apart from this, a good adjuvant effect (group immunity 75--100 per cent) was evoked in about one half and a moderate effect (group immunity 50--70 per cent) in about a quarter of the inoculated animals even if DEAE-D was separately injected locally and temporally from the inactivated virus. With regard to immunity it apparently does not matter whether DEAE-D or inactivated virus was given first, but an interval of 48 hours or 4 days between injection seemed to be more favourable than one of 24 hours. With regard to the formation of neutralizing antibodies the situation is comparable to that of immunity with the exception that a time interval of 24 hours between the application of DEAE-D and inactivated virus or vice versa was as good as that of 48 hours or 4 days. The results are discussed in regard to the possible mechanism of the adjuvant effect of DEAE-D on the cellular level.


Growth of SV 40, adeno 7 and SV 40-adeno 7 viruses in monkey and human cells at 29 degrees and 37 degrees C. Adeno 7 virus replicated well in human diploid (LEP) cells but only to a low degree in green monkey kidney (GMK) cells at 37 degrees C; it did not replicate in either system at 29 degrees C. At 37 degrees C SV 40 virus replicated well in GMK cells but only moderately in LEP cells; at 29 degrees C it did not replicate in either system. SV 40-adeno 7 hybrid grew in both GMK cells and LEP cells at 37 degrees C. At 29 degrees C this virus replicated in GMK cells but not in LEP cells. While the formation of V-antigen generally corresponded to the infectious virus production in the respective system, considerable differences were encountered in the T-antigens production. Adeno 7 T-antigen was detected earlier and in a higher percentage of GMK cells than in the fully permissive LEP cells and its formation was only slightly influenced by the incubation temperature. SV 40 T-antigen was more efficiently formed in GMK cells than in LEP cells. At 29 degrees C SV 40 T-antigen was only found in GMK cells and was detected later than at 37 degrees C. The difference in the formation of SV 40 T-antigen in GMK cells infected with SV 40 and SV 40-adeno 7 hybrid virus was further analyzed. The results obtained suggest that an early step of the virus-cell interaction, but neither virus attachment nor penetration, was involved.


Local cooling in myasthenia. Improvement of neuromuscular failure. Weak myasthenic muscles were tested by nerve stimulation both at normal temperature and after local cooling that was accomplished by exposing the skin to ice bags or cold paraffin oil. Reduction of intramuscular temperature from 35 C to 28 C increased the voltage of the bellytendon electrical response, the force of the tetanus elicited by 10/sec or 20/sec nerve stimulation. The myasthenic decrement of successive muscle responses was less marked after cooling, as were "delayed rundown" and "postactivation exhaustion." All these effects were reversed on rewarming the muscle. The abnormal neuromuscular jitter in motor-unit components was also reduced by local cooling. These observations may explain why diagnostic application of repetitive stimulation may be false-negative; muscle temperature must be controlled.


The mechanism of neuromuscular fatigue. A study of mammalian muscle using excitation-contraction uncoupling. Neuromuscular transmission failure was measured during rapid repetitive stimulation of the rat hemidiaphragm in vitro after excitation-contraction uncoupling by hyperosmolar glycerol, washing, and dantrolene sodium. Observations of resting membrane, miniature endplate, action, and endplate potentials (epps) showed that neuromuscular transmission failure occurred during stimulation because of a decrement of the epp. This decrement was due both to a reduced number of quanta released and to a variable reduction of quantal size. The time course of these events was studied, and limited observations were made on recovery. Observations of the difference between the epp decrement of cut muscle and of partially curarized muscle suggest a prejunctional action of curare.


Neurological sequelae of malignant external otitis. The neurological sequelae of malignant external otitis (MEO) form a characteristic syndrome. Following Pseudomonas external otitis, usually in an elderly, diabetic patient, either isolated facial nerve paralysis or multiple cranial nerve palsies develop. Once extensive neurological signs have developed, recovery rarely occurs. We saw a patient with MEO and multiple cranial nerve palsies who recovered following an extended course of gentamicin sulfate and carbenicillin disodium therapy.


Neurologic disorders following surgery for peptic ulcer disease. The neurologic status of 2,000 veterans who had had surgery for peptic ulcer between 1952 and 1957 was evaluated. In 1970, a total of 156 of these men were examined, 97 of whom had procedures that disrupted the normal continuity of the upper gastrointestinal tract. Twenty-one had neurologic disorders, including 17 patients with peripheral neuropathies. Procedures bypassing the ampulla of Vater were performed in 15 of these. The only detected factor associated with neurologic manifestations was weight loss since surgery. A mortality study of the total population revealed 865 patients had died by the end of 1973. There were seven deaths attributed to neurologic causes, one in a patient with amyotrophic lateral sclerosis and one in another with spinal paralysis. All seven were among the 70% of the deceased who had had surgery that disrupted the continuity of the upper gastrointestinal tract. Thus, we conclude that the type of surgery influenced the likelihood of neurologic complications, but at least for motor neuron disease, the increased risk was not appreciable.


Prognosis of malignant tumors of the parotid gland with facial paralysis. A parotid malignant tumor with spontaneous facial nerve involvement is a serious prognostic sign. Of 34 patients reported, 29 have been followed for at least five years. In this group, 20 are dead of disease (one at five years), one is dead of other causes (at seven years), one is living with disease, and seven are free of disease. Four of these seven cases have been followed for more than ten years, with two being free of disease for 12 years. Thus, it is not a hopeless situation, as some studies have shown, and not a contraindication to combined radical surgery and radiotherapy treatment for cure if not palliation.


Protein deficiency in carbon tetrachloride-induced hepatic lesions. The influence of protein deficiency on the hepatotoxicity of carbon tetrachloride (CCl4) was investigated in a group of rhesus monkeys.  Animals fed a protein-rich diet served as controls.  The results indicate that protein-deficiency protects the liver against acute hepatotoxicity of this drug.  The protective effect is abolished if the animals are administered phenobarbital prior to the administration of CCl4.  The protective action is due to a reduction, in protein deficiency, of the endoplasmic reticulum associated enzymes involved in hepatotoxicity of CCl4.  Repeated administration of CCl4, which induces hepatic regeneration, resulted in disappearance of fat from periportal cells in protein-deficient animals.  The regenerating cells (because of their better enzyme system) acquire "nutritional autonomy" and are thus able to synthesize adequate amounts of lipoproteins for mobilization of liver triglycerides.


Systemic lupus erythematosus.

Systemic lupus erythematosus. Recent investigations in systemic lupus erythematosus (SLE) have disclosed the presence of paramyxo-virus-like nucleocapsids in the cytoplasm of renal endothelial cells of patients with this disease.  Elaborate techniques have failed to demonstrate conclusively that these are indeed viruses.  Concomitantly, there is increasing evidence that SLE patients have defective cell-mediated-immunity.  A 3-year-old child with SLE exhibited clinical evidence of defective cellular immunity.  At postmortem, paramyxoviruslike tubular structures were demonstrated in her kidneys.  Additional findings included the presence of intracytoplasmic viral particles consistent morphologically with herpes simplex virus in neurons from the lateral geniculate body and numerous "nuclear bodies" in neurons and glial cells.  A dysplastic thymus gland demonstrated virtual absence of lymphocytes and was devoid of Hassall corpuscles, unlike the thymus usually seen in SLE.


Calcified mixed malignant tumor of the liver. Two adult patients died from mixed hepatic tumors of the liver with metastasis.  The outstanding findings in both patients were a long clinical course, roentgenologically identifiable calcification of the tumors, and, in one, hypertrophic osteoarthropathy and spider angiomas.  Mixed hepatic tumors are rare in adults, only 14 possible previous cases have come to our attention.  The mixed hepatic tumors of adults are morphologically different from the more common mixed hepatoblastomas of infancy and childhood.  Calcification in a slow-growing tumor in an adult without cirrhosis may indicate a mixed hepatic tumor.


Experimental egg-transmission of avian reovirus. Two experiments with an avian reovirus (FDO isolant), serologically identical to the Fahey-Crawley and WVU 2937 isolants, provided conclusive proof that egg-transmission can occur. In the first experiment, 4-day-old chicken embryos were inoculated into the yolk sac with serial dilutions of virus and subsequently hatched. High virus doses killed all embryos, whereas low doses (14 or less plaque-forming units) allowed some embryos to hatch normally although infected. In the second experiment, virus was administered by the nasal, esophageal, and tracheal routes to twenty 15-month-old breeders. Eggs from -6 to 21 days and 58 to 64 days postinoculation were incubated. Three newly hatched chicks from eggs laid on successive days (17, 18, and 19 days postinoculation) and one infertile egg (61 days postinoculation) were found infected. Active infection, as judged by virus isolations from cloacal swabs, was over by the 17th day postinoculation.


Horizontal transmission of turkey herpesvirus to chickens. IV. Viral maturation in the feather follicle epithelium. An experimental line of White Leghorns (WSU-VS) and a commerical line inoculated with turkey herpesvirus (HVT) at 1 or 8 weeks old did not differ in the occurrence of infectious cell-free HVT in their feather-tip preparations (FT-HVT). The birds examined at 2 weeks postinoculation (PI) were always positive for FT-HVT regardless of age at virus inoculation, dose of HVT inoculated, or genetic line of chickens, whereas only a few were positive at 4 weeks and none at 6 weeks PI.


Localization of avian reovirus (FDO isolant) in tissues of mature chickens. Mature hens were exposed to a reovirus (FDO isolant) serologically related to the viral arthritis agent. Examination of 23 tissues from the respiratory, alimentary, and reproductive tracts and tendons from the hock joint revealed widespread infection 4 days after inoculation via the respiratory and alimentary tracts. By 14-15 days, infection had largely subsided but was still detectable in the tendons, oviduct, and alimentary tract of some birds. No infection was found at 30 days. Virus isolation attempts in chick kidney cultures were more reliable than fluorescent-antibody tests for detecting infection. None of 156 chicks hatched from eggs laid between 12 and 33 days postinoculation were infected.


Characteristics of three stains of avian adenoviruses isolated in Queensland. I. Biological properties. Three strains of avian adenovirus were isolated and studied in cell cultures, chicken embryos, and chickens. One strain (ADAM-1) was isolated from the bursa of Fabricius of a chicken with Marek's disease, and the others (ADAM-2 and ADAM-3) from one-day-old chicks with respiratory signs. Experimentally, ADAM-1 produced no clinical signs or lesions except atrophy of the bursa of Fabricius. ADAM-2 caused mild respiratory signs in young chickens when inoculated intratracheally. The behavior of the three viruses in cell cultures and chick embryos was similar to that described for other avian adenoviruses (CELO). In a serological survey using the agar-gel precipitation test, 3-30% of sera tested gave a positive reaction.


Natural pox and herpes as a dual virus infection in chickens. A natural dual viral infection was confirmed in chickens of University of Illinois Poultry Farm by histopathologic, immunologic, and electron-microscopic examination of formalin-fixed tracheal tissue. Histopathologic examination of the tracheal mucosa revealed eosinophilic cytoplasmic inclusions characteristic of fowlpox and intranuclear inclusions suggestive of Marek's disease. Treatment of the formalin-fixed tracheal tissue with peroxidase and fluorescent-labeled antibody against Marek's disease virus revealed specificity of the reaction. Electron microscopy showed viral particles of two morphologic forms, i.e., pox and herpes. A single cell having dual infection with herpes virus in the nucleus and pox virus in the cytoplasm was also encountered on electron-microscopic examination.


[Biology of prostaglandins with reference to therapeutic aspects]. Prostaglandins are highly potent derivatives of unsaturated fatty acids with multiple biological activities.  They are synthesized and metabolized in almost all tissues studied so far.  The E- und F-type prostaglandins may be regarded as local modulators of hormonal effects on cell function and--in some cases (kidney, uterus-corpus luteum)--as regional or tissue hormones.  Thus they seem to be involved in the regulation of neurotransmission, kidney function, triglyceride metabolism in adipose tissue and progesterone biosynthesis.  Apart from their influence on renal blood flow prostaglandins of the A-type possibly have an additional function as circulatory hormones regulating blood pressure.  Second messenger-systems (cAMP, Ca++-cGMP) which mediate the effects of most non-steroidal hormones are also involved in the action of prostaglandins, at least of the E-and F-types.  Disturbances in prostaglandin metabolism (increased or decreased biosynthesis) are discussed to play a role in the pathogenesis of inflammation, pain, fever, hypertension, bronchial asthma and gastric or duodenal ulcer formation.  Drugs with antiinflammatory, analgesic and antipyretic activity have been shown to be potent inhibitors of prostaglandin formation.  The correlation of a local prostaglandin deficit or the therapeutic use of single effects of prostaglandins by administration of exogenous compounds (natural prostaglandins or modified derivatives) has so long been less satisfactory because of their large number of biological actions which lead to undesired side effects.  Extensive experience have been obtained in the successful induction of therapeutic abortion.  This effect is based on the stimulatory action of E- and F-type prostaglandins on the smooth muscles of the pregnant uterus which is resistent to the influence of other stimuli, e. g. oxytocin.  Here the incidence of side effects could be reduced by local administration of low doses of prostaglandins into the uterine cavity.  A general improvement of the therapeutic usefulness of prostaglandins will however only be achieved, if modified derivatives with more specific actions on the desired "target" tissues are available.


Hyperlipidaemia in children.

Hyperlipidaemia in children. Hyperlipidaemia in children is most commonly expressed as hypercholesterolaemia. "Normal values" for serum cholesterol, if defined statistically, vary between communities, and levels of cholesterol in childhood above which an increased risk of coronary heart disease in adult life may be expected have not been firmly established. It is suggested that serum cholesterol concentration over 250 mg/dl (6.47 mmol/l) in a child over 1 year of age merits detailed investigation, including full lipoprotein analysis, and levels of serum cholesterol between230 and 250 mg/dl (5.95-6.47 mmol/l) should be repeated with further studies if indicated. Secondary hyperlipoproteinaemia rarely presents diagnostic problems but must always be excluded. The only primary hyperlipoproteinaemia likely to be encountered in childhood is familial hyperbetalipoproteinaemia in its common heterozygous form. The most effective means to date of lowering serum cholesterol in this condition is cholestyramine, but the long-term consequences of therapy are not known and treatment should at present be limited to children from high-risk families. Long-term follow-up is essential and until results of such studies are available population screening is unjustified.


Priming of superhelical SV40 DNA by Escherichia coli RNA polymerase for in vitro DNA synthesis. When closed circular SV40 DNA containing 58 negative superhelical turns is used as a template for RNA synthesis with Escherichia coli RNA polymerase, a fraction of the RNA product remains complexed with the DNA. The RNA in the complex is resistant to ribonuclease in high salt, and the Tm indicates that it is hydrogen bonded to the DNA. The mole ratio of RNA to DNA nucleotides in the complex ranges from 0.01 to 0.08; the RNA ranges in length from 80 to 600 nucleotides. The formation of the complex is dependent on the circular DNA being topologically underwound since no complex is formed when closed circular DNA containing zero superhelical turns is used as the template. The DNA-RNA complex can serve as a primer-template combination for in vitro DNA synthesis by E. coli DNA polymerase I. After synthesis with (alpha-32P)-labeled deoxyribonucleoside triphosphates followed by alkaline hydrolysis, the isolation of 32P-labeled ribonucleotides is evidence for a covalent linkage between the RNA and the DNA synthesized. During the in vitro DNA synthesis, the template is nicked at a low rate, and the nicked molecules support extensive DNA synthesis. This observation indicates that only limited synthesis can occur on unnicked molecules possibly owing to the topological constraints against unwinding of the helix. Possible models for in vivo priming of double-stranded DNA by E. coli RNA polymerase are discussed.


Cell surface changes correlated with density-dependent growth inhibition. Glycosaminoglycan metabolism in 3T3, SV3T3, and con A selected revertant cells. A 35SO4-labeling/chromatography technique has been developed which facilitates quantitation of sulfated glycosaminoglycan (GAG) synthesis in mammalian cell cultures. The technique has been used to compare sulfated GAG biosynthesis, degradation, and turnover in three related cell lines with differing degrees of density-dependent inhibition of growth in vitro (Balb/c 3T3, SV3T3, and SV3T3 revertant cells). Viral transformation of Balb 3T3 cells is accompanied by a 2-5-fold decrease in cell associated sulfated GAG. SV3T3 revertant cells, which show partial reversion to low saturation density in vitro, show a 2.5-8-fold increase in cell-associated sulfated GAG compared to the parental SV3T3 cells from which they were selected. In addition, the distribution of 35SO4 and [3H]glucosamine among the different GAG species produced by SV3T3 revertant cells reverts so that it is similar to the distribution characteristic of untransformed 3T3 cells rather than SV3T3 cells. Mild trypsin treatment of 35SO4-labeled cells removed 68-84% of the cellular sulfated GAG, suggesting that at least this proportion of the total cellular sulfated GAG was located at the cell periphery. Removal of 35SO4-labeled cells from the Petri dish with a Ca2+ selective chelating agent revealed a fraction of the sulfated GAG that remained tightly bound to the Petri dish. A higher proportion of the total cell-associated sulfated GAG remained attached to the Petri dish in cultures of untransformed and revertant cells compared to that present in cultures of transformed cells. A role for sulfated GAG in density-dependent growth inhibition of fibroblast cultures is proposed and discussed in the light of the data obtained.


Relationship between fluorescence and conformation of epsilonNAD+ bound to dehydrogenases. This work reports on the interaction of the fluorescent nicotinamide 1,N6-ethenoadenine dinucleotide (epsilonNAD+) with horse liver alcohol dehydrogenase, octopine dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase from different sources (yeast, lobster muscle, and rabbit muscle). The coenzyme fluorescence is enhanced by a factor of 10-13 in all systems investigated. It is shown that this enhancement cannot be due to changes in the polarity of the environment upon binding, and that it must be rather ascribed to structural properties of the bound coenzyme. Although dynamic factors could also be important for inducing changes in the quantum yield of epsilonNAD+ fluorescence, the close similarity of the fluorescence enhancement factor in all cases investigated indicates that the conformation of bound coenzyme is rather invariant in the different enzyme systems and overwhelmingly shifted toward an open form. Dissociation constants for epsilonNAD+-dehydrogenases complexes can be determined by monitoring the coenzyme fluorescence enhancement or the protein fluorescence quenching. In the case of yeast glyceraldehyde-3-phosphate dehydrogenase at pH 7.0 and t = 20 degrees the binding plots obtained by the two methods are coincident, and show no cooperativity. The affinity of epsilonNAD+ is generally lower than that of NAD+, although epsilonNAD+ maintains most of the binding characteristics of NAD+. For example, it forms a tight complex with horse liver alcohol dehydrogenase and pyrazole, and with octopine dehydrogenase saturated by L-arginine and pyruvate. One major difference in the binding behavior of NAD+ and epsilonNAD+ seems to be present in the muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, no difference was found for epsilon NAD+ between the affinities of the third and fourth binding sites. The results and implications of this work are compared with those obtained recently by other authors.


Conformational and functional studies of chemically modified cytochromes: N-bromosuccinimide- and formyl-cytochromes c. N-bromosuccinimide-cytochromes c (Myer, Y. P. (1972), Biochemistry 11, 4195) and formyl-cytochrome c (Aviram, I and Schejter, A. (1971), Biochim. Biophys. Acta 229, 113) have been chromatographically purified, and the resulting components have been characterized in terms of their structure, conformation, and function. The activity measurements are considered in terms of the oxidizability, as the transference of an electron to solubilized cytochrome c oxidase, and reducibility, as the tendency to accept an electron from NADH-cytochrome c reductase. Conformational characterization has been carried out by absorption measurements, pH-spectroscopic behavior, circular dichroism, thermal denaturation, ionization of phenolic hydroxyls, the tendency to form the CO complex, and autoxidation with molecular oxygen. NBS-cytochrome c yields two major components, the relative proportions of which, with increasing modification of the protein, exhibit a pattern typical of the formation of the two in a consecutive manner. The first product contains the modification of the Trp-59 and Met-65 side chains, and the second contains the added modification of Met-80. The former in both valence states of iron is more or less like the native protein, except for an apparently slightly loosened heme crevice; the latter, as in other modifications involving modification of centrally coordinated Met-80, was found to be in a conformational state characteristic of the native protein with a disrupted central coordination complex, a loosened heme crevice, and small, but finite derangement of the polypeptide conformation. Functionally, the first component reflected 55% of the reducibility property and an unimpaired oxidizability property, while the latter exhibited derangement of both aspects of cytochrome c activity. Formyl-cytochrome c yielded a single component with modification of Trp-59. Conformationally, in both valence states, it is a molecular form with a disrupted central coordination complex, a loosened heme crevice, and gross derangement of the overall protein conformation. It exhibits a minimal reducibility property, 12%, whereas it retains a native-like tendency to transfer an electron to cytochrome c oxidase. The data from the NBS-cytochrome c components are analyzed with reference to the two forms in the earlier studies of the unpurified preparations. The results are found to be in agreement with one another. The selectivity between the reducibility and the oxidizability exhibited by the first NBS component and formyl-cytochrome c, irrespective of significant differences in the conformational and coordinational configurations of the two, has been viewed in light of a two-path, two-function model for oxidoreduction, as well as with reference to conformational and structural requirements for the oxidizability and reducibility properties of the molecule.


Metabolic properties of substrate-attached glycoproteins from normal and virus-transformed cells. Balb/c 3T3, SV40-transformed 3T3 (SVT2), and Con A revertant variants of transformed cells leave a layer of glycoprotein on the culture substrate upon EGTA mediated removal of cells. The metabolic properties of this substrate-attached material (glycoprotein) have been examined. Pulse and cumulative radiolabeling experiments with glucosamine and leucine precursors established that this substrate-attached material accumulates on the substrate in growing cultures until cells have completely covered the substrate. The synthesis and/or deposition of the material diminished dramatically in cultures whose substrates had been completely covered with cells as observed microscopically, even though the contact-inhibited cell lines continued to make cell-associated and medium-secreted glycoproteins and transformed cells continued to divide and form multilayered cultures. Pulse-chase analysis using long periods of pulsing with radioactive leucine demonstrated that these glycoproteins are deposited directly on the substrate by cells and not subsequent to secretion into the medium. The substrate-attached material accumulated during long pulses was stably adherent to the substrate and displayed little appreciable turnover during 3 days of chasing of either sparse or dense cultures. Short-term pulse-chase analysis with leucine revealed two metabolically different pools of material-one which turns over very rapidly with a half-life of 2-3 hr (observed in both low-density and high-density cultures) and a second pool which is stably deposited on the substrate and whose proportion increased with the length of the radiolabeling period. No appreciable differences in the metabolic properties of substrate-attached material were observed in the three cell types studied during growth on a plastic substrate. These results are discussed with regard to the implicated roles of these glycoproteins in in mediating adhesion of normal and virus-transformed cells to the substrate.


Synthesis P1-dolichyl P2-alpha-D-mannopyranosyl pyrophosphate. The acid and alkaline hydrolysis of polyisoprenyl alpha-D-mannopyranosyl mono- and pyrophosphate diesters. P1-Dolichyl P2-ALPHA-D-mannopyranosyl pyrophosphate (9) has been chemically synthesized by a method developed for the corresponding citronellyl derivative, which also contains a saturated alpha isoprene residue. In each case, the P1-polyisoprenyl P2-diphenyl pyrophosphate was treated with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate to give a fully acetylated pyrophosphate diester, which was purified chromatographically and subsequently deacetylated. The citronellyl and dolichyl pyrophosphate diesters were compared with the previously synthesized citronellyl and dolichyl alpha-D-mannopyranosyl phosphate, respectively, by chromatography and by hydrolysis experiments. Good separations of the monophosphate from the corresponding pyrophosphate were achieved by silica gel tlc in a variety of solvent systems. Brief dilute acid hydrolysis of both the mono- and pyrophosphate diesters gave D-mannose and no alpha-D-mannosyl phosphate, the other products being polyprenyl phosphate and pyrophosphate, respectively. When the polyprenyl alpha-D-mannopyranosyl mono- and pyrophosphate diesters were treated with hot dilute alkali, the major products were polyprenyl phosphate and substances arising from the breakdown of D-mannose, indicating that the alpha-D-mannosyl phosphate bond was the most labile linkage in both compounds. However, the formation of a small proportion of free dolichol indicated that alpha-D-mannosyl phosphate was also formed to a minor extent. The interpretation of the results of the alkaline hydrolysis was complicated by the instability of D-mannose under basic conditions, it being almost completely degraded by even a brief treatment.


Glucocorticoid receptors in mouse mammary tumors: Specific binding to nuclear components. The specific interaction of glucocorticoids with nuclei of mouse mammary tumor was studied in vitro by incubation of the tissue with [3H]dexamethasone at 25 degrees. It was demonstrated that the mammary tumors contain a limited number of specific nuclear binding sites which were saturated with low hormone concentrations (10-8 M)9 The concentrations of specific binding sites in the nuclei were related to the concentration of cytoplasmic binding sites of unincubated tissues and varied between individual tumors. The binding component in the nuclei appeared to be a protein and was easily solubilized with 0.4 M KCl containing buffers. The ability of various corticoids to block the nuclear localization of the steroid correlated well with their glucocorticoid potency. Estradiol and progesterone at concentrations of 10-6 M were also effective in competing for the glucocorticoid receptor binding sites. However, while the glucocorticoids such as hydrocortisone and corticosterone translocated to nuclear sites also specific for dexamethasone, estradiol and progesterone competed for the cytoplasmic binding sites and did not translocate to the nucleus. The possible significance of the interaction of various steroids with the glucocorticoid receptors in mammary tumors is discussed.


An investigation of the role of the copper in galactose oxidase. Galactose oxidase is a metalloenzyme containing a single copper atom per molecule. The mechanism of action of galactose oxidase is studied in this paper by investigating substrate specificity and activation by peroxidase, and probing the copper site by electron spin resonance (ESR) spectroscopy. Line-shape simulation of ESR spectra are also reported and a comparison is made between observed and simulated spectra for galactose oxidase. A comparison is also reported for the enzyme from various commercial sources and enzyme isolated from a fungus in this laboratory. The results of this investigation suggest that the copper is in an environment of four in-plane nitrogens with axial symmetry.


Mandelate racemase from Pseudomonas putida. Magnetic resonance and kinetic studies of the mechanism of catalysis. The interactions of mandelate racemase with divalent metal ion, substrate, and competitive inhibitors were investigated. The enzyme was found by electron paramagnetic resonance (EPR) to bind 0.9 Mn2+ ion per subunit with a dissociation constant of 8 muM, in agreement with its kinetically determined activator constant. Also, six additional Mn2+ ions were found to bind to the enzyme, much more weakly, with a dissociation constant of 1.5 mM. Binding to the enzyme at the tight site enhances the effect of Mn2+ on the longitudinal relaxation rate (1/T1p) of water protons by a factor of 11.9 at 24.3 MHz. From the frequency dependence of 1/T1p, it was determined that there are similar to 3 water ligands on enzyme-bound Mn2+ which exchange at a rate larger than or equal to 10-7 sec-1. The correlation time for enzyme-bound Mn2+-water interaction is frequency-dependent, indicating it to be dominated by the electron spin relaxation time of Mn2+. Formation of the ternary enzyme-Mn2+-mandelate complex decreases the number of fast exchanging water ligands by similar to 1, but does not affect tau-c, suggesting the displacement or occlusion of a water ligand. The competitive inhibitors D,L-alpha-phenylglycerate and salicylate produce little or no change in the enzyme-Mn2+-H2O interaction, but ternary complexes are detected indirectly by changes in the dissociation constant of the enzyme-Mn2+ complex and by mutual competition experiments. In all cases the dissociation constants of substrates and competitive inhibitors from ternary complexes determined by magnetic resonance titrations agree with K-M and K-i values determined kinetically and therefore reflect kinetically active complexes. From the paramagnetic effects of Mn2+ on 1/T1 and 1/T2 of the 13C-enriched carbons of 1-[13C]-D,L-mandelate and 2-[13C]-D,L-mandelate, Mn2+ to carboxylate carbon and Mn2+ to carbinol carbon distances of 2.93 plus or minus 0.04 and 2.71 plus or minus 0.04 A, respectively, were calculated, indicating bidentate chelation in the binary Mn2+-mandelate complex. In the active ternary complex of enzyme, Mn2+, and D,L-mandelate, these distances increase to 5.5 plus or minus 0.2 and 7.2 plus or minus 0.2 A, respectively, indicating the presence of at least 98.9% of a second sphere complex in which Mn2+, and C1 and C2 carbon atoms are in a linear array. The water relaxation data suggest that a water ligand is immobilized between the enzyme-bound Mn2+ and the carboxylate of the bound substrate. This intervening water ligand may polarize or protonate the carboxyl group. From 1/T2p the rate of dissociation of the substrate from this ternary complex (larger than or equal to 5.6 times 10-4 sec-1) is at least 52 times greater than the maximal turnover number of the enzyme (1070 sec-1), indicating that the complex detected by nuclear magnetic resonance (NMR) is kinetically competent to participate in catalysis. Relationships among the microscopic rate constants are considered.


Effect of salts and polyamines on T4 polynucleotide kinase. The activity of T4 polynucleotide kinase (EC 2.7.1.78) was found to be greatly stimulated by salts, such as NaCl and KCl, and polyamines such as spermine and spermidine. Up to a sixfold increase in initial rates was observed with a variety of different single-stranded DNAs and mono- and oligonucleotides. The optimal concentrations of salts were 0.125 M, corresponding to a total ionic strength of mu equals 0.19. For polyamines the optimal concentrations were found to be at approximately 2 mM. With low enzyme concentration and in the absence of activators complete phosphorylation was not achieved for a number of substrates. In the presence of salts or polyamines or high concentration of enzyme the phosphorylation proceeded to completion. Addition of salt led to an increase in both the apparent V-max and the Michaelis constant for the DNA substrate whereas the Michaelis constant of ATP remained unchanged. Polyamines had a similar influence on the kinetic constants for the DNA substrate whereas a decrease was found for the apparent Michaelis constant for ATP. The overall mechanism in the presence of activators was found to be sequential but probably of a rapid equilibrium random type. Of the inorganic anions tested both P-i and PP-i inhibited the enzyme in a competitive manner with both substrates.


Binding of manganese(II) to DNA and the competitive effects of metal ions and organic cations. An electron paramagnetic resonance study. The binding of manganese(II) to DNA was studied by monitoring the concentration of free Mn2+ by electron paramagnetic resonance (EPR). It was found that the association constnat of the Mn-DNA complex depends upon the degree of saturation. The competitive effects of magnesium, calcium, sodium, and a number of organic cations including the antibiotic drug daunomycin were analyzed and the parameters describing the cation-DNA interaction were evaluated. It was found that the association constant as well as the parameter describing its dependence upon the degree of saturation decrease along the series Mn, Mg, Ca, Na. Differences in the extent of interaction with the base nitrogens (N-7) are suggested as the possible mechanisms leading to these observations. The EPR spectrum of the manganese-DNA complex was found to be similar to that of manganese-nucleotide complexes suggesting a similar mode of coordination. A comparison of the results of competitive and direct binding studies reveals some salient features of the small molecule-DNA interaction and leads to the conclusion that manganese binds at the major groove of the DNA helix.


Evidence for glucocorticoid transport into AtT-20/D-1 cells. Glucocorticoid uptake by AtT-20/D-1 mouse pituitary adenocarcinoma cells grown in tissue culture was examined. The binding of triamcinolone acetonide, a potent synthetic glucocorticoid, by intact cells and by cell cytosol was studied at both 4 and 25 degrees. Specific binding of [3H]triamcinolone acetonide by intact cells was markedly different from cell-free cytosol binding at 4 degrees. Intact cells bound a relatively small amount of labeled steroid within 2 min, after which no further binding was observed. In contrast, the receptor in a cell-free cytosol preparation was capable of binding steroid progressively at 4 degrees, indicating that the limited binding by intact cells was not a consequence of receptor characteristics. At 25 degrees, uptake by intact cells and cytosol was nearly identical and appeared to be limited only by the binding kinetics of the cytosol receptor. Estradiol-17 beta, a nonglucocorticoid steroid, was not bound by the AtT-20/D-1 cell at 4 degrees. Triamcinolone was not bound significantly at 4 or 25 degrees by an adrenal carcinoma cell that does not appear to be a glucocorticoid target cell. An Arrhenius plot of cell steroid uptake vs. the reciprocal of absolute temperature revealed an abrupt change in slope at 16 degrees, which is compatible with the temperature-dependent mechanism involved in glucocortidoid uptake being associated with lipid constituents of the cell membrane. These data suggest that glucocorticoid uptake by this target cell involves a mechanism of specific, temperature-dependent transport through the cell membrane.


Stimulus-secretion coupling in platelets. Effects of drugs on secretion on adenosine 5'-triphosphate. The mechanism of stimulus-secretion coupling in platelets was investigated by observing the effects of drugs on the kinetics on ATP secretion induced by either thrombin or the divalent cation ionophore A23187. The actual secretion is the same with either of these agents, since the rate constants and activation energies of secretion are the same and since drugs that affect the final, enzyme-independent steps of thrombin-induced secretion have the same effect on ionophore-induced secretion. Drugs that affect early steps of thrombin-induced secretion have no effect on ionophore-induced secretion. Drugs that act through cAMP (PGE1, theophylline, dibutyryl-cAMP) slow an early step in the mechanism of thrombin-induced secretion and completely block at higher levels, with the required concentration of inhibitor dependent on thrombin concentration. The inhibition of rate appears to be all-or-none, with no intermediate rates observed. By replacing thrombin with trypsin, which makes it possible to observe a complete change in rate-determining step from an enzyme-dependent to an enzyme-independent platelet step, it was found that these drugs slow the rate only when the enzyme-independent step is rate determining. These drugs have no effect on A23187-induced secretion. It was concluded that cAMP inhibits at a step after the enzyme step but before the final step by interfering with transmission of the stimulus-secretion coupling signal. Disruption of microfilament function by cytochalasin B (10 muM) accelerates the rate of secretion induced by either thrombin or ionophore. The microtubule agents colchicine, vinblastine, and vincristine had effects only at concentrations above those usually considered necessary for the specific inhibition of microtubule function. Drugs that inhibit prostaglandin synthesis (aspirin, indomethacin, eicosatetraynoic acid), drugs that block ATP production (antimycin A, deoxyglucose), or several other drugs previously reported to inhibit platelet function had no effect on secretion.


The outer membrane of Proteus mirabilis. II. The extractable lipid fraction and electron-paramagnetic resonance analysis of the outer and cytoplasmic membranes. 1. The lipid fraction extracted from the outer and cytoplasmic membranes of Proteus mirabilis with chloroform/methanol consisted almost entirely of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. 2. The phospholipid content of the cytoplasmic membrane was more than twice that of the outer membrane (38% as against 18% of the total dry weight) and the proportions of the three phospholipids differed somewhat in the two membranes. Yet, the fatty acid composition of the extractable lipids was essentially the same in both membranes. 3. The freedom of motion of spin-labeled fatty acids in the outer membrane of P. mirabilis depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe is an associate lipid structure with the properties of a bilayer. Nevertheless, the mobility of the probe was more restricted in the outer membrane than in the cytoplasmic membrane, indicating a higher viscosity of the outer membrane. 4. Chloroform/methanol completely removed the phospholipids from the outer membrane, leaving the lipopolysaccharide moiety intact. The motion of spin-labeled fatty acids in the extracted membranes was, however, highly restricted, suggesting that, in the native outer membrane, the local environment of the probe is composed of phospholipids rather than lipopolysaccharide. Aqueous acetone extraction removed only 75-80% of the phospholipids of the outer membrane. Nevertheless, the mobility of the spin-labeled fatty acid remained highly restricted, suggesting the existence of two phospholipid environments in the outer membrane differing in the nature of their association with the lipopolysaccharide and protein moieties.


The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae. Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both promitochondria and mitochondria obtained from O2-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N2) to elaidic (O2)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O2-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.


Kinetics of glycerol uptake by the perfused rat liver. Membrane transport, phosphorylation and effect on NAD redox level. The kinetics of glycerol uptake by the perfused rat liver were determined according to a model which includes membrane transport, intracellular  phosphorylation and competitive inhibition of glycerol phosphorylation by L-glycerol 3-phosphate. The membrane transport obeys first-order kinetics at concentrations below 10 mM in the affluent medium. The K-m of the glycerol phosphorylation was 10 muM and the K-i of the L-glycerol 3-phosphate inhibition was 50 muM. The maximum activity (V) was 3.70 mumoles/min per g liver wet wt. These results are similar to in vitro kinetics of the glycerol kinase, except that K-i was found to be somewhat lower in the intact organ. At low glycerol concentrations, a steep concentration gradient exists across the liver cell membrane. The increase in the lactate to pyruvate concentration ratio during glycerol metabolism is related to the actual concentration of L-glycerol 3-phosphate, not to the rate of glycerol uptake.


Binding of Au(CN)2- and Pt(CN)4-2- to horse liver alcohol dehydrogenase. A 35C1NMR relaxation study. The binding of Au(CN)2- and Pt(CN)4-2- ions to the coenzyme binding site of horse liver alcohol dehydrogenase (alcohol : NAD+ oxidoreductase EC 1.1.1.1) has been studied by 35C1 nuclear magnetic relaxation. Longitudinal relaxation rates were analyzed in terms of a simple model and binding constants for Au(CN)2-, Pt(CN)4-2- and C1- were estimated. From a comparison between transverse and longitudinal relaxation rates the correlation time and the quadrupole coupling constant of bound chloride ion were obtained. The quadrupole coupling constant estimated from a simple electrostatic model for chloride ion interacting with an arginine group agrees with the experimental value.


Cyclic 3',5'-AMP phosphodiesterase of rabbit aorta. Cyclic AMP and cyclic GMP phosphodiesterase activities (3' : 5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca2+ in the presence of the activator. However, the activity of the cyclic AMP phosphodiesterase was not significantly stimulated by Ca2+ up to 100 muM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60-70%) of the media enzyme was found in 105 000 times g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 times g supernatant the main kinetic parameters were specified as follows: 1) The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg2+ was achieved at 4mM of MgC12. 2) High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3) Activity-substrate concentration relationship revealed a high affinity (Km equals 1.0 muM) and low affinity (Km equals 45 muM) for cyclic AMP. The homogenate and 105 000 times g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent Km values for cyclic AMP hydrolysis were 1.2 muM and 36-40 muM and an enzyme extracted by sonication from 105 000 times g precipitate also exhibited non-linear kinetics (Km equals 5.1 muM and 70 muM). 4) Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I5 o at 0.62 muM, I5 o of the media at 0.62 muM and I5 o of the adventitia at 1.0 muM) than on the brain (I5 o at 8.5 muM) and serum (I5 o at 20 muM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.


Mammalian carbamyl phosphate : glucose phosphotransferase and glucose-6-phosphate phosphohydrolase: extended tissue distribution. Carbamyl phosphate : glucose phosphotransferase and glucose-6-phosphate (Glc-6-P) phosphohydrolase activities have beeh demonstrated in pancreas, adrenals, brain, testes, spleen, and lung. Catalysis of these activities by classical multifunctional glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) has been firmly established for the first four of these tissues on the basis of characteristic catalytic properties of the transferase pH-activity profiles, apparent Km values for carbamyl phosphate and glucose, substrate specificity, susceptibility to inhibition by molybdate, and activation by deoxycholate. Additional such activity due to non-specific acid (and alkaline) phosphatase action also is indicated at very high glucose concentrations. The possible physiological significance of the newly-elucidated presence of glucose-6-phosphatase-phosphotransferase in these various tissues, in addition to previously extensively studied liver, kidney, and mucosa of small intestine, is discussed briefly.


The attack mechanism of an exo-1,3-beta-glucosidase from Basidiomycete sp. QM 806. The attack mechanism of a purified exo-1,3-beta glucosidase (1,3-beta-D-glucan glucohydrolase, EC 3.2.1.58) was investigated by using as a substrate a mixture of two structurally characterized periodate-oxidized and reduced unbranched 1,3-beta-D-glucans which differed only at the reducing terminal. The substrates, derivatives of laminarin, were altered only at the terminals due to resistance of the internal (1 leads to 3)-linked glucosyl residues to periodate oxidation. Each glucan has only a single and identical altered non-reducing terminal per molecule. Upon enzymatic hydrolysis, one molar equivalent of glycerol was produced from the altered non-reducing terminal of each substrate molecule attacked. Using glycerol as an indication of the number of chains acted upon, the quantity of D-glucose produced from the internal residues was used to determine the extent to which a chain was initially attacked. The glucose to glycerol ratio during the course of the hydrolysis indicates that the enzyme proceeds by a multiple-attack mechanism where four glucosyl residues are successively removed per encounter from the non-reducing terminal of each substrate molecule.


Cold inactivation of glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle. Inactivation of apo-glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase(phosphorylating) (EC 1.2.1.12) from rat skeletal muscle at 4 degrees C in 0.15 M NaC1, 5 mM EDTA, 4 mM 2-mercaptoethanol pH 7.2 is a first-order reaction. The rate constant of inactivation depends on protein concentration. With one molecule of NAD bound per tetrameric enzyme, a 50 per cent loss in activity is observed and the rate constant of inactivation becomes independent of the protein concentration over a 30-fold range. Two moles of NAD bound per mole of enzyme fully protect it against inactivation. NADH affords a cooperative effect on enzyme structure similar to that of NAD. Inactivation of 7.8 S apoenzyme is reflected in its dissociation into 4.8-S dimers. In the case of enzyme-NAD1 complex, no direct relationship between the extent of inactivation and dissociation is observed, suggesting that these two processes do not occur simultaneously; we may say that dissociation is slower than inactivation. A mechanism in which the rate-limiting step for inactivation is a conformational change in the tetramer occurring prior to dissociation and affecting only the structure of the non-liganded dimer, is consistent with the experimental observations. Inorganic phosphate protects apoenzyme against inactivation. Its effect is shown to be due to the anion binding at specific sites on the protein with a dissociation constant of 2.6 plus or minus 0.4 mM. The NaC1-induced cold inactivation of glyceraldehyde-phosphate dehydrogenase is fully reversible at 25 degrees C in the presence of 20 mM dithiothreitol and 50 mM inorganic phosphate. The rate of reactivation is independent of protein concentration. Inactivated enzyme retains the ability to bind specific antibodies produced in rabbits, but diminishes its precipitating capability.


Electron-electron double resonance measurements on xanthine oxidase. Electron-electron double resonance measurements were carried out on milk xanthine oxidase (xanthine:oxygen oxidoreductase EC 1.2.3.2) and the spectra obtained supported a previous model, based on EPR data, proposing a spin-spin interaction between unpaired electrons associated with Fe-S and Mo. The technique demonstrated that the additional apparently isotropic, splitting in the Mo EPR spectra observed at low temperature is produced by a single site giving two spectra interconverting at a rate consistent with the Fe-S spin lattice relaxation time. Other data concerning the model and the relaxation behaviour of the species are discussed.


Spin label and lanthanide binding sites on glyceraldehyde-3-phosphate dehydrogenase. The electron spin resonance spectrum of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl has two components. One component is due to a spin label highly immobilized on the enzyme surface and the other to a nitroxyl group able to tumble more rapidly. The spin-labelled enzyme is inactive. Selective modification of the active site cysteine residue (149) and determinations of total sulphydryl content implicate this residue as the site of the immobile spin-label. The mobile spin label is attached to another sulphydryl group. Crystallographic studies on the human muscle enzyme (Watson, H.C., Duee, E. and Mercer, W.D. (1972) Nat. New Biol., 240, 130) have located a binding site for samarium ion in the active centre. Addition of the paramagnetic gadolinium ion to spin-labelled enzyme reduces the intensity of both the spin label signals (by 72% for the mobile and by 11% for the immobile component). This indicates that the metal ion site (Kd equals 0.7 mM) is close to both types of spin label. Measurements of the effect of gadolinium-protein binding on the relaxation rate of solvent water protons enable the enzyme-bound spin label-metal ion distances to be tentatively estimated as 15 angstrom.


Demonstration by EPR spectroscopy of the functional role of iron in soybean lipoxygenase-1. 1. The EPR spectrum at 15 degrees K of soybean lipoxygenase-1 in borate buffer pH 9.0 has been studied in relation to the presence of substrate (linoleic acid), product (13-L-hydroperoxylinoleic acid) and oxygen. 2. The addition of 13-L-hydroperoxylinoleic acid to lipoxygenase-1 at pH 9.0 gives rise to the appearance of EPR lines at g equals 7.5, 6.2, 5.9 and 2.0, and an increased signal at g equals 4.3. 3. In view of the effect of the end product on both the kinetic lag period of the aerobic reaction and the fluorescence of the enzyme, it is concluded that 13-L-hydroperoxylinoleic acid is required for the activation of soybean lipoxygenase-1. Thus it is proposed that the enzyme with iron in the ferric state is the active species. 4. A reaction scheme is presented in which the enzyme alternatingly exists in the ferric and ferrous states for both the aerobic and anaerobic reaction.


Non-allosteric regulation of the uridine kinase from seeds of Zea mays. Uridine kinase (ATP: uridine 5'-phosphotransferase, EC 2.7.1.48) has been partially purified from ungerminated hybrid corn seed. It is associated with a soluble high molecular weight fraction from which it apparently cannot be dissociated without loss of activity. The stability of the enzyme is enhanced by the addition of dithiothreitol, glycerol and nucleotide substrate. The nucleoside specificity of the enzyme is limited to nucleosides containing pyrimidine and ribose moieties, such as uridine and cytidine. High concentrations of nucleosides cause substrate inhibition, however. The Km values for uridine and cytidine are 53 muM and 125 muM, respectively, and under subsaturating conditions uridine is phosphorylated about five times faster than cytidine. The reaction follows an ordered Bi Bi kinetic pattern, with ATP and ADP in competition for the free form of the enzyme. Purine, but not pyrimidine, nucleoside triphosphates serve as phosphate donors without regard to the sugar moiety. However, all of these triphosphates appear to compete for the same site on the enzyme. (Km ATP equals 590 muM, Km (app) GTP equals 61 muM, and CTP and UTP are linear competitive inhibitors against ATP, with Ki values of 60 muM and 240 muM, respectively.) Therefore, end product control of uridine kinase apparently does not involve allosteric sites, but instead is envisioned as simple competition between relatively effective or ineffective phosphate donors for a position on the enzyme.


Comparative study of two ATP : L-arginine phosphotransferases of molecular weight 84 000. Solen ensisensis muscle arginine kinase (ATP : L-arginine phosphotransferase, EC 2.7.3.3) was isolated in an homogeneous state. Its molecular weight was found to be about 80 000. The properties of this enzyme were compared with those of arginine kinase from Sipunculus nudus, an enzyme which also has a molecular weight of about 80 000. Both enzymes have several reactive thiol groups (8 thiol groups in the Solen kinase and 12 in the Sipunculus enzyme were titrateable with 5,5'-dithio-bis-(2-nitrobenzoic) acid and histidine residues (both enzymes have 6 reactive histidine residues). These kinases were, therefore, highly susceptible to oxidation. Both enzymes show the same pH optimum and absolute specificity towards the guanidine substrate, L-arginine. The reaction kinetics of both enzymes are of the sequential type. In the presence of alpha-aminoacids of Mg2+-ADP, similar spectral effects were obtained. The enzymes differ in their enzymic activities and in their rate of recovery following urea denaturation. The most important difference that appeared to be a special feature of the Sipunculus enzyme is that the spectrum of the Mg2+-ADP-enzyme complex is strongly intensified by L-arginine.


Kinetic properties and related changes of molecular weight in a fructokinase from Streptomyces violaceoruber. 1. A study of the initial reaction rates at variable substrate concentrations and of the molecular weight of the enzyme in the presence of different effectors, has been carried out using fructokinase (ATP: fructose 6-phosphotransferase, EC 2.7.1.4) from Streptomyces violaceoruber. 2. Saturation curves for MgATP or CoATP are sigmoidal and they change to hyperbolic in the presence of 10 mM Mg2+ or Co2+ in excess over the nucleoside triphosphate. 3. Saturation cuvves for fructose show intermediary plateaux at high (but not at low) concentrations of ATP or Mg2+. 4. The molecular weight of the enzyme in the presence of high concentrations of MgATP is 80 000. In the presence of fructose, and/or Mg2+,  the molecular weight is 20 000. 5. The effects of MgADP, uncomplexed ADP or ATP, and low concentrations of detergent on the kinetics have been studied. The results are interpreted as showing the existence of cooperative effects.


Properties of cyclic nucleotide phosphodiesterase in the central nervous system of Manduca sexta. The properties of cyclic nucleotide phosphodiesterase were studied in soluble and particulate fractions from the central nervous system of Manduca sexta (Lepidoptera: Sphingidae). It was determined that: (1) The highest levels of phosphodiesterase occur in nervous tissue. (2) The total and specific enzyme activities of larval and adult brains are greater than those of the remaining ganglia. (3) Specific central nervous sy  stem phosphodiesterase activities of the adult are lower than those of the larva, but both protein and total phosphodiesterase contents are considerably greater in the adult central nervous system. (4) Mg2+ is not absolutely required for either cyclic AMP-phosphodiesterase or cyclic GMP-phosphodiesterase activity. (5) Phosphodiesterase is inhibited by a variety of physiological and non-physiological compounds, nucleoside triphosphates being particularly effective; Some potent inhibitors of mammalian phosphodiesterase are comparatively ineffective toward Manduca sexta phosphodiesterase. (6) Kinetic analyses of soluble and particulate phosphodiesterase revealed non-linear double-reciprocal plots for the hydrolysis of both cyclic AMP and cyclic GMP, with Michaelis constants of approximately 10 mu M and 20 mu M; (7) The hydrolysis of both cyclic nucleotides appears in part to be the function of a single enzyme or related enzymes in the insect central nervous system. It follows that the intracellular level of one cyclic nucleotide may influence the concentration of the other by inhibiting its DEGRADATION.


Cyclic nucleotide-stimulable protein kinases in the central nervous sytem of Manduca sexta. Cyclic nucleotide-stimulable protein kinase (EC 1.7.1.37) has been studied in crude extracts from the central nervous system of the tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae). The insect kinase was fulfhydryl-sensitive and required Mg-2+ for optimal activity. Polyacrylamide gel electrophoresis of supernatants demonstrated the presence of multiple kinases in the larval nerve cord. At low concentrations, cyclic AMP was a much more potent activator of soluble and particulate activities than was cyclic GMP. The specific activity of coluble kinase and the magnitude of its activations by cyclic AMP were greater in the adult than in the larval central nervous system. The exogenous protein substrate specificity of the insect enzyme was similar to that of rat brain kinase with the sole exception that protamine was more readily phosphorylated than histone by nerve cord kinase. It was observed that cyclic AMP lowered the Km of Manduca sexta kinase for ATP, a phenomenon which is apparently nervous tissue=specific in mammals. An effective inhibitor of cyclic AMP-dependent protein kinase was prepared from the larval central nervous system.


Interaction of 4,5-dibromo-2,7-di-(acetatomercuri)-fluorescein with DNAs of different base composition. The changes in absorption spectra in the visible region observed on adding different naturally occurring and synthetic DNA duplexes to solutions of 4,5-dibromo-2,7-di-(acetatomercuri)-fluorescein indicate that the mercurial reacts with polynucleotides of this type. The reaction is reversible as proved by adding excess of KCN which restores the original spectra of the free dye. The interaction is characterised also by quenching of the fluorescence of the dye and the induction of optical activity in it. The extent of these spectral effects depends strongly on the (A+T) content of the complexed DNA and decreased in the order: poly [d(A-T)], Clostridium perfringens DAN, Escherichia coli DNA, Micrococcus luteus DNA and poly(dC). From equilibrium-dialysis experiments the same order in affinity is obtained when these poly-nucleotides are at equilibrium with the same concentration of 4,5-dibromo-2,7-di-(acetatomercuri)-fluorescein. From the changes produced by different mercurials in the ORD spectra and viscosity of a DNA solution it has been concluded that 4,5-dibromo-2,7-di(acetatomercuri)-fluorescein does not cause any drastic alteration of the secondary structure of DNA.


The purification and properties of superoxide dismutase from a blue-green alga. Soluble extracts of Plectonema boryanum have been shown to contain a single, electrophoretically distinct, superoxide dismutase. The enzyme has been isolated and has been found to be an iron-containing enzyme similar to that described from the periplasm of Escherichia coli. It contains 1 Fe3+/mole of enzyme. The molecular weight was approximately 36 500, and the enzyme appeared to be composed of two  subunits of equal size joined by non-covalent interactions. ESR data are presented, as are the results of amino acid analysis.


Studies on the conformational changes of metalloproteins induced by electrons in water-ethylene glycol solutions at low temperatures. III. Adrenal ferredoxin. The reduction of adrenal ferredoxin (adrenodoxin) at low temperatures was investigated in order to separate local modifications of the active centre of the protein on its reduction, from the conformational transition which seems to accompany the change of the redox state of the irons; The ESR spectra of the states of the protein, where the reduced active centre is to be found by the "oxidized" conformation of the apoprotein, were obtained. The transition from the states of the protein to the state which occurs on its chemical reduction at room temperature was also investigated. The results of the work support the view that conformational changes in proteins (enzymes) which take place while they are functioning proceed after modifications of the active centres (change of the redox state, adsorption of a substrate, etc.), and are essentially caused by them. Adrenal ferredoxin was the third subject in our studies of the intermediate states of proteins which appear after reduction of their active centres by means of electrons trapped in water-ethylene glycol mixtures at the temperature of liquid nitrogen [1, 2]. In the reduced state, the active centre of the protein has an ESR signal with a g-factor of 1.94 [3, 4] which is convenient for our purposes.


Studies on plasma membranes. XXII. Fatty acid profiles of lipid classes in plasma membranes of rat and mouse livers and hepatomas. Plasma membranes were isolated from rat and mouse livers, a transplanted rat hepatoma, two transplanted mouse hepatomas and spontaneous mouse hepatomas. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine, free fatty acids and triglycerides were separated and their fatty acid profiles determined. The various lipid classes of rat and mouse liver plasma membranes each demonstrated more or less specific fatty acid profiles. The number of double bonds decreased in the order: phosphatidylserine greater than or equal to phosphatidylethanolamine greater than phosphatidylinositol greater than phosphatidylcholine greater than or equal to sphingomyelin and lysophosphatidylcholine. Small species differences were noted in most lipid classes. A marked sex difference was observed in sphingomyelin of mouse liver membranes but in none of the phospholipids of rat liver membranes. The increased cholesterol content of all hepatoma versus liver plasma membranes was accompanied by a decrease of fatty acyl poly-unsaturation in most lipid classes of the rat hepatoma but not of the mouse hepatoma membranes. The fatty acid profiles of the mouse hepatoma membranes deviated much less from those of mouse liver than did the pattern of rat hepatoma versus rat liver. The results were discussed in relation to lipid fluidity of which fatty acyl unsaturation and cholesterol are the main parameters.


The removal of cholesterol from aortic smooth muscle cells in culture and Landschutz ascites cells by fractions of human high-density apolipoprotein. Ascites cells were labeled by intraperitoneal injection of [3H]cholesterol and aortic smooth muscle cells by addition of [3H]cholesterol to the serum component of the culture medium. The release of cholesterol from cells into a serum-free medium supplemented with the various "acceptors" was studied using ascites cells in suspension and aortic smooth muscle cells in a multilayer culture. Unfractionated human high-density apolipoprotein was somewhat more effective in the removal of labeled cellular free cholesterol, in both cell types, than apolipoprotein derived from rat high-density lipoprotein. Following separation of human high-density apolipoprotein into four fractions by Sephadex chromatography, the effect of each fraction on the removal of cellular cholesterol from ascites cells was studied. The individual fractions had a lower capacity for cholesterol removal than the original unfractionated high-density apolipoprotein and the lowest activity was detected in Fraction II which comprised 75% of the total apolipoprotein. The effectiveness to remove cholesterol could be restored to all the fractions, as well as enhanced, by addition of sonicated suspensions of lecithin or sphingomyelin, which by themselves promoted a more limited removal of cellular cholesterol. Negatively stained preparations of mixtures of the four fractions and sonicated dispersion of lecithin were shown to consist of vesicles and discs of various sizes. Addition of the apolipoprotein fractions (especially Fractions II and IV) to sonicated dispersion of sphingomyelin resulted in a pronounced formation of discs which showed a high tendency towards stack formation. Mixtures of Fraction II and lecithin or sphingomyelin were effective in the release of cellular cholesterol from multilayers of aortic smooth muscle cells in culture. These results show the feasibility of net removal of cholesterol from cells which grow in a form resembling a tissue and thus provide a model to study the role of apolipoprotein-phospholipid mixtures in cholesterol removal from cells and tissues in vivo.


A method for the quantitative determination of glycerolipids containing O-alkyl and O-alk-1-enyl moieties. We have developed a spectrophotometric procedure, based on a combination of established methods, for the quantitative determination of aklyl and alk-1-enyl (plasmalogens) ether-linked glycerolipids. It depends upon the release of alkylglycerols and alk-1-enylglycerols from phospholipids by phosphlipase C (Bacillus cereus) followed by saponification or by Vitride reduction the phospholipids; aldehydes are subsequently formed and measured colorimetrically after reacting them with a fuchsin reagent. The total alkyl and alk-1-enyl content of glycerolipids is determined oxidation of the sample withperiodate to form aldehydes and alkylglycolic aldehydes. The O-alk-1-enyl lipid content is determined on a separate sample by measuring the aldehydes produced after acid hydrolysis. The quantity of O-alkyl lipids is calculated from the difference between the values obtained for the total ether-lipid content and that of the O-alk-1enyl lipid content. Alternately, direct determination of alk-1-enylglycerols and alkylglycerols can be made if these hydrolytic products are first separated by thin-layer chromatography.


NMR studies of pig low- and high-density serum lipoproteins. Molecular motions and morphology. 1. NMR spectra of porcine high- and low density lipoproteins (d 1.120--1.210 and 1.019--1.070, respectively) and their extracted lipids were obtained as functions of temperature, frequency and solution viscosity, and from solutions to which paramagnetic species had been added. 2. About one-third of the N(CH3)3 groups in low-density lipoproteins are so immobile that they do not give a sharp resonance at any temperature up to 65 degrees C, unless the particles are disrupted with sodium dodecylsulphate. Most of the protein residues also undergo little segmental motion. 3. A marked restriction of motion of acyl chain terminal CH3 groups suggests that chain interdigitation occurs in low-density lipoprotein. Apart from this, there is a general ordering of the lipids without a decrease in the rate of rotation about bonds, suggesting that the protein organizes the lipids by controlling the molecular packing rather than by direct strong interactions. The lipids are more ordered in low-density than in high-density lipoprotein. 4. All phospholipids with mobile N(CH3)3 groups are at the particle surfaces, in patches separated by protein. In low-density lipoprotein the patches are raised proud of the protein, whereas in high-density lipoproteins the protein and lipid polar groups are coplanar. 5. The high-density lipoprotein results are consistent with literature models for the structure. The low-density lipoprotein results suggest a new model, which is basically a trilayer. The centre consists of a monolayer of phospholipid with tightly-packed polar groups in contact with a protein core. The outer monolayer of phospholipid contains the rest (most) of the protein; the central layer contains the neutral lipid (cholesterol esters and triglycerides), interdigitated into both the inner and outer monolayers. Unesterified cholesterol is distributed through all three layers.


Localization of enzymes involved in polyphosphoinositids metabolism on the cytoplasmic surface of the human erythrocyte membrane. 1. Impermeable inside-out and right-side-out vesicles were prepared from membranes of human erythrocytes. During preparation of each kind of impermeable vesicle, permeable vesicles were also obtained. 2. Incubation of vesicles with [gamma-32P]ATP at 37 degrees C for periods of up to 1 hr did not change the topography or the permeability of the vesicles. 3. Vesicles incorporated labeled phosphate from [gamma-32P]ATP into both diphosphoinositide and triphosphoinositide, but impermeable inside-out vesicles incorporated significantly more nuclide than did right-side-out vesicles. 4. Permeable vesicles derived during the preparation of inside-out vesicles were as active as impermeable inside-out vesicles in the incorporation of labeled phosphate into the polyphosphoinositides. However, permeable vesicles derived during the preparation of right-side out vesicles were not as active. 5. Impermeable right-side-out vesicles, treated with 0.01 percent saponin, incorporated labeled phosphate into the polyphosphoinositides at a level comparable to that of impermeable inside-out vesicles. 6. These data show that the enzymes involved in metabolism of diphosphoinositide and triphosphoinositide are located on the cytoplasmic surface of the erythrocyte membrane.


Changes in phosoholipid susceptibility toward phospholipases induced by ATP depletion in avian and amphibian erythrocyte membranes. About half of the sphingomyelin content of fresh and ATP-depleted chicken erythrocytes is hydrolysed by sphingomyelinase. Removal of spingomyelin exposes the rest of the membrane phospholipids to hydrolysis by phospholipase C only in ATP-depleted but not in fresh cells. Addition of both sphinogomyelinase and phospholipase C to ATP-depleted cells causes about 60-70 percent hydrolysis of the total phospholipids accompanied by extensive (90 percent) hemolysis. The phospholipids of toad erythrocytes are partially available to phospholipase C activity in fresh cells (17-25 percent hydrolysis) without prior sphingomyelinase treatment. However, in ATP-depleted toad cells phospholipase C hydrolyses 66 percent of phospholipids and causes extensive lysis. Treatment of either fresh or ATP-depleted toad erythrocytes by sphingomyelinase together with phospholipase C induces hydrolysis of most of the phospholipds with complete lysis. Restoration of ATP to ATP-depleted cells endows them with resistance to the attack of phospholipase C. The correlation between changes in ATP level and membrane organization as revealed by increased susceptibility toward phospholipases is discussed.


Evidence for constrained lipid mobility in the erythrocyte ghost. A spin label study. Our data show that the ESR spectrum of 5-nitroxide stearate bound to erythrocyte membranes varies with the amount of label bound and suggest that, at high binding, a significant proportion of label molecules lie within approximately equal to 15-10--8 cm; this gives rise to spin-exchange (ambient temperature) and dipole-dipole interactions. We find that these spectral manifestations due to label clustering can be abolished by reduction of pH and the conjoint action of lysolecithin and trypsin, although both perturbations increase 5-nitroxide stearate binding. Both perturbations are known to mobilize intramembranous particles by modifying or extracting some membrane proteins. We accordingly suggest that the lipids and proteins of erythrocyte membranes exist in a relatively fixed mosaic, and that the mobility of both components is restricted by some membrane-associated protein framework.


ESR spectral analysis of the molecular motion of spin labels in lipid bilayers and membranes based on a model in terms of two angular motional parameters and rotational correlation times. Electron spin resonance (ESR) spectral line shapes are calculated for a nitroxide spin-labeled molecule undergoing rapid restricted rotations (twisting) about its long molecular axis while simultaneously tumbling within a cone. Explicit expressions are derived for the hyperfine splittings and g-values, as well as for the secular contributions to the motionally modulated linewidths. The present model is useful for analyzing the restricted twisting and tumbling motions, and rotational correlation times, of spin-labeled molecules in bilayers. Simulated spectra compare well with experimental spectra of lecithin bilayers marked with cholestane spin label, over a wide temperature range.


Organization of enzymes of glycolysis and of glutathione metabolism in human red cell membranes. 1) The activities of 16 enzymes of glycolysis and of glutathione metabolism were determined in intact human red cell membranes (ghosts) which were prepared by hypotonic hemolysis. 2) Enzymes and hemoglobin of the ghosts were resolved by two toluene extractions. Only the four enzymes hexokinase, fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase and pyruvate kinase could not be released completely from the ghosts. 3) The residual membrane fraction, which was obtained after the toluene extraction of ghosts prepared at 30 imOsM, contained 0.02% of the original hemoglobin content of the red cell. Between 6.5 and 23% of the hemolysate activities of glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase and fructose-bisphosphate aldolase were detected in this fraction after mechanical disruption. 4) Sonication of intact ghosts increased the activities of fructose-bisphosphate aldolase, pyruvate kinase and phosphoglycerate kinase. 5) In "white" ghosts prepared at 5 imOsM phosphate buffer which contained 0.5% of the original hemoglobin the activities of fructose-bisphosphate aldolase and glyceraldehyde-phosphate dehydrogenase were detected at high levels. The activities of pyruvate kinase and phosphoglycerate kinase were low in these preparations. 6) The results indicate that one part of all enzymes is loosely attached to the inner surface of the membrane as is hemoglobin. A second part, the "cryptic enzyme activity", is available after resolving by toluene. A residual part of four enzymes is firmly bound to the membrane. Two of them (fructose-bisphosphate aldolase and glyceraldehyde-phosphate dehydrogenase) are oriented toward the inner surface of the membrane, whereas pyruvate kinase and phosphoglycerate kinase are hidden in the lipid core of the membrane.


Solubilized DNA-dependent nuclear RNA polymerases from the mammary glands of late-pregnant rats. Mammary gland nuclear extracts from late-pregnant Wistar or Sprague-Dwaley rats, analyzed by DEAE-Sephadex chromatography, were found to contain DNA-dependent RNA polymerases, cyclic nucleotide-binding proteins, and cyclic AMP-independent protein kinases. 1. The fractions from chromatographed nuclear extracts which contained nucleolar enzymes or nucleoplasmic enzyme II, bound radioactive cyclic AMP and cyclic GMP. Other fractions also bound cyclic nucleotides, some preferentially associating with one or the other compound. 2. Cyclic AMP increased the amount of RNA formed by several alpha-amanitin-insensitive fractions containing nucleolar enzymes. 3. Cyclic AMP reduced the amount of RNA formed by column fractions which included nucleoplasmic enzyme II. 4. Cyclic GMP increased the amount of RNA synthesized by column fractions containing enzyme II. 5. Two major cyclic AMP-independent protein kinases which did not elute with enzymes Ib and II, and several minor protein kinases were present. These findings may have important implications for understanding the proximate control of transcription. A relationship between them is not established, but is under study.


Mitochondrial protein synthesis and the stimulation of steroidogenesis by cyclic adenosine 3',5'-monophosphate in isolated rat adrenal cells. The stimulation by cyclic AMP of steroidogenesis in rat adrenal cells isolated by trypsin treatment was inhibited by D-threo-chloramphenicol and by its L-threo-isomer. The former is an inhibitor of mitochondrial protein synthesis while the latter is not. Both substances, at concentrations which inhibit steroidogenesis, inhibit amino acid incorporation into the proteins of microsomes. Inhibition in other subcellular fractions also occurs depending on the isomer and its concentration. In no case was there a preferential inhibition of amino acid incorporation into mitochondrial proteins. Carbomycin, another inhibitor of mitochondrial protein synthesis, gave similar results. In addition, subfractionation of mitochondria in these experiments revealed no preferential inhibition of amino acid incorporation into the proteins of either the soluble of membrane fractions of this organelle. The above results were obtained at several concentrations of the inhibitors when only partial inhibition of steroidogenesis was present. Both isomers of chloramphenicol inhibited steroidogenesis in a cell-free system to an extent equal to that found with cyclic AMP-stimulated steroidogenesis in intact cells. It is concluded that these inhibitors of mitochondrial protein synthesis have multiple metabolic effects in adrenal cells.


Alterations in SVT2 cell transfer RNAs in response to cell density and serum type. The chromatographic elution profiles of tRNA-A sn, tRNA-A sp, tRNA-H is and tRNA-T y r from SV40-transformed BALB-3T3 cells grown in fetal calf serum or cald serum-supplemented media have been examined. The relative proportions of certain of the isoaccepting species of these four tRNAs are altered in a similar fashion depending on the serum type. It is suggested that the elution profile alterations reflect the extent of modifications of a specific G residue to the minor nucleoside Q, and that this process differs between untransformed and transformed cells. In addition, cell density appears to influence the Q content of these tRNAs, though other density-dependent tRNA modifications also appear to occur.


Influence of reserpine on all-night sleep pattern in nonlobotomized and lobotomized chronic schizophrenic patients. This study was performed on two groups of schizophrenic patients. One group consisted on nine nonlobotomized patients and the other of nine lobotomized ones. The groups were matched for age, sex, duration of illness, clinical symptoms, type and dose of psychopharmacological treatment. The patients of both groups were administered 1 mg of reserpine half an hour before bedtime, for three successive days. Before reserpine administration the mean percentage time of the NREM stage 4 was significantly higher in the lobotomized group. There was no significant difference in the REM parameters. After three days of reserpine administration in the nonlobotomized group, there was no significant difference in the mean percentage of the NREM stage 4, whereas the mean REM percentage significantly increased and REM latency decreased. In the lobotomized group the same procedure, i.e., three days of reserpine administration, provoked a significant decrease in the mean percentage of the NREM stage 4 and no significant changes in the REM parameters. This difference in reserpine action on sleep in the lobotomized group is discussed.


The behavioral effects of L-methionine and related compounds in rats and mice. Several groups of investigators have reported that the administration of L-methionine, with or without a monoamine oxidase inhibitor, induced an acute florid psychotic reaction in 40 percent of schizophrenics tested. The mode of action of L-methionine in brain is unknown, but may be via one or more of three mechanisms: the excess methionine (i) may lead to the production by transmethylation of some psychotomimetic methylated derivative of dopamine or serotonin, or (ii) could result in an increase in the levels of a metabolite of methionine (e.g., homocysteine, cystathionine, or cysteine), or (iii) may effect the cellular uptake of other amino acids. In order to test the first two hypotheses, L-methionine, betaine (another metyl group donor), L-methionine plus L-serine, L-cysteine, L-serine, and saline (as a control) were studied on the sleep-wake cycles of random-bred Swiss mice and on the avoidance behavior or rats. L-methionine plus L-histidine, L-methionine plus nicotinamide, L-histidine, and nicotinamide were also tested in the mice. Daily injections of 250 mg/kg of these compounds were administered for at least 21 consecutive days. Schedule performance in the rat and the sleep-wake cycles of the mice were monitored during this period and compared to controls. L-Methionine induced behavioral and sleep cycle disturbances which were removed by the simultaneous administration of L-serine but not by the addition of L-histidine or nicotinamide. These data suggest that the disruption may be due to an increase in the levels of one of the metabolities of methionine, homocysteine, rather than to an increase in the number of available methyl groups.


Somatosensory cerebral evoked potentials after vascular lesions of the brain-stem and diencephalon. Clinical and electrophysiological observations are described in 7 patients with clinically well-identified vascular lesions of the brain-stem or diencephalon. In the patients of Group A with a thalamic syndrome, the somatosensory cerebral evoked potentials had a reduced voltage and increased latency on the affected side. No significant anomalies were recorded in the patients of Group B with a Wallenberg or Weber syndrome. In patients of group C with a locked-in syndrome, the cerebral evoked potentials presented marked bilateral anomalies which provided interesting data about the extension of the pontine vascular lesions into the tegmentum. The pathophysiological mechanisms involved in the changes of average cerebral evoked potentials and in the slowing of corticipetal conduction are discussed.


Physiological studies of the dying-back phenomenon. Muscle stretch afferents in acrylamide neuropathy. (1) The responses of 1,001 medial gastrocnemius afferents with conduction velocities of 24-126 m/sec have been studied in cats with experimental acrylamide neuropathy. (2) Many units which conducted at velocities indicating that they innervated muscle stretch receptors failed to discharge during muscle stretch or contraction. In animals with mild neuropathy 10 per cent of 366 units were non-responsive. In animals with moderate or severe neuropathy the proportion of non-responsive units was 68 per cent of 315 fibres and 89 per cent of 320 fibres respectively. Group I fibres were involved to a greater extent than Group II fibres. (3) The distributions of identified functioning units with respect to conduction velocity were normal, indicating that acrylamide did not produce conduction slowing before failure of function. (4) Electrophysiological and histological findings indicate that acrylamide initially produces failure of impluse conduction and subsequent fibre breakdown in the terminal axon while normal impulse conduction is preserved more proximally.


Proteins of rat brain myelin in neonatal hypothyroidism. The amount of myelin and its protein composition was studied in hypothyroid rats during the first 30 days after birth. Although the brain weight was 92% of that in the controls, the yield of myelin in hypothyroid animals was only 60% of that in controls. The protein and glycoprotein ocmposition of the isolated myelin was similar in hypothyroid and control rats. The 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity in whole brain from hypothyroid animals was only 60% of the controls and reflected the decrease in myelin formation. In isolated myelin the specific activity of CNP was not different in hypothyroid and control animals with the exception of very immature animals (10 days). The major fucose-labeled glycoprotein in myelin of the hypothyroid rats had a slightly higher apparent molecular weight than that in myelin from age matched controls, probably reflecting a retardation of brain maturation and myelin formation.


Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section. Chemical properties of substance P in the spinal cord and dorsal roots of the cat were examined by gel chromatography and paper electrophoresis. The results suggest that the substance is identical with the undecapeptide, hypothalamic substance P, recently isolated from bovine hypothalamus. Distribution of substance P in the cat spinal cord (L5-S1) was investigated. Substance P was highly concentrated in the dorsal horn, where the highest level was found in the dorsal part. After the unilateral ligation and/or section of the dorsal roots, the level of substance P in the dorsal horn, particularly in its dorsal part, was markedly lowered. In the ligated and sectioned dorsal root, substance P was highly accumulated on the ganglion side, whereas its level was lowered on the central side of the ligature. These results suggest that hypothalamic substance P synthesized in the spinal ganglia is transported in the dorsal root toward their intraspinal axon terminals, where the substance is concentrated and serves as a neurotransmitter.