Showing papers in "Molecular Pharmacology in 1968"

The Influence of Chemical Agents on the Accumulation of Adenosine 3',5'-Phosphate in Slices of Rabbit Cerebellum

Abstract: Incubation of slices of rabbit cerebellum with norepinephrine for a few minutes resulted in large increases in the tissue content of adenosine 3',5'-phosphate. Exposure of slices to norepinephrine for more than 6 min led to a progressive disappearance of this nucleotide. Theophylline, a known inhibitor of cyclic-3',5'-ribonucleotide phosphodiesterase, had relatively little effect by itself upon the slice content of adenosine 3',5'-phosphate, but potentiated the effects of norepinephrine severalfold. In a survey of substances known to occur in the central nervous system, histamine, and to a lesser extent serotonin, were observed to produce effects similar to those of norepinephrine. The effects of histamine and norepinephrine were additive; phenoxybenzamine and diphenhydramine were observed to inhibit the effects of histamine at concentrations having relatively little influence on the action of norepinephrine while dichloroisoproterenol specifically prevented the effects of norepinephrine. Prolonged exposure of slices to norepinephrine desensitized slices to the readdition of norepinephrine, but not to histamine and vice versa. ACKNOWLEDGMENTS This work was supported in part by Grants AM-06141, NB-05716, and GM-661 from the U.S. Public Health Service. The authors also wish to acknowledge the skillful technical assistance of Mrs. Arleen Maxwell Haley and Mr. George Thorne in carrying out this work.

Studies on the interaction of ouabain and other cardio-active steroids with sodium-potassium-activated adenosine triphosphatase.

Abstract: The combination of ouabain with (Na+ + K+)-ATPase is essentially irreversible at physiological temperature and pH. The rate of inhibition of the enzyme by different cardioactive steroids varies widely. The amount of steroid bound parallels the inhibition of enzyme activity. Mg2+, Na+, K+, nucleotides, and orthophosphate markedly influence the rate of interaction. In the presence of nucleotides, the rate is accelerated by Mg2+ and Na+ and retarded by K+. In the presence of orthophosphate, Mg2+ increases, K+ slows, and Na+ markedly decreases the rate of interaction. The ratio of ouabain binding to Na+-dependent phosphorylation is 0.50 for Electrophorus electric organ ATPase. In contrast to the native enzyme, the ouabain-treated enzyme rapidly incorporates orthophosphate. Cardioactive steroids appear to inhibit (Na+ + K+)-ATPase by reducing the difference between the conformational energies ot the phosphorylated and nonphosphorylated forms of the enzyme.

Studies on adenosine 3',5'-phosphate in rabbit cerebral cortex.

Abstract: Incubation of slices of rabbit cerebral cortex with histamine resulted in large accumulations of adenosine 3',5'-phosphate. Maximal levels of the nucleotide required about 15 min exposure to histamine and represented more than an 8-fold increase. Inclusion of theophylline (0.5 mM) in incubation media had little effect by itself on the accumulation of adenosine 3',5'-phosphate but increased the effect of histamine 3-fold. Norepinephrine increased the nucleotide level only about 70%, but only 2 min exposure to the agent was required to produce maximal values. This pattern of responses is in sharp contrast to that observed in cerebellar slices, but appears to be similar to that of other areas of rabbit brain. The content of adenosine 3',5'-phosphate in the cerebral cortex of rabbit heads frozen rapidly at intervals after decapitation rose about 8-fold within 90 sec after decapitation. Although phosphorylase a levels increased very rapidly after decapitation, no changes in phosphorylase a content of either cerebellar or cerebral cortex slices was detected under conditions producing large increases in adenosine 3',5'-phosphate. ACKNOWLEDGMENTS This work was supported in part by U.S. Public Health Service Grants AM-06141, NB-05716, and GM-661. The authors also wish to acknowledge the skillful technical assistance of Mrs. Arleen Maxwell Haley, Mr. George Thorne, and Mr. Neil Joebchen.

The proton relaxation of benzyl alcohol in erythrocyte membranes.

Abstract: The mobility of benzyl alcohol molecules in erythrocyte membranes has been measured by nuclear magnetic resonance relaxation. At low concentrations the alcohol molecules are strongly immobilized by the membrane structure, this decreases as the concentration rises but starts to increase again at 60 mM. Suspensions of membrane lipid produce similar effects on relaxation at low concentrations but show no upswing at high concentrations. Separated membrane protein has a large effect on benzyl alcohol relaxation rate as have erythrocyte membranes pretreated with 300 mM benzyl alcohol. These results suggest that as a result of insertion of time anesthetic in the membrane the lipid becomes disordered and finally exposes the protein, making available binding sites in the protein which are occult in the untreated membrane.

Mutations and decreases in density of transforming DNA produced by derivatives of the carcinogens 2-acetyl-aminofluorene and N-methyl-4-aminoazobenzene.

Abstract: A series of metabolites and synthetic derivatives of the carcinogens 2-acetylaminofluorene (AAF) and N -methyl-4-aminoazobenzene (MAB) was tested at pH 7.5 and room temperature for interaction with biologically active DNA isolated from Bacillus subtilis . Previous studies have indicated that AAF and MAB are converted in rat liver, via N -hydroxy metabolites, to reactive carcinogenic esters which can attack guanine residues in nucleic acids. In the present work the reactive synthetic esters N -acetoxy-AAF, N -benzoyloxy-AAF, AAF- N -sulfate, and N -benzoyloxy-MAB caused: (a) severe reductions in transforming activity, (b) up to 100-fold increases in the frequencies of mutations in transforming DNA, and (c) decreases in the buoyant density of DNA as measured in a CsCl gradient. MAB, AAF, and certain metabolites of AAF less carcinogenic than the reactive esters, viz., N -hydroxy-AAF, the glucuronide of N -hydroxy-AAF, 2-aminofluorene (AF), and N -hydroxy-AF, neither inactivated nor caused mutations or density changes in the transforming DNA. 2-Nitrosofluorene-treated transforming DNA exhibited a marginal increase in mutational frequency. The high carcinogenicities of N -acetoxy-AAF, N -benzoyloxy-AAF, and N -benzoyloxy-MAB, especially at the sites of subcutaneous injection in rats, correlate well with their high reactivities and mutagenicities for transforming DNA. Similarly, the lack of activity of the nonester derivatives in the transforming DNA system is in accord with their lack of reactivity with nucleophiles at neutrality. In DNA samples reacted with esters of N -hydroxy-AAF, the frequencies of induced mutations were directly proportional to the decreases in buoyant density. Furthermore, experiments with N -acetoxy-AAF-9-14C demonstrated that the decrease in buoyant density was also directly proportional to the number of fluorene molecules covalently bound to the DNA. The decrease in density appears to result from two opposing effects: binding of the highly buoyant AAF residues and slight denaturation of the reacted DNA. Severely inactivated DNA, after denaturation, showed CsCl and Cs2SO4 gradient sedimentation patterns compatible with the occurrence of crosslinks between the complementary strands. Bacterial strains capable of repairing lesions in DNA caused by ultraviolet radiation restored up to 50% of the transforming ability inactivated by these reactive esters. The induced mutations were spontaneously reversible and thus appeared to be caused by single base-pair changes. ACKNOWLEDGMENT This investigation was supported by grants from the National Science Foundation (B-14976), the National Cancer Institute, USPHS (CA-07175 and CRTY-5002), the Jane Coffin Childs Memorial Fund for Medical Research, and by the Alexander and Margaret Stewart Trust Fund. We would like to acknowledge the valuable suggestions and frequent help of our colleagues Dr. H. Kubinski, Dr. H. A. Lozeron, and Dr. W. C. Summers. Mr. D. Zuhse has provided expert assistance with the ultracentrifuge, and Mr. J. T. Fiandt and Mr. T. E. Noll assisted with other technical aspects of the study.

Mechanism of p-nitrobenzoate reduction in liver: the possible role oc cytochrome P-450 in liver microsomes.

Abstract: The following evidence suggests that the reduction of p -nitrobenzoate to p -aminobenzoate by liver microsomes is mediated by cytochrome P-450: (a) carbon monoxide blocks nitroreduction, and the degree of inhibition is proportional to the amount of cytochrome P-450 bound as the carbon monoxide complex; (b) the rate of nitroreduction is proportional to the amount of cytochrome P-450 in liver microsomes from animals previously treated with either phenobarbital or carbon tetrachloride.

The role of potassium and calcium ions in the effect of epinephrine on cardiac cyclic adenosine 3',5'-monophosphate, phosphorylase kinase, and phosphorylase.

Abstract: The effect of epinephrine on the biochemical sequence of events leading to the activation of glycogen phosphorylase was studied in the isolated, perfused rat heart. In arrested preparations, perfused with a medium either devoid of Ca++ or containing a depolarizing concentration of K+, the epinephrine-induced activation of phosphorylase was markedly diminished. In the absence of Ca++, however, the epinephrine-induced rise in cyclic adenosine 39,59-monophosphate (cyclic AMP) concentration and the activation of phosphorylase kinase were not greatly altered. This suggested that the effect of omission of Ca++ occurred at the phosphorylase b to a conversion step. The effect of epinephrine on phosphorylase activation was blocked simultaneously with the cessation of contraction following perfusion with a Ca++-deficient medium. The store of Ca++ required for the maintenance of contractile activity appeared to be similar to that necessary for the conversion of phosphorylase b to a. Elevation of Ca++ in the perfusion medium resulted in activation of phosphorylase but not of phosphorylase kinase; nor was there an increase in cyclic AMP concentration. This observation also suggested that Ca++ is required for the catalytic activity of phosphorylase kinase. In the heart depolarized with K+, only a small and transient increase in cyclic AMP concentration occurred in response to epinephrine. Activation of phosphorylase kinase was considerably less than that observed in the beating heart. This suggested that an excess of K+ interfered with the action of epinephrine at a site at or before adenyl cyclase. Thus the activation of phosphorylase in cardiac muscle can be the consequence of one of the following events: (a) the activation of phosphorylase kinase as a result of a rise in cyclic AMP concentration; (b) an increase in the catalytic activity of phosphorylase kinase as a result of an increase in the concentration of Ca++ available to the enzyme; or (c) a combination of these two mechanisms.

Inhibition of drug metabolism. V. Inhibition of drug metabolism by steroids.

Abstract: In a study employing hepatic microsomes from rats, estradiol-17β, testosterone, androsterone, progesterone, and hydrocortisone inhibited competitively the oxidation of ethylmorphine and hexobarbital. Inhibitor constants for each steroid were the same whether ethylmorphine or hexobarbital served as substrates. Results are consistent with the concept that certain drugs and steroids are alternative substrates for a common microsomal mixed function oxidase system. The inhibitory effects of steroids on chlorpromazine metabolism were both qualitatively and quanitatively different from those observed when ethylmorophine and hexobarbital metabolism were studied. Not only were the steroids less potent inhibitors of chlorpromazine oxidation, but inhibition was not competitive.

Hydrogen peroxide detoxication by glutathione peroxidase and catalase in rat liver homogenates.

Abstract: The activity of glutathione peroxidase (H2O2:glutathione oxidoreductase, EC 1.11.1.9) has been studied in the soluble supernatant fractions prepared from rat liver homogenates. Enzyme activity may be demonstrated in the presence of active as well as inhibited catalase. The findings are compatible with an essential function for hepatic glutathione peroxidase in the detoxication of intracellular hydrogen peroxide, and with the view that hydrogen peroxide is the common toxic agent in the drug-induced anemia associated with a genetic deficiency of erythrocyte glucose 6-phosphate dehydrogenase. The experiments also suggest a potential role for glutathione peroxidase in regulating cellular metabolism via pentose shunt control.

Effects of epinephrine on cardiac cyclic 3',5'-AMP, phosphorylase kinase, and phosphorylase.

Abstract: The concentration of cyclic 3',5'-adenosine monophosphate (cyclic AMP) and the activities of phosphorylase kinase and phosphorylase were determined in biopsy samples from dog hearts and in whole rat hearts frozen in situ. Cyclic AMP was measured by the activation of muscle phosphorylase kinase employing a preincubation at 0° to improve the sensitivity of the assay. Dog heart phosphorylase kinase was measured on extracts of homogenates by comparing the activity of the enzyme at pH 6.0 to that at 8.2. Epinephrine markedly increased the activity at pH 6.0 at a dose that did not significantly elevate the percentage of phosphorylase α. The time course of the change in kinase activity was consistent with the hypothesis that activation of this enzyme was necessary for time formation of cardiac phosphorylase α. No significant increase in the concentration of cyclic AMP was demonstrable in the dog heart at a dose of epinephrine that produced a marked inotropic effect. The possibility of an artifact in the biopsy technique was suggested by the finding that a rapid increase in cyclic AMP occurred in rat hearts frozen in situ , but not when samples were excised and frozen. Phosphorylase kinase activity increased as rapidly as did the cyclic AMP concentration and before any change in the phosphorylase activity occurred. Pronethalol blocked the epinephrine-induced rise in the cyclic AMP concentration. These results are consistent with the hypothesis that the effect of catecholamines on cardiac phosphorylase is mediated through an action of cyclic AMP on phosphorylase kinase. ACKNOWLEDGMENTS The authors wish to thank Mrs. Margaret Maltbie for her excellent technical assistance. This study was supported by Research Grant HE 04626 from the National Heart Institute, Career Development Award GM 6257 (S.E.M.), and the Interdepartmental Cardiovascular Training Program of Emory University School of Medicine sponsored by the National Heart Institute Training Grant 5T1 HE 5380.

Purine nucleoside phosphorylase from human erythrocytes. 3. Inhibition by the inosine analog formycin B of the isolated enzyme and of nucleoside metabolism in intact erythrocytes and sarcoma 180 cells.

Abstract: Formycin B, an analog of inosine, is a potent competitive inhibitor of purified erythrocytic purine nucleoside phosphorylase. The inhibition constant, Ki (1 x 10-4 M) is of the same order of magnitude as the Km for inosine (5 x 10-5 M). Formycin B also inhibits the degradation of nucleosides by intact and hemolyzed human erythrocytes and by Sarcoma 180 ascites cells.

Reactions in Vitro of Some Tissue Nucleophiles with the Glucuronide of the Carcinogen N-Hydroxy-2-acetylaminofluorene

Abstract: The metabolically formed glucuronide of N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) is cleaved at the N—O bond in reactions in vitro at pH 7 with methionine, tryptophan, and guanosine. These reactions of the glucuronide (N-GlO-AAF) are similar to, but considerably slower than, those with esters of N-hydroxy-AAF such as N-acetoxy-AAF. At pH 7 the major product of either N-acetoxy-AAF or N-GlO-AAF with methionine is 3-CH3S-AAF. At pH levels greater than 7, N-GlO-AAF also yields considerable amounts of 3-CH3S-2-aminofluorene (3-CH3S-AF). Neither N-hydroxy-AAF nor the triacetyl methyl ester of N-GlO-AAF gives significant reaction with methionine at pH 5-9; with N-hydroxy-2-aminofluorene the reaction with methionine to yield 3-CH3S-AF increases markedly below pH 5.5. The reaction of N-GlO-AAF with guanosine appears to yield a mixture of N-(guanosin-8-yl)-2-acetylaminofluorene (the predominant product with N-acetoxy-AAF) and N-(guanosin-8-yl)-2-aminofluorene. The uncharacterized products formed by reaction of N-GlO-AAF or N-acetoxy-AAF with tryptophan appear to be similar. Tumor development did not occur within 12 months after repeated subcutaneous injections of either the sodium or cupric salt or of the triacetyl methyl ester of N-GlO-AAF into female rats. Under the same conditions N-hydroxy-AAF induced high incidences of tumors in the subcutaneous tissue, mammary glands, and ear duct glands. Whether or not metabolically formed N-GlO-AAF is involved in the formation of protein- and nucleic acid-bound fluorene derivatives in vivo and in tumor induction by AAF and N-hydroxy-AAF requires further investigation.

Increased synthesis of catecholamines in the intact rat following administration of alpha-adrenergic blocking agents.

Abstract: The present study was undertaken to evaluate the effects of α-blocking agents on the rate of catecholamine synthesis in vivo . It was found that phentolamine and phenoxybenzamine increase the formation of radioactive norepinephrine and epinephrine in rat tissues when L-tyrosine-14C is used as a precursor but not when L-dopa-3H is the precursor. These findings indicate that α-blocking agents increase catecholamine synthesis by stimulating tyrosine hydroxylase activity.

Lethal Action and Metabolic Effects of Streptonigrin on Escherichia coli

Abstract: Streptonigrin is lethal to cultures of Escherichia coli at concentrations that allow synthesis of DNA, RNA, and protein to continue. At higher concentrations DNA synthesis is preferentially inhibited. The lethal event is accompanied by DNA degradation, an effect enhanced if protein synthesis is also inhibited by witholding a required amino acid or by adding chloramphenicol. The degradation products, which are released into the culture medium, include nucleotides and bases, but no unusual products were detected. Both an electron source and oxygen are required for streptonigrin to exert its greatest lethal effect. This suggests that a reaction product of oxygen and intracellularly reduced streptonigrin is the lethal agent. This agent is likely to be a peroxide or peroxy free radical but is not hydrogen peroxide.

Methanol Metabolism in the Monkey

Abstract: The peroxidative system involving hepatic catalase plays a major role in the oxidation of methanol in the rat (1), but in the monkey the peroxidative mechanism does not appear to be important. This conclusion is based on the following observations: (a) ethanol and methanol were about equally reactive with the peroxidative system, but ethanol was much more reactive with the alcohol dehydrogenase system than methanol. Ethanol was a much more effective inhibitor of methanol oxidation in the intact monkey than it was in the rat, which is what would be expected if methanol is oxidized by the alcohol dehydrogenase system in the monkey, but by the peroxidative system in the rat. (b) By similar reasoning, 1-butanol was a stronger inhibitor of methanol oxidation in the monkey than it should have been if the peroxidative system was involved. (c) 3-Amino-1,2,4-triazole, a potent inhibitor of hepatic catalase, greatly reduced methanol oxidation in the rat, but had no measurable effect on methanol oxidation in the monkey. (d) Ethylene glycol stimulated the rate of methanol oxidation in the rat, probably as a result of an increased H2O2 production that occurs when glycolic acid, a metabolite of ethylene glycol, is oxidized to glyoxylic acid (6,7); no such stimulation was seen in the monkey. Studies in vitro which measured the methanol-oxidizing activity of hepatic alcohol dehydrogenase isolated from monkeys also support the view that this enzyme is largely responsible for methanol oxidation in this species.

Metabolic Studies with Carbocyclic Analogs of Purine Nucleosides

Abstract: In carbocyclic analogs of nucleosides a methylene group replaces the ring oxygen atom of the ribofuranosyl moiety. When evaluated for cytotoxicity against H. Ep. #2 cells in culture, the carbocyclic analog of adenosine (C-Ado) was highly cytotoxic (ED50 0.7 µM), whereas the carbocyclic analogs of inosine, 6-mercaptopurine ribonucleoside, and 6-methylthiopurine ribonucleoside were not cytotoxic at concentrations up to 150 µM. Sublines of H. Ep. #2 cells that lacked adenosine kinase were resistant to C-Ado, but the degree of resistance was less than that to other cytotoxic adenosine analogs. In H. Ep. #2 cells in culture C-Ado inhibited an early step of de novo purine biosynthesis, as determined by its ability to reduce the amount of formylglycinamide derivatives accumulating in azaserine-treated cells. In crude cell-free supernatants from H. Ep. #2 cells, C-Ado interfered with the phosphorylation of 6-methylthiopurine ribonucleoside; but none of the four carbocyclic analogs interfered with the phosphorylation or deamination of adenosine or the cleavage of inosine to hypoxanthine. In crude supernatants supplemented with ATP and Mg2+, C-Ado was phosphorylated and, to some extent, deaminated. In intact cells 3H-C-Ado was converted to mono-, di-, and triphosphates and, to a small extent, deaminated; no compound migrating like NAD was detected, and little radioactivity was found in polynucleotides. C-Ado was a substrate for a partially purified adenosine kinase from H. Ep. #2 cells (Km = 7.8 x 10-5 M) and for calf intestinal adenosine deaminase (Km = 3.3 x 10-3 M).

Thyroid hormone and activities of drug-metabolizing enzymes and electron transport systems of rat liver microsomes.

Abstract: Drug-metabolizing activities of liver microsomes and the activities of microsomal electron transport systems were investigated in male and female rats with altered thyroid states. The administration of thyroxine decreased the N-demethylation of aminopyrine and hydroxylation of hexobarbital by liver microsomes in male rats. In contrast, the same treatment increased the metabolism of aminopyrine and hexobarbital in female rats. The hydroxylation of aniline and reduction of p-nitrobenzoic acid were increased in both male and female rats. The administration of thyroxine increased the activity of microsomal NADPH oxidase, NADPH-cytochrome c reductase, and NADPH-neotetrazolium reductase in both male and female rats, but the magnitude of increase was much greater in female rats than in male rats. The content of P-450 was decreased in male rats, but this content was not significantly altered in female rats. The metabolism of aminopyrine, hexobarbital, aniline, and p-nitrobenzoic acid and the activities of NADPH oxidase, NADPH-cytochrome c reductase, NADPH-neotetrazolium reductase, and NADH oxidase were decreased in the thyroidectomized male and female rats, but the activity of NADH-cytochrome c reductase and cytochrome b5 and the P-450 content were not significantly altered. The administration of triiodothyronine completely restored all values in the thyroidectomized female rats, but the activities of aminopyrine N-demethylase, hexobarbital hydroxylase, and NADPH-neotetrazolium reductase and P-450 content remained low in the male rats. The stimulative effect of phenobarbital administration on the microsomal enzymes was observed in the thyroxine-treated rats and thyroidectomized rats as well as in normal rats.

Kinetics of enzyme reactions with competing alternative substrates.

Abstract: When two alternative substrates are present simultaneously in an enzyme reaction, and one is a better substrate (higher Vmax) than the other, the combined rate of the two reactions may be greater than, equal to, or less than the rate observed with the better substrate alone. At some concentration of the better substrate, the poorer substrate will appear to have no effect. On a plot of the combined velocity against the concentration of the more effective substrate, the family of curves obtained, each representing a different level of the poorer substrate, will have a common point of intersection. This phenomenon can be exploited effectively to determine whether or not a single enzyme catalyzes two similar reactions, or to determine whether or not two drugs act on the same receptor. In examining the applicability of these conclusions to more complex enzyme mechanisms, six variations of two major bi-bi (two-substrate, two-product) mechanisms have been studied. It was found that in every case, the formal theory based on the simple Michaelis-Menten mechanism holds, provided that apparent Michaelis constants are used.

Kinetics of Oligomycin Inhibition of Sodium- and Potassium-Activated Adenosine Triphosphatase from Beef Brain

Abstract: Oligomycin and rutamycin inhibited the activity of a beef brain adenosine triphosphatase that required both sodium and potassium, but these antibiotics were without effect on the potassium-requiring p-nitrophenyl phosphatase or acetyl phosphatase obtained from the same preparation. Several other macrolide antibiotics believed to be structurally similar to oligomycin were without appreciable effect on either the adenosine triphosphatase or the p-nitrophenyl phosphatase. Kinetic studies indicated that oligomycin is an uncompetitive inhibitor of the sodium- and potassium-requiring adenosine triphosphatase. The antibiotic was an uncompetitive inhibitor of sodium activation and a noncompetitive inhibitor of the potassium activation of this enzyme. Alteration of the Mg++: ATP ratio from 0.1 to 5.0 did not affect the inhibition by oligomycin. It would appear that oligomycin is an even more selective inhibitor than the cardiac glycosides of the hydrolytic function that requires both sodium and potassium.

A possible mechanism of release of posterior pituitary hormones involving adenosine triphosphate and an adenosine triphosphatase in the neurosecretory granules.

Abstract: Neurosecretory granules were isolated from bovine posterior pituitary glands and incubated at 30 or 37° in a medium containing KCl and MgCl2. On exposure to ATP the granules released vasopressin, oxytocin, and protein. Vasopressin release in response to ATP was potentiated by phosphoenolpyruvate + pyruvate kinase, was inhibited by AMP, and was accompanied by a fall in the optical density (at 540 mµ) of the granule suspension. The neurosecretory granules possessed ATPase activity and ATP. It is suggested that ATP and ATPase may participate in the release of hormones from the intact posterior pituitary gland.

Regulation of histidine decarboxylase in rat stomach by gastrin: the effect of inhibitors of protein synthesis.

Abstract: Histidine decarboxylase activities in the stomachs of freely feeding rats or fasted rats treated with gastrin are 3 and 2 times, respectively, greater than enzyme activity of fasted rats. Administration of gastrin produces a marked increase of gastric histidine decarboxylase in 30 min with maximal effects between 2 and 3 hr, and a decline of enzyme activity to fasting levels after 8 hr. Puromycin and cycloheximide completely prevent the rise of gastric histidine decarboxylase activity induced by gastrin, whereas actinomycin D tends to stimulate this rise in activity. When cycloheximide is administered to freely feeding rats there is an exponential fall in gastric histidine decarboxylase activity with a half-life of 2.1 hr. Cycloheximide treatment accelerates the decline of gastric histidine decarboxylase activity after enhancement by gastrin. ACKNOWLEDGMENT This research was supported by USPHS grants TO 1-MH-11267 and 1 RO1 NB 07275. Solomon H. Snyder is a recipient of NIMH Research Career Development Award 5 KO3-MH-33128.

Studies of the binding of tetracycline to ribosomes in vitro.

Abstract: Ribosomes, isolated from Bacillus megaterium or from rat liver, were found capable of binding considerable quantities of tetracycline. The amount bound depended upon the concentrations of tetracycline and of Mg2+ and K+ present. The highest level of binding observed was equivalent to about 300 molecules of tetracycline per ribosome. Binding of the antibiotic was largely reversible, but, using tetracycline-3H of high specific activity, it was possible to detect a low level of irreversible binding, amounting to less than one molecule per ribosome under the conditions used. This binding was observed to take place to both 30 S and 50 S ribosome subunits. The possible relevance of the binding to inhibition of protein synthesis by tetracycline is discussed. ACKNOWLEDGMENTS The author wishes to thank members of the Subdepartment of Chemical Microbiology, in particular Dr. Kenneth McQuillen, for much valuable discussion. Thanks are due to the Medical Research Council for a Scholarship for Training in Research Methods and to Merck Sharp and Dohme for a generous gift of actinomycin.

Inhibition of brain sodium- and potassium-stimulated adenosine triphosphatase activity by chlorpromazine free radical.

Abstract: Ultraviolet light was observed to have a profound effect on the inhibition in vitro of rat brain microsomal sodium- and potassium-stimulated ATPase activity [Mg2+-dependent, (Na+ + K+)-stimu1ated ATP phosphohydrolase, EC. 3.6.1.3] by chlorpromazine. The inhibition of the enzyme activity was minimal when the experiments were carefully performed, avoiding exposure to light. However, a significant inhibition of (Na+ + K+)-ATPase activity was observed when the chlorpromazine free radical was added to the enzyme mixture after it had been generated either by ultraviolet irradiation or by chemical oxidation (with sulfuric acid) of chlorpromazine solutions. The concentration required for 50% inhibition of enzyme activity (I50) was 40 µM. Even greater inhibition was observed when the chlorpromazine free radical was generated by ultraviolet irradiation in a mixture containing enzyme. Under these experimental conditions, the I50 was 3.5 µM. The ultraviolet irradiation of the drug-enzyme mixture failed to enhance the inhibition of (Na+ + K+)-ATPase activity when inhibitory concentrations of ouabain or p-hydroxymercuribenzoate were used. A positive correlation was observed between the amount of chlorpromazine transformed by ultraviolet irradiation and the enhancement of inhibition of (Na+ + K+)-ATPase activity by a corresponding amount of irradiation of the chlorpromazine-enzyme mixture. An enhanced inhibition (I50 = 15 µM) was also observed when the chlorpromazine free radical was generated in the ATPase assay system in the simultaneous presence of peroxidase and hydrogen peroxide. The inhibition of (Na+ + K+)-ATPase activity by chlorpromazine sulfoxide was minimal even in the presence of peroxidase and hydrogen peroxide. It is concluded that a semiquinone free radical of chlorpromazine, rather than chlorpromazine itself, may be responsible for the inhibition of sodium- and potassium-stimulated ATPase activity in vitro.

Hydrolysis of succinyldicholine and succinylmonocholine in human serum.

Abstract: The enzymatic hydrolysis of the muscle relaxant suxamethonium (succinyldicholine) and the first product of its degradation, succinylmonocholine, in sera of different pseudocholinesterase phenotypes have been investigated. Kinetic data were obtained, using 14C-labeled substrates, thus making the method very sensitive and accurate at concentrations of pharmacologic interest. Radioactivity of substrates and split products were estimated after separation by high voltage electrophoresis on paper. The results give much evidence that in human serum both esters are hydrolyzed by pseudocholinesterase. ACKNOWLEDGMENT This investigation was supported by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie.

A Molecular Orbital Calculation of the Preferred Conformation of Nicotine

Abstract: Using extended Huckel theory molecular orbital calculations, two equally preferred conformations of the nicotinium ion have been predicted from total energy minimization as a function of geometry. The calculated conformations are in good agreement with reported nuclear magnetic resonance (NMR)-predicted conformations of several nicotine derivatives. In only one predicted nicotinium ion conformation are there found to be two atoms, including the quaternary nitrogen atom, of comparable charge and interatom distance to those found in acetylcholine. Specifically these atoms in the nicotinium ion relate very closely to the quarternary nitrogen and carbonyl oxygen atoms of acetylcholine in its calculated preferred conformation. This observation permits time prediction that the two principal atoms necessary for nicotinic activity in the nicotinium ion are a quaternary nitrogen atom and a negatively charged atom about 4.85 A removed. As an adjunct to the study, an extended Huckel theory population analysis of acetylcholine in its calculated preferred conformation indicates a slightly negative total (σ + π) charge density on the ether oxygen atom.

Conformational changes of acetylcholinesterase.

Abstract: The conformation of highly purified acetylcholinesterase was measured with a spectropolarimeter. It was demonstrated that heat, strong base, a substrate, and anticholinesterase agents induce alterations in the conformation of this enzyme.

In vitro inhibition of aldosterone-stimulated sodium transport by steroidal spirolactones.

Abstract: The spirolactones SC 9420 and SC 14266 both are effective inhibitors of the aldosterone-induced increase in in vitro sodium transport as measured in the isolated toad bladder. These compounds appear to be specific antagonists of aldosterone as the shortcircuit current response to exogenous vasopressin and glucose repletion were not altered by concentrations of spirolactone sufficient to inhibit the electrophysiologic effects of aldosterone. The results of kinetic analysis of aldosterone-spirolactone interaction fulfilled the criteria for competitive inhibition. Assuming that the tissue receptors for aldosterone are homogeneous, the relative affinity for these receptors was estimated by the ratio of the dissociation constants of aldosterone (KD) and spirolactone (Ki). Using two separate derivations, each from independent data, the resulting ratios for aldosterone: SC 14266 were in reasonable agreement, i.e., 1:235 and 1:336. The average maximal effective aldosterone concentration (2 x KD) was calculated to be 1 x 10-8 M, while the inhibitor constant (Ki) for the spirolactone SC 14266 was 1 x 10-6 M by direct plot and 2.6 x 10-6 M when derived from a Lineweaver-Burk plot. The Ki for SC 9420 by direct plot was 5.25 x 10-6 M and the KD:Ki ratio was 1:681.

Tryptophan Derivatives as Inhibitors of Tyrosine Hydroxylase in Vivo and in Vitro

Abstract: Certain typtophan analogs were found to be potent inhibitors of tyrosine hydroxylase and to act by a mechanism that is not competitive with substrate. The most active compounds in these studies were those with a hydroxyl group at the 5 position on the indole ring. The most potent inhibitor was α-methyl-5-hydroxytryptophan. Amines or acids resulting from metabolism of the parent compounds were found to be inactive. Tyrosine hydroxylase activity was inhibited in vivo after a 50 mg/kg dose of α-methyl-5-hydroxytryptophan; a single dose of 200 mg/kg inhibited the enzyme up to 48 hr. Administration of this compound resulted in depletion of tissue stores of catecholamines as well as in sedation.

The effects of nitrogen mustard on DNA template activity in purified DNA and RNA polymerase systems.

Abstract: The effects of nitrogen mustard (HN2) on DNA and RNA synthesis in preparations utilizing purified DNA templates and polymerase enzymes have been investigated in order to more completely define the mechanism of action of antineoplastic alkylating agents at the molecular level. Pretreatment of native calf thymus DNA with 5 x 10 -7 M HN2 produced a significant inhibition of DNA template activity in a purified RNA polymerase system from Escherichia coli . Concentrations of HN2 20-100 times higher were required to achieve an equivalent amount of inhibition of DNA template function utilizing a DNA polymerase system from E. coli . The template activities of both native and denatured DNA were inhibited by HN2. The quantity of alkylation of the DNA templates was determined by measuring the binding of 14 C-HN2, and the percent inhibition of DNA template activity was correlated with the number of alkylations per 10 4 nucleotide units. It was found that the template function of DNA as it exists in a nucleoprotein complex isolated from E. coli is relatively insensitive to treatment with HN2. The possible relationships of these findings to the cytotoxic effects of HN2 are discussed.

Kinetics of the carbamylation of cholinesterase.

Abstract: The kinetics of inhibition of acetylcholinesterase by three methylcarbamates was explored, and it was shown that the observations of both short-term and steady-state inhibitions were satisfactorily accounted for if one assumed that a reversible complex was formed prior to carbamylation of the enzyme. New kinetic evidence for the existence of such a complex was also provided. Both the complex formation and the carbamylation steps were inhibited by the substrates acetylthiocholine and 1-naphthyl acetate. The decarbamylation step was not affected by 2-pyridine-aldoxime methiodide. 1-Naphthyl methylcarbamate did not affect a muscle preparation containing acetylcholine receptor.