The regulation of rat liver tryptophan pyrrolase activity by reduced nicotinamide-adenine dinucleotide (phosphate). Experiments with glucose and nicotinamide. 1. Chronic administration of glucose or nicotinamide in drinking water inhibits the activity of rat liver tryptophan pyrrolase, and subsequent withdrawal causes an enhancement. The enzyme activity is also inhibited by administration in drinking water of sucrose, but not fructose, which is capable of preventing the glucose effect. 2. The inhibition by glucose or nictinamide is not due to a defective apoenzyme synthesis nor a decreased cofactor availability. 3. The inhibition by nicotinamide is reversed by regeneration of liver NAD+ and NADP+ in vivo by administration of fructose, pyruvate or phenazine methosulphate. Inhibition by glucose is also reversed by the above agents and by NH4Cl. Reversal of inhibition by glucose or nicotinamide is also achieved in vitro by addition of NAD+ or NADP+. 4. Glucose or nicotinamide increases liver [NADPH]. [NADP+] is also increased by nicotinamide. [NADPH] is also increased by sucrose, but not by fructose, which prevents the glucose effect. Phenazine methosulphate prevents the increase in [NADPH] caused by both glucose and nicotinamide. 5. It is suggested that the inhibition of tryptophan pyrrolase activity by glucose or nicotinamide is mediated by both NADPH and NADH.


Basic ethers of cyclohexylphenols with beta-blocking activity: synthesis and pharmacological study of exaprolol. The paper describes the preparation and study of the pharmacological properties of a series of derivatives with the general formula. (see article) where R is a cyclohexyl or cyclohexenyl radical and R1 is a hydrogen, methyl or cyclohexyl. The compounds with a single cycloaliphatic radical ortho to the basic chain, and in particular the one with a cyclohexyl (exaprolol), were found to be particularly active in blocking the beta-adrenergic receptors, as antiarrhythmics and local anesthetics, while the introduction of a second radical or the shift of the cycloaliphatic radical to meta or para position caused the said pharmacological activities to disappear almost entirely, with the exception of the local anesthetic action.


General pharmacological properties of a new non-opiate antitussive: zipeprol (3024 CERM). I. Action on respiratory function and acute toxicity. 1-(2-Methoxy-2-phenyl)-ethyl-4-(2-hydroxy-3-methoxy-3-phenyl)-propyl-iperazine-dihydrochloride (zipeprol, Respilene) is a substance of non-phenanthrenic chemical structure. In the cat, it antagonised cough induced by stimulation of the superior laryngeal nerve or by direct mechanical excitation of the sensitive tracheo-bronchial receptors. The efficacy of zipeprol after enteral administration made it possible both to establish good intestinal absorption and to rank it favourably in relation to several major antitussive reference products; codeine, codethyline, dextromethorphan, diphenhydramine and pentoxyverine. The activity of zipeprol was superior or equal to that of all these substances, excdept codeine. The antitussive properties appeared to be due to a central action. Other properties have been demonstrated which suggest at least a supplementary mechanism in the inhibition of cough, in addition to the central action. These consisted of slight antihistamine and anticholinergic properties, marked local-anesthetic potency and bronchospasmolytic activity. This latter property was demonstrated by the inhibition of histamine and serotonin induced bronchospasm in the guinea-pig. In vitro, using human sputum, zipeprol had a mucolytic action, shown by a decrease in sputum vis viscosity and lysis of DNA and AMPS fibrils. In the dog, at high doses, zipeprol unlike codeine, did not inhibit central stimulation of respiration by hypercapnia, in addition no modification of ventilatory dynamics or blood gases was seen. On the basis of these results, zipeprol can be considered as possessing no respiratory depressant effect even in the upper ranges of its antitussive doses.


[Bronchospasmolytic activity of aqueous aerosols in anesthetized dogs (author's transl)]. A modification of the method of Konzett and Rössler is described. This modification allows the administration of atomised drug solutions to anaesthetised dogs. Information is thus obtained on the bronchodilatory activity of a drug and the onset and duration of effect. Two anticholinergics (atropinsulfate and ipratropiumbromide) and three beta-adrenoceptor stimulants (isoprenaline, orciprenaline and salbutamol) were investigated. After local administration as aerosols the anticholinergics proved to be as potent or even more so than the beta-adrenoceptor stimulants.


Actions of narcotics on brain dopamine metabolism and their relevance for "psychomotor" effects. A review is given about the effects of narcotic analgesics, particularly of morphine, on the dopamine metabolism in the corpus striatum and about the relations of these effects to motility and "psychomotor" phenomena. In rats, acute doses of morphine decrease the dopaminergic neurotransmission in brain, without blocking postsynaptic dopamine receptors. Chronic treatment of rats with morphine reverses these acute effects of morphine and induces symptoms of an increased dopaminergic neurotransmission in brain. In mice and cats on the other hand, acute doses of morphine apparently increase dopaminergic neurotransmission. The effects of morphine on striatal dopamine metabolism seem to be a model well suited to study opioid-specific effects on a cellular level. Furthermore, they might also be responsible for some narcotic-specific effects on behaviour observed in animals and man.


Metabolic deacetylation: in vitro and in vivo studies in man, rat, dog and rhesus monkey with isomeric tetrahydroisoquinolyl derivatives of 3,4-dimethylbenzyl acetate. The pharmacological activities of a racemic mixture of tetrahydroisoquinolyl derivatives of 3,4-dimethylbenzyl acetate, (+/-) Ro 03-4661 and the corresponding resolved isomers (+) Ro 03-4661 and (--) Ro 03-4661 have been studied in rat and rhesus monkey. The racemate and the (--) isomer showed narcotic analgesic activity by the oral route in both species. Drug metabolism studies indicated that the activity was probably due to metabolic Deacetylation to the corresponding carbinol Ro 03-4632. deacetylation did not occur in the dog, but was observed in the rat and rhesus monkey. In both these species the hydrolysis was more extensive after oral than parenteral administration. In vitro studies also showed considerable species variation in the ability of blood and tissue esterases to hydrolyse the different stereochemical isomers.


Purification and characterization of lipoprotein lipase from human heart. Human heart lipoprotein lipase was purified by affinity chromatography on heparin-Sepharose 4B. When crude extracts of heart acetone powder were applied to columsn, about 40% of total lipase activity was bound to the gel and then eluted with 1.5 M NaCl. At this stage the eluted enzyme was purified 1900-fold. Disc gel electrophoresis yielded a single protein band corresponding with lipolytic activity. Minimum molecular weight of the protein was 60,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme was highly unstable; however, its activity could be partially stabilized at --20C by bovine serum albumin, glycerol, or ethylene glycol. The activity of the purified enzyme (i) had a pH optimum between 7.8 and 8.0; (ii) required serum for full enzymatic activity; apoC-II could be substituted for serum; (iii) was inhibited by by apoC-I in the presence of activated substrate; (iv) was markedly inhibited by NaCl; and (v) was stimulated by heparin.


Neurogenic hypercholesterolemia: influence of autonomic drugs. Eleven substances capable of either augmenting or depleting the alpha- and - beta-adrenergic capacities of the autonomic nervous system were administered to rats exhibiting hypothalamic hypercholesterolemia and to normal controls. Only the beta-adrenergic blocking agents propranolol and possibly 6-OH dopamine were observed to alter (raise) the serum cholesterol concentration, and this occurred in both experimental and control animals. Neither atropine, nor the serotonin-depleting agent, rho-chlorophenylalanine, nor the serotonin-antagonist cyproheptadine, were observed to alter serum cholesterol level. Such absence of effect was also noted with metaraminol, phenoxybenzamine, isoproterenol, epinephrine, reserpine, and alpha-methyl tyrosine.


Effects of some benzodiazepine derivatives on Triton WR-1339-Induced hyperlipidaemia in rats. Oral administration of the benzodiazepines (diazepam, lorazepam, chlordiazepoxide, bipotassium chlorazepate) in Triton WR-1339-induced (200 mg/kg, blood collection 18 h later) hyperlipidaemia in rats elicited marked decrease of serum total lipids, total cholesterol and triglyceride levels, and alterations in free fatty acid and free glycerol content. The optimal doses for diazepam, lorazepam, chlordiazepoxide and bipotassium chlorazepate were estimated to be 5 mg/kg. Other benzodiazepines, namely oxazepam, medazepam and nitrazepam, elicited only minor changes in serum lipids levels, while with grandaxin no change was observed. The optimal doses of diazepam and lorazepam brought about the same changes in serum lipid content as did clofibrate (90 mg/kg, p. o.). If diazepam, lorazepam, chlordiazepoxide and bipotassium chlorazepate were administered in doses of 5 mg/kg in Triton WR-1339-treated rats (blood collection taken 3 h later), a significant decrease of total lipids and triglyceride levels was observed. The free glycerol level only altered after the administration of chlordiazepoxide, which brought about a significant reduction.


Clinical studies of induction agents XLIII: Flunitrazepam. Flunitrazepam (Ro 5-4200) has been studied as an induction agent in 220 volunteers or patients. It was assumed to be 10 times as potent as diazepam. The maximum soporific effect did not occur until 90-120 s after injection. There was great individual variation in response to flunitrazepam and some patients did not lose consciousness even after receiving 6 mg (approximately 0.1 mg/kg). Opiate premedication enhanced its action, but delayed recovery. There was a dose-related increase in minor respiratory upset with flunitrazepam in unpremedicated patients and a high frequency of arterial hypotension following large doses given to patients who had received opiate premedication. Venous sequelae were no more frequent than after comparable doses of diazepam. Flunitrazepam was not a very satisfactory drug for the induction of anaesthesia, and recovery was too prolonged for routine use.


Salmefamol and Salbutamol in exercise-induced asthma in children. A double-blind controlled study is reported of salmefamol 200 mug and salbutamol 200 mug administered by aerosol before exercise tests in asthmatic children. Both drugs prevented exercise-induced asthma. There was no significant difference between them.


The role of divalent cations in epidermolysis. In experiments designed to elucidate the role of divalent cations in maintaining the integrity of the epidermis, newborn mouse skin was incubated in ethylenediamine tetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA). In EDTA, epidermolysis occurred and was confirmed by rubbing the specimen to demonstrate that a sheet of epidermis could be split off. After 30 min incubation in 0-01 mol/EDTA the split occurred in the lower granular-upper spinous layer; after 45 min, it was in a spinous-suprabasilar location and at 60 min and later at the dermal-epidermal junction. Ultrastructurally, a clear zone of apparent intracellular oedema occurred along the cell membranes. The split then occurred intracytoplasmally through this clear zone and the adjacent cell membrane was lost. Since incubation in EGTA at pH 7-4 did not result in epidermolysis, we suggest that the removal of magnesium rather than calcium is responsible for epidermolysis. Thirty min after the addition of either calcium or magnesium to EDTA-treated specimens, epidermolysis could no longer be demonstrated.


Planned pregnancy in a renal transplant recipient. A scheme is described for the investigation and management of a renal transplant recipient before conception, over conception and during the subsequent pregnancy, delivery and puerperium. The patient showed the physiological changes in homeostasis of normal pregnancy. Methods of monitoring maternal, fetal and renal well-being are discussed. The patient was delivered of a live male child and the neonatal problems are described. The puerperium was uneventful.


Effect of oral 17-beta-oestradiol on the liver in women with intrahepatic cholestasis (hepatosis) during previous pregnancy. The effects of 17-beta-oestradiol in 14 women who had had intrahepatic cholestasis of pregnancy (hepatosis) were studied. This natural oestrogen was given orally in a daily dose of 5 mg for two 20-day periods with a 7-day interval between courses. The bromsulphthalein (BSP) retention rose during the treatment, but only occasionally to levels above the upper limit of normal. Other conventional liver function tests did not usually become abnormal. In a previous investigation in the same type of patients, we studied the effects of mestranol (a synthetic 17-alkylated oestrogen) in a daily dose of 0.1 mg with the same dose-schedule as the present investigation and 17-beta-oestradiol appeared to impair liver function less than mestranol. It might thus be safer to use 17-beta-oestradiol as a therapeutic agent.


Structural elucidation and properties of 8alpha-(N1-histidyl)riboflavin: the flavin component of thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase. In addition to 8alpha-(N3-histidyl)riboflavin, 8alpha-(N1-histidyl)riboflavin is also formed during the reaction of Nalpha-blocked histidine with 8alpha-bromotetraacetylriboflavin in a yield of 20-25% of the total histidylflavin fraction. The properties of 8alpha-(N1-histidyl)riboflavin are inditical with those of the histidylflavin isolated from thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase but differ from those of 8alpha-(N3-histidyl)riboflavin. These properties include pKa of fluorescence quenching, electrophoretic mobility at pH 5.0, stability to storage, and reduction by NaBH4. Proof for 8alpha substitution is shown by the electron paramagnetic resonance and electron-nuclear double resonance spectra of the cationic semiquinone form, as well as by the proton magnetic resonance spectrum of the oxidized form. The site of histidine substitution by the 8alpha-methylene of the flavin moiety was shown by methylation of the imidazole ring with methyl iodide, cleavage of the methylhistidine-flavin bond by acid hydrolysis at 150 degrees C, and identification of the methylhistidine isomer by electrophoresis. 3-Methylhistidine is the product from the N1-histidylflavin isomer, while 1-methylhistidine is produced from the N3 isomer. The flavin product from reductive Zn cleavage of either isomer has been identified as riboflavin. The compound obtained on acid treatment of 8alpha-(N3-histidyl)riboflavin (previously thought to be the N1 isomer) differs from the parent compound only in the ribityl side chain, since chemical degradation studies show 1-methylhistidine as a product and a flavin product which differs from riboflavin only in mobility in thin-layer chromatography, but not in absorption, fluorescence, and electron paramagnetic resonance spectral properties. Proof that acid modification involves only the ribityl chain has come from the observations that alkaline irradiation of this flavin yields lumiflavin, that the proton magnetic resonance spectrum of the compound differs from that of riboflavin in the region of the ribityl proton resonance, and that its periodate titer is lower than that of authentic riboflavin. The identity of 8alpha-(N1-histidyl)riboflavin with the histidylflavin from thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase shows that both isomeric forms of 8alpha-histidylflavin occur in nature.


Energetics of primary processes in visula escitation: photocalorimetry of rhodopsin in rod outer segment membranes. A sensitive technique for the direct calorimetric determination of the energetics of photochemical reactions under low levels of illumination, and its application to the study of primary processes in visula excitation, are described. Enthlpies are reported for various steps in the bleaching of rhodopsin in intact rod outer segment membranes, together with the heats of appropriate model reactions. Protonation changes are also determined calorimetrically by use of buffers with differing heats of proton ionization. Bleaching of rhodopsin is accompanied by significant uptake of heat energy, vastly in excess of the energy required for simple isomerization of the retinal chromophore. Metarhodopsin I formation involves the uptake of about 17 kcal/mol and no net change in proton ionization of the system. Formation of metarhodopsin II requires an additional energy of about 10 kcal/mol and involves the uptake on one hydrogen ion from solution. The energetics of the overall photolysis reaction, rhodopsin leads to opsin + all-trans-retinal, are pH dependent and involve the exposure of an additional titrating group on opsin. This group has a heat of proton ionization of about 12 kcal/mal, characteristic of a primary amine, but a pKa in the region of neutrality. We suggest that this group is the Schiff base lysine of the chromophore binding site of rhodopsin which becomes exposed on photolysis. The low pKa for this active lysine would result in a more stable retinal-opsin linkage, and might be induced by a nearby positively charged group on the protein (either arginine or a second lysine residue). This leads to a model involving intramolecular protonation of the Schiff base nitrogen in the retinal-opsin linkage of rhodopsin, which is consistent with the thermodynamic and spectroscopic properties of the system. We further propose that the metarhodopsin I leads to metarhodopsin II step in the bleaching sequence involves reversible hydrolysis of the Schiff base linkage in the chromophore binding site, and that subsequent steps are the result of migration of the chromophore from this site.


Characterization of 20S component in tubulin from mammalian brain. Tubulin from porcine brain, purified by at least two cycles of assembly and disassembly, was characerized at different pH values by sedimentation velocity analysis and turbidimetric measurements. At pH 6.4 the depolymerized material was composed of two major species sedimenting with so20,w values of 6 and 36 and a minor one of 20S. By raising the pH, the amount of the 20S component increased and that of the 36S decreased, whereas that of the 6S component was unaltered. At pH 7.6 the mixture contained 20S and 6S components but hardly any 36S. The 20S species can be separated from the 6S ones by gel filtration on agarose A-15m at pH 7.6. On electron microscopic examination this preparation contains far fewer double rings compared to the material at pH 6.4, but single rings could often be seen. Sodium dodecyl sulfate gel electrophoresis of the 20S and 36S components showed that they consist almost entirely of tubulin and some higher and lower molecular weight fractions. Turbidity measurements showed that the minimal protein concentration necessary for polymerization increases with increasing pH. The turbidity plateau reached at a given pH can be raised by decreasing the pH. From these results it is suggested that the 20S component is an intermediate of the 36S species. The results further indicate the existence of a pH-dependent equilibrium between the 20S species and the 36Soligomers.


Histone interactions in solution and susceptibility to denaturation. Histone interactions in solution may depend upon treatments used for purification. Optical rotatory dispersion and sedimentation-velocity measurements have been made in a reference solvent, before and after exposure to various treatments, to investigate histone susceptibility to irreversible denaturation. Some acid conditions and urea and guanidine solutions may denature. Interaction studies performed on nondenatured histones indicate that the dimer, (H4)(H3), and tetramer, (H4)2(H3)2, dissociate to monomers at low ionic strength. Sedimentation-velocity experiments suggest a model for the (H4)2(H3)2 tetramer, with a compact semispherical center and four protruding amino-terminal regions. Fractions H2a and H2b interact to form the mixed dimer in equilibrium with monomers. Fraction H2a self-associates readily to dimers, tetramers, and octamers, while fraction H1 associates only weakly to form dimers.


Structural analysis of O2'-methyl-5-carbamoylmethyluridine, a newly discovered constituent of yeast transfer RNA. A compound tentatively identified as O2-methyl-5-carboxymethyluridine (cm5Um) was recently isolated in this laboratory from bulk yeast transfer RNA (Gray, M. W. (1975), Can, J. Biochem. 53, 735-746). Alkaline hydrolysis of yeast tRNA releases this nucleoside as part of an alkali-stable dinucleotide, cm5Um-Ap, from which sufficient cm5Um was prepared in the present investigation for a detailed examination of its properties. The ultraviolet absorption spectra and chromatographic and electrophoretic properties of cm5Um were consistent with the proposed structure, which was confirmed by characterization of the base and sugar moieties as 5-carboxymethyluracil and 2-O-methylribose, respectively. Snake venom hydrolysis of yeast tRNA releases cm5Um in the form of a carboxyl-blocked 5'-nucleotide, designated pU-2. Identification of the alkali-labile blocking group in pU-2 as an amide was based on quantitative assay for ammonia released upon acid hydrolysis of the corresponding nucleoside, U-2, and by chromatographic comparison of U-2 with the semisynthetic methyl ester and amide derivatives of cm5Um (mcm5Um and ncm5Um, respectively). Quantitative analysis has indicated that ncm5Um may be confined to a single species of yeast tRNA. In view of the unique localization (the "Wobble" position of the anticodon sequence) and coding properties (pairing with A but not with G) of other cm5U  derivatives in transfer RNA, the dinucleotide cm5Um-Ap may be derived from the first two positions of the anticodon sequence of a yeast tRNA species recognizing an NUA codon. This predicts that O2-methyl-5-carbamoylmethyluridine will be found in an isoleucine, leucine, or valine isoacceptor.


Mechanisms of electron transfer from sulfite to horseradish peroxidase-hydroperoxide compounds. Using a rapid-scan spectrophotometer equipped with a stopped-flow apparatus, reactions of sulfite with compounds I and II of two horseradish peroxidase isoenzymes A and C were investigated. The direct two-electron reduction of peroxidase compound I by sulfite occurred at acidic pH but the mechanism gradually changed to the two-step reduction with the intermediate formation of compound II as the pH increased. The pH at which the one- and two-electron changes occurred at the same speed was 4.5 for peroxidase A and 7.7 for peroxidase C. A new peroxidase intermediate was found in the reaction between peroxidase compound II and sulfite. The sulfite compound showed a characteristic absorption band at 850 nm and the optical spectrum was similar to that of isoporphyrins but was quite different from that of sulfhemoproteins. The rate (k) of conversion from the sulfite-compound II complex to the sulfite compound was proportional to the concentration of H+ and the log k vs. pH plot for peroxidase A moved to the acidic side by 1.1 pH unit from that for peroxidase C.


Vinylglycine and proparglyglycine: complementary suicide substrates for L-amino acid oxidase and D-amino acid oxidase. Proparglyglycine (2-amino-4-pentynoate) and vinylglycine (2-amino-3-butenoate) have been examined as substrates and possible inactivators of two flavo enzymes, D-amino acid oxidase from pig kidney and L-amino acid oxidase from Crotalus adamanteus venom. Vinylglycine is rapidly oxidized by both enzymes but only L-amino acid oxidase is inactivated under assay conditions. The loss of activity probably involves covalent modification of an active site residue rather than the flavin adenine dinucleotide coenzyme and occurs once every 20000 turnovers. We have confirmed the recent observation (Horiike, K, Hishina, Y., Miyake, Y., and Yamano, T. (1975) J, Biochem. (Tokyo), 78, 57) that D-proparglglycine is oxidized with a time-dependent loss of activity by D-amino acid oxidase and have examined some mechanistic aspects of this inactivation, The extent of residual oxidase activity, insensitive to further inactivation, is about 2%, at which point 1.7 labels/subunit have been introduced with propargly[2-14C]glycine as substrate. L-Proparglyclycine is a substrate but not an inactivator of L-amino acid oxidase and the product ahat accumulats in the nonnucleophilic N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer is acetopyruvate. In the presence of butylamine HCl, a species with lambdaman 317 nm (epsilon = 15 000) accumulates that may be a conjugated eneamine adduct. The same species accumulates from D-amino acid oxidase oxidation of D-propargylglycine prior to inactivation; the inactivated apo D-amino acid oxidase has a new peak at 317 nm that is probably a similar eneamine. A likely inactivating species is 2-keto-3,4-pentadienoate arising from facile rearrangement of the expected initial product 2-keto 4 pentynoate. Vinylglycine and proparglyglycine show inactivation specificity, then, for L-and D-amino acid oxidase, respectively.


Properties of crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from bovine adrenocortical mitochondria. II. Essential histidyl and cysteinyl residues at the NADPH binding site of NADPH-adrenodoxin reductase. The binding site of NADPH in NADPH-adrenodoxin reductase was examined using crystalline enzyme from bovine adrenocortical mitochondria by studies on the effects of photooxidation and chemical modifications of amino acid residues in the reductase. (1) Photoxication decreased the enzymatic activity of NADPH-adrenodoxin reductase. Photooxidation of the reductase was prevented by NADP+, adrenodoxin, or reduced glutathione, but not NAD+. Photoinactivation caused loss of a histidyl residue, but not of tyrosyl, tryptophanyl, cysteinyl, or methionyl residues of the reductase. It did not affect the circular dichroism spectrum of the reductase appreciably. (2) NADPH-adrenodoxin reductase activity was inhibited by diethyl pyrocarbonate and the inhibition was partially reversed by addition of hydroxylamine. The inhibition was prevented by NADP+, but not NAD+. (3) NADPH-adrenodoxin reductase activity was inhibited by 5,5'-dithiobis(2-nitrobenzoate) and the inhibition was reversed by reduced glutathione. It was also protected by NADP+, but not NAD+. The results indicate that a histidyl residue and a cysteinyl residue of NADPH-adrenodoxin reductase are essential for the binding of NADPH by the reductase.


Steric effects on penicillin-sensitive peptidoglycan synthesis in a membrane-wall system Gaffkya homari. Residues 4 and 5 of the pentapeptide moiety, R-Ala1-DGlu2-Lys3-DAla4-DAla5, of peptidoglycan play an important role in the donor phase of cross-linked glycan synthesis. To assess the role of these residues in this phase, a series of UDP-MurNAc-peptides were biosynthesized with residues 4 and 5 replaced singly by either D-alpha-amino-n-butyric acid, D-norvaline, or D-valine. The six nucleotides were compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla (reference) in nascent (penicillin-insensitive) peptidoglycan synthesis and in penicillin-sensitive peptidoglycan synthesis. The synthesis of penicillin-sensitive peptidoglycan is catalyzed by membrane-walls isolated from Gaffkya homari and would appear to require the concerted action of transglycosylase and transpeptidase. The membrane-wall system shows a high degree of discrimination for the steric substituents, -CH3 and -CH2CH3, in residue 4. For example, for UDP-MurNAc-Ala-DGly-Lys-DAbu-DAla and -Ala-DGlu-Lys-DAla-DAbu, Vmax/km is 0.19 and 0.95 and Vmax is 0.03 and 0.52, respectively, of the value for the reference nucleotide. In contrast, for the synthesis of nascent peptidoglycan with these nucleotides Vmax/Km is 0.75 and 0.80, and Vmax is 0.71 and 1.0, respectively, of the value for the reference nucleotide. This trend was also illustrated with the other nucleotides in the time course experiments. These results indicate that the penicillin-sensitive enzyme(s), presumably the transpeptidase, has a higher degree of specificity in the donor phase for D-alanine in residue 4 than for D-alanine in residue 5 in the cross-linking stage of peptidoglycan synthesis.


Role of ribosomal RNA methylases in the regulation of ribosome production in mammalian cells. The activity of rRNA methylases was stimulated by high-energy precursors of RNA (ribonucleoside triphosphates) and inhibited by degradation products of RNA (ribonucleotides and oligoribonucleotides). The response of methylases from rat Novikoff ascites tumor and liver to these metabolites was strikingly different. The highly active tumor enzymes responded preferentially to inhibition by catabolic metabolites, whereas the less active liver enzymes responded exclusively to stimulation by anabolic metabolites. When the activity of rRNA methylases was assayed in response to increasing concentration of S-adenosylmethionine, the tumor enzymes responded with a hyperbolic substrate dependence curve and the liver enzymes with a sigmoidal curve. In the presence of an inhibitory dinucleotide, ApA, the tumor enzymes responded with a sigmoidal curve; in the presence of a stimulator, adenosine 5'-triphosphate, the liver enzymes responded with a hyperbolic substrate concentration curve. When normal rats were subject to a series of treatments by thioacetamide, a hepatocarcinogen, the liver nucleolar rRNA methylases became responsive to inhibition by ApA and relatively unresponsive to stimulation by adenosine 5'-triphosphate. When tumor-bearing rats were treated with polyinosinate:polycytidylate, an antitumor agent, the tumor nucleolar rRNA methylases became unresponsive to inhibition by ApA and more responsive to stimulation by adenosine 5'-triphosphate. A correlation was noted between increased methylation efficiency in vivo and increased stability of nucleolar RNA during incubation in vitro, or vice versa. These results are interpreted to indicate that rRNAmethylases are regulated by cellular metabolites during the nucleolar biosynthesis of ribosomes and that rRNA methylases may provide a favorable site for selective action by cancer chemotherapeutic agents.


Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase. The monophoton counting technique was used to obtain the fluorescence decay kinetics of NADH (dihydronicotinamide adenine dinucleotide) bound to LADH (HORSE LIVER ALCOHOL DEHYDROGENAS). It was found that the fluorescence decay of the enzyme complex did not follow a single exponential decay law but that the data could be well described as a sum of two exponentials. The decay parameters of the enzyme complex do not depend on the degree of binding-site saturation. These results are interpreted in terms of a reversible excited-state reaction forming a nonfluorescent product. Fluorescence decay kinetics are also reported for NADH and related molecules in solution. The decay parameters, fluorescence emission maxima, and fluorescence intensities depend on solvent polarity and viscosity.


pH dependence of the hydrolysis of hippuric acid esters by carboxypeptidase A. The pH dependence (pH 4.5-10.5) of the hydrolysis of seven hippuric acid esters (C6H5CONHCH2C-O2CR1R2CO2H: 1a: R1 = R2 = H; 1b: R1 = R2 = CH3; 1c: R1 = H, R2 = p-ClC6H4; 1d: R1 = H, R2 = C2H5; 1e: R1 = H, R2 = (CH3)2CHCH2; 1f: R1 = H, R2 = C6H5; 1g: R1 = H, R2 = C6H5CH2) by bovine carboxypeptidase A has been investigated, and the pH dependence of the substrate activation of 1a-c and the substrate inhibition of 1d-g have been compared. For all seven esters the catalytically productive binding of the first substrate molecule depends on enzymatic pKa values of 6.0 and 9.1. For 1d, 1e, and 1g the rate of hydrolysis (k2app) of this complex is pH independent, whereas for 1f k2app depends on a pKa of 5.9. The rate of hydrolysis (k3app) of the 1:2 enzyme-substrate complex (ES2) is pH independent for 1d-g, but for 1a-c k3app depends on the following pKa values: 1a, 6.1 and 9.1; 1b, 5.4; 1c, 6.6. The pH dependences of k2app for 1f and k3app for 1c are rationalized by the presence of catalytically nonproductive species. Equivalent ES2 species are believed to be productive for 1c-g; however, the productive ES2 species for 1b must be quite different.


Pulse fluorimetry study of beef liver glutamate dehydrogenase reduced nicotinamide adenine dinucleotide phosphate complexes. Single photon counting pulse fluorimetry has been used in order to study the two ternary complexes GDH-GTP-NADPH and GDH-L-glutamate-NADPH and the quaternary complex GDH-GTP-L-glutamate-NADPH. The fluorescence decay of the enzyme-bound NADPH is not monoexponential in any of these complexes. Moreover, it does not seem to be dependent on the coenzyme concentration. The experimental curves can be satisfactorily fitted with the sum of two exponentials, the relative amplitudes of which significantly depend on the complex studied. Thus, for dihydronicotinamide two possible environments  might exist in the enzyme active sites. It is also shown that the fluorescence decay times of the enzyme are shortened by the bound NADPH.


Binding of phosphorylcholine to an IgM Waldenström as studied by fluorescence spectroscopy and circular dichroism. The binding characteristics of an IgM Waldenström(FR) for the ligand phosphorylcholine has been studied by fluorescence spectroscopy. Upon phosphorylcholine addition, IgM FR exhibited 83% enhancement of the tryptophanyl fluorescence, which was associated with a red shift of the emission maximun (5nm). The same properties were observed with the 7S IgM subunits. The association constant KA for phosphorylcholine was 6X10(4) M-1 FOR IgM FR and the 7S subunit, as determined by fluorescence titration, a value in agreement with the obtained by equilibrium dialysis. No significant decrease in the KA value was found in the presence of 3 M urea; in 6 M urea, the increase in fluorescence intensity was 36% of the value obtained in the absence of denaturing agent. In contrast, only 4% of fluorescence enhancement was noted upon binding in 3 M GuHC1 and no enhancement could be seen when the concentration of  GuHC1 was increased to 5 M, thus suggesting complete unfolding of the protein and subsequent loss of binding activity. The pH dependence study of the phosphorylcholine binding to IgM FR indicated no significant differences in the fluorescence enhancement between pH 5 and 8, whereas at more acidic or alkaline pH values, the enhancement became smaller. At pH 3.0 and 10.0, no enhancement was seen suggesting no binding of the ligand, a fact confirmed independently by equilibrium dialysis. When the spectroscopic properties of the IgM FR were compared with those of murine myeloma proteins that bind the same ligand large differences were recorded in the amplitude of the  phosphorylcholine induced enhancement of the fluorescnece and in the shift of the emission maximum wavelength. This suggests that the human and murine proteins interact differently with the small ligand phosphorylcholine thus implying that the variable domains of these molecules are not identical


pH dependence of tritium exchange with the C-2 protons of the histidines in bovine trypsin. At pH 8.9 and 37 degrees C the half-times for tritium exchange with the C-2 protons of the histidines of trypsin are 73 days for His-57, and greater than 1000 days for His-40 and His-91. These half-times are much longer than the half-life of exchange for the C-2 proton of free histidine (2.8 days at pD 8.2), and longer than any previously reported half-time of exchange at pH greater than 8. These very low rates of exchange are discussed with reference to the refined structure of trypsin. The tritium exchange of His-57 depends on an apparent pKa of 6.6. This pKa may represent the pKa of the imidazole of His-57 in an inactive conformation of the enzyme.


Multiple acyl-coenzyme A carboxylases in Pseudomonas citronellolis. Pseudomonas citronellolis was shown to contain four different acyl-coenzyme A carboxylases, including acetyl-, propionyl-, 3-methylcrotonyl-, and geranyl-CoA carboxylases, when grown on the appropriate carbon sources. Acetyl-CoA carboxylase activity in crude extracts was stimulated approximately 40-fold by inclusion of 0.4-0.5 M ammonium sulfate in the assay. Unexpectedly high levels of propionyl-CoA carboxylase activity, also stimulated by ammonium sulfate, were found in acetate-grown cells. That these acetyl- and propionyl-CoA carboxylase activities were due to different enzymes was shown by their resolution during purification by a procedure that stabilized acetyl-CoA carboxylase as a complex and separated propionyl-CoA carboxylase into two required protein fractions. Propionate- or valine-grown cells contained a propionyl-CoA carboxylase activity that was strongly inhibited by ammonium sulfate in the assay, and which may represent an inducible form of the enzyme. Geranyl- and 3-methylcrotonyl-CoA carboxylases that catalyze the carboxylation of the 3-methyl groups of homologous acyl-CoA acceptors, were induced by growth on the monoterpenes, citronellic or geranoic acid; only 3-methylcrotonyl-CoA carboxylase was induced by growth on leucine or isovaleric acid. Induction of either carboxylase was associated with the appearance of similar high-molecular-weight, biotin-containing proteins as measured by gel filtration. These two carboxylases are probably distinct enzymes since 3-methyl-crotonyl-CoA carboxylase from isovalerate-grown cells does not carboxylate geranyl-CoA, while geranyl-CoA carboxylase will carboxylate both acyl-CoA homologues. P. citronellolis appears to be a useful system for studying the structural aspects of pairs of homologous acyl-CoA carboxylases.


Role of bound calcium ions in thermostable, proteolytic enzymes. Separation of intrinsic and calcium ion contributions to the kinetic thermal stability. The total kinetic thermal stability of a protein molecule, expressed as the total free energy of activation in thermal denaturation reactions, can be separated into an intrinsic contribution of the polypeptide chain and a contribution due to the binding of calcium ions. The theory for this procedure is applied to thermal denaturation data, obtained at the pH of optimum stability, for the serine proteases, thermomycolase and subtilisin types Carlsberg and BPN', and for the zinc metalloendopeptidases, thermolysin and neutral protease A. The results, obtained from Arrhenius plots at high and low free calcium ion concentrations, reveal a considerable variation in the calcium ion contribution to the total kinetic thermal stability of the various enzymes. In the serine protease group, at 70 degrees C, the stability is largest for thermomycolase, mainly due to a relatively high intrinsic contribution. For the metalloendopeptidases the total kinetic thermal stability is largest for thermolysin, the difference between thermolysin and neutral protease A being dominated by bound calcium ion contributions. The intrinsic kinetic thermal stability of the polypeptide chain of thermolysin is considerably smaller than that of any of the serine proteases and is probably of the same order of magnitude as that of neutral protease A. Thus, the well known total kinetic thermal stability of thermolysin is due mainly to a single calcium ion (Voordouw, G., and Roche, R. S. (1975), Biochemistry 14, 4667) that binds with high affinity even at very high temperatures (K congruent to 6 X 10(7) M-1 at 80 degrees C).


Reinvestigation of the phenacyl bromide modification of alpha-chymotrypsin. The modification of alpha-chymotrysin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl alpha-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-chymotrypsin. In addition, incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been determined, using [carbonyl-14C]phyenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl alpha-chymotrypsin produced at neutral pH, alkylation reactions of modified alpha-chymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt.


Evidence for more than one Ca2+ transport mechanism in mitochondria. The active transport and internal binding of the Ca2+ analogue Mn2+ by rat liver mitochondria were monitored with electron paramagnetic resonance. The binding of transported Mn2+ depended strongly on internal pH over the range 7.7-8.9. Gradients of free Mn2+ were compared with K+ gradients measured on valinomycin-treated samples. In the steady state, the electrochemical Mn2+ activity was larger outside than inside the mitochondria. The observed gradients of free Mn2+ and of H+ could not be explained by a single "passive" uniport or antiport mechanism of divalent cation transport. This conclusion was further substantiated by observed changes in steady-state Ca2+ and Mn2+ distributions induced by La3+ and ruthenium red. Ruthenium red reduced total Ca2+ or Mn2+ uptake, and both inhibitors caused release of divalent cation from preloaded mitochondria. A model is proposed in which divalent cations are transported by at least two mechanisms: (1) a passive uniport and (2) and active pump, cation antiport or anion symport. The former is more sensitive to La3+ and ruthenium red. Under energized steady-state conditions, the net flux of Ca2+ or Mn2+ is inward over (1) and outward over (2). The need for more than one transport system inregulating cytoplasmic Ca2+ is discussed.


The metal ion catalyzed decomposition of nucleoside diphosphate sugars. The metal ion catalysed decomposition of the nucleotide diphosphate sugars, uridine diphosphate glucose, uriding diphosphate galactose, uridine diphosphate N-acetylglucosamine, guanosine diphosphate mannose, and guanosine diphosphate fucose (UDPGlc, UDPGal, UDPGlc-NAc, GDPMan, and GDPFuc, respectively), has been studies as a function of pH. UDPDlc and UDPGal decompose readily to the a,2-cycle phosphate derivative of the sugar and uridine 5'-phosphoric acid (UMP) in the presence of Mn2+. Under all conditions tested, UDPGal decomposes two to three times more rapidly than does UDPGlc. GDPFuc is slowly degraded to free fucose under similar conditions; the other nucleotide diphosphate sugars are stable. The rate of reaction increases with increasing hydroxide ion concentration from pH 6.5 to 7.9 and with metal ion concentration from 10 to 200 mm. Several metal ions are effective catalysts; at pH 7.5 WITH 20 mM UDPGal and 20 mM metal ion, the following apparent first-order rate constants (min-1 x 10(4)) were obtained: Eu3+ 700; Mn2+, 70; Co2+ 27; Zn2+, 22; Ca2+, 3.0; Cu2+, 2.4; and Mg2+, 0. It appears that Mn2+ concentrations that have been used in studies with nucleotide diphosphate sugars at neutral pH can catalyze significant decomposition leading to erroneous interpretation of kinetic and incorporation experiments.


The aromatic and heme chromophores of rabbit hemopexin. Difference absorption and fluorescence spectra. Spectrophotometric and fluorimetric techniques were employed to charcterize the environment of the heme chromophore of rabbit hemopexin and to monitor changes in the environment of aromatic amino acid residues induced by the interaction of hemopexin with porphyrins and metalloporphyrins. Difference spectra showed maxima at 292 and 285 nm when hemopexin binds heme or deuteroheme but not deuteroporphyrin. These maxima are attributed to alterations in the local environment of tryptophan and tyrosine residues. Spectro-photometric titrations of the tyrosine residues of hemopexin, heme-hemopexin and hemopexin in 8 M urea showed apparent pK values at 11.4, 11.7, and 10.9 respectively. Perturbation difference spectra produced by 20% v/v ethylene glycol are consistent with the exposure of 6-8 of the 14 tyrosine residues and 6-8 of the 15 tryptophan residues of rabbit hemopexin to this perturbant. Only small differences were found between the perturbation spectra of apo- and heme-hemopexin near 290 nm, suggesting that slight or compensating changes in the exposure to solvent of tryptophan chromophores occur. In the Soret spectral region, the exposure of heme in the heme-hemopexin complex to ethylene glycol was 0.7, relative to the fully exposed heme peptide of cytochrome c. The fluorescence quantum yields of rabbit apo- and heme-hemopexin were estimated to be 0.06 and 0.03, respectively, compared to a yield of 0.13 for L-tryptophan. Iodide quenched 50% of the fluorescence of the deuteroheme-hemopexin complex. Cesium was not an effective quencher. Modification of approximately, 4 tryptophan residues with N-bromosuccinimide also decreased the relative fluorescence of apo-hemopexin by 50% and concomitantly reduced the heme-binding ability of the protein by 70%. The existence of sterically unhindered tryptophan residues in either apo- heme-hemopexin is unlikely since no charge transfer compelxes between these proteins and N-methylnicotinamide were detected.


Effect of temperature on tryptophan fluorescence of beta-lactoglobulin B. The effect of heat on the conformation of bovine beta-lactoglobulin has been studied using intrinsic fluorescence spectroscopy. Changes in the intensity, wave-length of maximum emission and emission peak width at half height of tryptophan fluorescence over the range 15-90 degrees C at pH 6.4-6.5 has allowed the environments of the two tryptophans in the molecule to be discriminated. At 20 degrees C both tryptophans are in hydrophobic environments. As the temperature is raised the conformation changes such that at about 50 degrees C one of the tryptophans is transferred to a more polar environment accessible to solvent. Conformational changes appear to be reversible if the protein is cooled to 20 degrees C after heat treatments up to 70 degrees C. Above 70 degrees C the second tryptophan residue becomes exposed to solvent. Complete exposure of one residue occurs at 80 degrees C while the other is still partially buried even at 90 degrees C. When the protein is then cooled to 20 degrees C the conformational changes appear to be irreversible with only one tryptophan residue returning to the hydrophobic interior of the molecule.


Structure and fluctuation of a Streptomyces subtilisin inhibitor. The kinetics of the hydrogen-deuterium exchange reaction in a subtilisin inhibitor from Streptomyces albogriseolus has been examined by infrared absorption measurement in aqueous solutions at various pH values and temperatures. In the analysis of each piece of kinetic data, it was assumed that the total 104 peptide hydrogen atoms are classified into three kinetic classes A, B1, and B2, and that the sizes of these classes are 72, 15, and 17, respectively at every pH and at every temperature examined. On the basis of the peak position determined for the amide II band in each stage of the exchange reaction, an approximate assignment was suggested of the A, B1 and B2 respectively to an unordered structure, a beta-structure,and an alpha-helical structure in the molecule. This assignment was supported by infrared absorption measurement of a film of this protein and by circular dichroic study of the solutions. On the basis of the temperature effect on the hydrogen-exchange rate constants and on the basis of ultraviolet absorption study in the higher temperature region (40 to 90 degrees C), a discussion has been made on the nature of the fluctuation of the molecular structure of this protein.


Conformation in aqueous medium of the neutral, protonated and anionic forms of 9-beta-D-arabinofuranosyladenine. Proton magnetic resonance spectroscopy was employed to study the solution conformations of the neutral, protonated and dissociated forms of the therapeutically active 9-beta-D-arabinofuranosyladenine (araA). In particular, in strongly basic medium, increasing alkalinity led to pronounced changes in chemical shifts and coupling constants of some pentose protons, due to ionization of the pentose hydroxyls, especially the 2'-OH. The neutral form of araA may be characterized as approx. 25% C(2')endo and approx. 60% gauche-gauche, hence somewhat different from that of the therapeutically active 1-beta-D-arabinofuranosylcytosine (araC). By contrast, the conformations of the anionic forms of both of these are identical, predominantly (greater than 80%) C(2')endo and gauche-gauche. With the aid of the 3'-O-methyl derivatives of araA and araC, where only the 2'-OH ionizes, and the accompanying conformational changes are similar, it follows that the conformation C(2')endo and gauche-gauche for all the foregoing is constrained to this form via a strong intramolecular hydrogen bond, viz. O(5')H...O(2')(-). The influence of the foregoing hydrogen bond on the chemical shifts of the adenine H(8) in the araA anion points to the existence of the latter in the form anti. A similar effect of the doubly ionized phosphate group on H(8) in 5'-araAMP shows the nucleotide to also prefer the form anti, as previously demonstrated for 5'-AMP. The conformations of the sugar rings of the neutral forms of araA and adenosine in aqueous medium differ appreciably, whereas in the solid state they are very similar. PMR spectroscopy is shown to be an effective method for following sugar hydroxyl dissociation. The extent of ionization of a given hydroxyl is provided by the resulting chemical shifts of neighbouring (geminal and vicinal) protons. When ionization is accompanied by a change in conformation, the process may be followed also by changes in proton-proton vicinal coupling constants.


Regulation of nitrogen fixation by Rhizobia. Export of fixed N2 as NH+4. The metabolic fate of gaseous nitrogen (15N2) fixed by free-living cultures of Rhizobia (root nodule bacteria) induced for their N2-fixation system was followed. A majority of the fixed 15N2 was found to be exported into the cell supernatant. For example, as much as 94% of the 15N2 fixed by Rhizobium japonicum (soybean symbiont) was recovered as 15NH+4 from the cell supernatant following alkaline diffusion. Several species of root nodule bacteria also exported large quantities of NH+4 from L-histidine. Evidence is presented that overproduction and export of NH+4 by free-living Rhizobia may be closely linked to the control of several key enzymes of NH+4 assimilation. For instance, NH+4 was found to repress glutamine synthetase whereas L-glutamate repressed glutamate synthase. Assimilation of NH+4 as nitrogen source for growth of Rhizobia was inhibited by glutamate. The mechanism of regulation of NH+4 production by root nodule bacteria is discussed.


Amino acids as repressors of nitrogenase biosynthesis in Klebsiella pneumoniae. Nitrogenase biosynthesis in Klebsiella pneumoniae including mutant strains, which produce nitrogenase in the presence of NH+4 (Shanmugam, K.T., Chan, Irene, and Morandi, C. (1975) Biochim. Biophys. Acta 408, 101--111) is repressed by a mixture of L-amino acids. Biochemical analysis shows that glutamine synthetase activity in strains SK-24, SK-28, and SK-29 is also repressed by amino acids, with no detectable effect on glutamate dehydrogenase. Among the various amino acids, L-glutamine in combination with L-aspartate was found to repress nitrogenase biosynthesis completely. In the presence of high concentrations of glutamine (1 mg/ml) even NH+4 repressed nitrogenase biosynthesis in the strains SK-27, SK-37, SK-55 and SK-56. Under these conditions, increased glutamate dehydrogenase activity was also detected. Physiological studies show that nitrogenase derepressed strains are unable to utilize NH+4 as sole source of nitrogen for biosynthesis of glutamate for biosynthesis of glutamate, whereas back mutations leading to NH+4 utilization results in sensitivity to repression by NH+4. These findings suggest that amino acids play an important role as regulators of nitrogen fixation.


Mechanism of inhibition of Chromatium D growth by L-methionine. Regulation of L-threonine biosynthesis by the intracellular level of S-adenosylmethionine. (1) An unusual accumulation of S-adenosyl-L-methionine in Chromatium D was associated with a marked growth inhibition by L-methionine. The inhibition was overcome by L-isoleucine, L-leucine, L-phyenylalanine, L-threonine, L-valine and putrescien. Based on their effects, these compounds are classified into 3 types. (2) L-Isoleucine, L-leucine, L-phyenylalanine and L-valine (Type I) inhibited the L-methionine uptake and consequently prevented the bacterium from the unusual accumulation of S-adenosyl-L-methionine even in the presence of L-methionine in the medium. Putrescine (Type II) stimulated the consumption of S-adenosyl-L-methionine, but did not influence the L-methionine uptake. Hence, the effect of putrescine would be explained by the action to diminish the intracellular level of S-adenosyl-L-methionine. L-Threonine (Type III) neither inhibited the L-methionine uptake nor affected the content of S-adenoxyl-L-methionine due to the addition of L-methionine. (3) The specific activity of homoserine kinase (EC 2.7.1.39) was greatly lowered by the addition of L-methionine under conditions in which Chromatium D unusually accumulates S-adenoxyl-L-methionine. Homoserine dehydrogenase (EC 1.1.1.3) activity was inhbitied by S-adenosyl-L-methionine (50% inhibition index, 3.5 mM). These facts strongly suggest that the growth inhibition by L-methionine is associated with the L-threonine deficiency caused by the unusual accumulation of S-adenosyl-L-methionine.


The ionic control of 1,25-dihydroxyvitamin D-3 synthesis in isolated chick renal mitochondria. The role of potassium. In previous studies it was found that change in the concentrations of Ca2+, H+, and HPO2-4 in the incubation medium altered the rates of synthesis of 1,25-dihydroxyvitamin D-3 (1,25(OH)2D-3) by isolated renal mitochondria obtained from D-deficient chicks. The present studies demonstrate that raising the medium concentration of K+ from 1 to 50 mM leads to a 6-fold increase in rate of 1,25(OH)2D-3 synthesis by isolated chick mitochondria; that the magnitnitude of this K+-dependent stimulation is enhaced by optimal concentrations of calcium (pCa = 5) and phosphate (pPi = 3) (3 mM) but not by pH (from 6.8 to 7.4); that the effect is not produced by similar changes in media Na+ concentration; and that the stimulatory effect of K+ is not blocked by ruthenium red, and inhibitor of calcium transport and of the calcium-dependent stimulation of mitochondrial 1,25(OH) 2D-3 synthesis. It was also found that valinomycin, a K+-specific ionophore, enhanced the sensitivity of the mitochondrial 1 alpha-hydroxylase activity to K+. In the presence of valinomycin, an increase of pK+ to 3 was sufficient to cause a significant stimulation of 1,25(OH)2D-3 synthesis. It was concluded that changes in the ion content of the mitochondrial matrix space regulated the activity of the 1 alpha-hydroxylase.


Properties of mouse alpha-galactosidase. alpha-Galactosidase has been examined in various murine tissues using the substrate 4-methylumbelliferyl-alpha-galactoside. Mouse liver appears to contain a single major form of the enzyme, as judged by chromatography and electrophoresis. The enzmye was purified 467-fold with a yield of about 40% by a method involving chromatography on Concanavalin A-Sepharose. It has maximal activity at pH 4.2, a Km value of 1.4 mM, and energy of activation of 16 400 cal/mol, and a molecular weight of 150 000 at pH 5.2. It is inhibited at high concentrations of myoinositol and appears to contain N-acetylneuraminic acid. In these characteristics it resembles human alpha-galactosidase A. The enzyme from various tissues differs in electrophoretic mobility. After treatment with neuraminidase, however, the enzyme from all tissues comigrates as a single band of activity. By this criterion the alpha-galactosidase of liver is most heavily sialylated and that from kidney the least. As estimated by gel filtration, the enzyme from liver and kidney exists as species of molecular weight 320 000, 150 000 and 70 000, depending upon pH and ionic strength. This appears to be the result of aggregation of the enzyme, since the forms are interconvertible and under some conditions a single molecular weight species is observed. The liver enzyme is primarily lysosomal, while the kidney enzyme is distributed approximately equally between lysosomal and microsomal fractions.


Insulin and glucagon degradation by the kidney. I. Subcellular distribution under different assay condition. Insulin and glucagon degradation by rat kidney homogenates and subcellular fractions was examined under a variety of conditions including high and low substrate concentrations, at pH 4 and pH 7, with and without glutathione. At high insulin concentration (4.1 - 10(-5) M) insulin degradation by the homogenate was greatest at pH 4 but at low insulin concentration (1 - 10(-10) M) insulin degradation was greatest at pH 7. At either high or low glucagon concentration glucagon degradation by the homogenate was greatest at pH 7. Glutathione at pH 7 stimulated insulin degradation at high insulin concentrations and inhibited insulin degradation at low concentrations; Glucagon degradation at pH 7 was inhibited at both high and low concentrations of glucagon by glutathionemseparation of kidney into cortex and medulla prior to homogenation produced a pattern of insulin and glucagon degradation identical to the whole homogenate but glucagon degradation by the medulla was greater than by the cortex. Examination of degradation by subcellular fractions revealed that at high concentration at neutral pH most insulin was degraded by the 100 000 X g pellet but at low insulin concentrations over 90% of the activity was in the 100 000 X g supernatant; At pH 7, at both high and low concentrations, most glucagon-degrading activity was in the 100 000 X g pellet, although the cytosol also had activity; At pH 4 most degradation occurred in the lysosomal fractions. Separation into cortex and medulla again showed similar distribution of activity as the whole gland with the medulla having more glucagon-degrading activity than the cortex. With low insulin concentrations the cortex 100 000 X g supernatant had higher relative specific activities than the medulla supernatant. Examination of recoveries of enzyme activity revealed that the subcellular fractions consistently had markedly less insulin-degrading activity than the original homogenate. This loss of activity was only discernible when insulin degradation was performed at pH 7 at low substrate concentrations. Comparable losses of glucagon-degrading activity were not seen.


Insulin and glucagon degradation by the kidney. II. Characterization of the mechanisms at neutral pH. Examination of insulin and glucagon degradation by rat kidney subcellular fractions revealed that most degrading activity was localized to the 100 000 X g pellet and 100 000 X g supernatant fractions. Further characterization of the degrading activities of the 100 000 X g pellet and supernatant suggested that three types of enzymatic activity were present at neutral pH. From the cytosol an enzyme with characteristics of the insulin glucagon protease of skeletal muscle was purified. This enzyme appeared to be responsible for insulin degradation by the kidney at physiological insulin concentrations. This enzyme also contributed to glucagon degradation but was not the most active mechanism for this. In the 100 000 X g pellet at least two separate enzymatic activities were present. One of these had properties consistent with those described for glutathione insulin transhydrogenase and appeared to be responsible for insulin degradation at high insulin concentration. The other enzyme was associated with the brush border and had properties consistent with the brush border neutral protease. This enzyme appeared responsible for glucagon degradation at both low and high substrate concentrations. An apparent marked synergism between the 100 000 X g pellet and the 100 000 X g supernatant was noted for insulin degradation at physiological insulin concentrations. Pellet glucagon-degrading activity and soluble insulin-degrading activity were necessary for this. The mechanism was found to be limited insulin degradation by the soluble enzyme resulting in both trichloroacetic acid-precipitable trichloroacetic acid-soluble fragments followed by further degradtion of the fragments by the glucagon-degrading enzyme resulting in an additional increase in trichloroacetic acid-soluble products.


Human pancreatic-type ribonucleases with activity against double-stranded ribonucleic acids. Purified acid-thermostable ribonuclease (Ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) from human pancreas degrades double-stranded RNA at 2% the rate for single-stranded RNA. The activities against single-stranded RNA and double-stranded RNA were shown to be due to a single enzyme with properties similar to bovine pancreatic RNAase A. For purposes of comparison the activities against double-stranded RNA of crystalline ribonucleases of the whale, rat and cow were assayed and found to be 0.4%, 0.03% and 0.003%, respectively, of their activities against single-stranded RNA. Both human serum and urine contain RNAse components of pancreatic origin which hydrolyze double-stranded RNA at 2% and 0.4%, respectively, of the rates against single-stranded RNA. By contrast, purified acid-thermostable RNAases from human spleen and liver hydrlyze double-stranded RNA at least 20-fold more slowly than human pancreatic RNAase, relative to the corresponding rates against single-stranded RNA. The human pancreatic and serum enzymes exhibit appreciable activity against the poly(C) component of the double-stranded poly(I)-poly(C); they also attack poly(C) itself at approximately 25 times the rate for poly(U) and at more than 50 times the rate for single-stranded RNA.


Purification and characterization of rat liver microsomal beta-glucuronidase. Beta-Glucuronidase (EC 3.2.1.31) has been isolated from rat-liver microsomes by a novel chromatographic method employing antibody to rat preputial gland beta-glucuronidase coupled to Sepharose. The purified enzyme, homogeneous by several methods, was purified some 1700-fold. The microsomal beta-glucuronidase has been characterized with respect to catalysis, stability, and molecular weight. The purified enzyme is a tetramer of 290 000 daltons. Comparative studies with lysosomal beta-glucuronidase indicate that while these two enzymes are electrophoretically distinct, they are catalytically and immunologically identical and have indistinguishable molecular dimensions. The results suggest that microsomal and lysosomal beta-glucuronidase are charge isomers.


Purification and some properties of a neutral protease from human leukocyte granules and its comparison with pancreatic elastase. 1. A cationic protease has been purified from the granule fraction of blood-donor leukocytes by a preparative method including precipitation by acetone and chromatography on Bio-Gel A 1.5 m, CM-Sephadex C-50 and Sephadex G-G-75. 2. The pH optimum against denatured bovine hemoglobin is 7.4. Gel chromatography indicated a molecular weight close to 23 000. 3. This neutral protease (EC 3.4.-.-) is able to split the synthetic esters Z-Ala-NPh and AcAla3OMe, its activity on the former substrate being 2.2 times greater than that of pancreatic elastase, on the latter the same. It differs crucially from pancreatic elastase in having small elastinolytic activity. 4. In cationic disk electrophoresis, neutral protease resolves into three protein bands with lower mobility than lysozyme: all bands exhibit esterolytic activity against 2-acetoxy-3-naphthoic acid o-toluidide, strongly suggesting that they represent isoenzymes. 5. The enzyme is completely inhibited by iPr2P-F, partially so by soybean trypsin inhibitor and Trasylol. Cysteine, EDTA and TosLysCH2Cl have no effect. 6. During chromatography on CM-Sephadex C-50 a more positively charged enzyme(s) was identified. This had hemoglobinolytic activity at pH 7.4 but only a small esterolytic effect on Z-Ala-NPh; it showed only traces of activity against AcAla3OMe.


Novel activity of potato nucleotide pyrophosphatase. The classical Kornberger-Pricer procedure for purification of potato nucleotide pyrophosphatase (EC 3.6.1.9) has been modified to yield a preparation purified 2500-fold. In addition to the known activity against pyrophosphate linkages in pyrophosphates located at the 5'-OH of nucleosides, and phosphodiester linkages in aryl esters of nucleoside-5'-phosphates, the enzyme has now been shown to catalyze the cleavage of: (a) aryl esters of nucleoside-3'-phosphates and orthophosphates, (b) nucleotide pyrophosphate linkages of the type (3')-pp-(3'), and (c) pm7G from m7GpppGm-terminated fragments of viral mRNA. Activities against aryl esters of nucleoside-3'- and 5'-phosphates, and NAD, were shown to be due to the same protein by three criteria: (a) constant ratio of activities during purification and gel electrophoresis, (b) identical chromatographic properties in various systems, and (c) similarities in pH-dependence, heat inactivation, and the effects of cations and other substances. Since potato nucleotide pyrophosphatase does not exhibit exonuclease or phosphatase activities against natural substrates for the latter enzymes, but does cleave synthetic aryl esters of nucleotide-3'- and 5'-phosphates and of orthophosphate, it follows that these substrates are not suitable for detection of such activities in higher plants.


Urea amidolyase of Candida utilis. Characterization of the urea cleavage reactions. Evidence is presented that the enzymes catalyzing the three reactions involved in urea cleavage in Candida utilis, biotin carboxylation, urea carboxylation, and allophanate hydrolysis occur as a complex of enzymes. The allophanate-hydrolyzing activity could not be separated from the urea-cleaving activity using common methods of protein purification. Further, urea cleavage and allophanate hydrolysis activities are induced coordinately in cells grown on various nitrogen sources. The reactions involved in urea cleavage can be distinguished from one another on the basis of their sensitivities to (a) heat, (b) pH, and (c) chemical inhibitors. Evidence is presented for the product of the first reaction in urea cleavage, biotin carboxylation. Production of carboxylated enzyme is ATP dependent and avidin sensitive. Carboxylated enzyme is not observed in the presence of 1 mM urea.


Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom. By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon acutus venom was separated into twelve fractions. The fibrinolytic activity was concentrated in Fraction 9. This fraction was rechromatographed on Sephadex G-75 three times and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 2.44 S was obtained by ultracentrifugation, the molecular weight of which was estimated to be 24 100, and the isoelectric point 3.8. The specific activity was four times higher than that of crude venom. The optimal pH value on fibrinolysis was 7.4. In addition to fibrinolytic activity, the purified principle also had fibrinogenolytic and caseinolytic activities. The purified fibrinolytic principle had a specific action on the a(A) chain subunit of fibrinogen, leaving the beta(B) chain and the gamma chain unaffected.


Specific fluorescent derivatives of macromolecules. Reaction of dansyl fluoride with serine proteinases. 5-Dimethylaminonaphthalene-1-sulfonyl fluoride was evaluated as a reagent for the selective labeling of proteins. In a comparative study with Dns-chloride a greatly increased selectivity of the fluoride was found with a number of proteins. The reaction of Dns-fluoride with alpha-chymotrypsin, subtilisin Carlsberg and trypsin was found to be highly specific, resulting in a stoichiometric incorporation of the Dns label with concomitant loss of enzymatic activity. The reaction of Dns-chloride with the same proteinases is unspecific. Evidence was obtained to indicate that reaction of the serine esterases with Dns-fluoride occurs exclusively at the active serine residue. The stability of Dns-fluoride labeled chymotrypsin was investigated. The conjugate was found to be fairly stable in the pH range from 3 to 9 at 25 degrees C and is therefore suitable for fluorescence investigations of the chymotrypsin active-site. Molar extinction coefficients for Dns-labeled serine proteinases were determined using radiocative label.


Hemoglobin Athens-Georgia, or alpha 2 beta 2 40(C6)Arg replaced by Lys, a hemoglobin variant with an increased oxygen affinity. A new hemoglobin variant, termed hemoglobin Athens-Georgia, has been found in a 23-year-old Caucasian student and three members of her family. The electrophoretic mobility of this variant at pH 9.0 is slightly less than that of hemoglobin-A. Arginyl residue in position 40 of the beta chain, corresponding to position 6 of the C helix, has been replaced by a lysyl residue. This amino acid substitution is at the alpha1-beta2 contact and slightly affects the oxygen binding properties of the hemoglobin molecule. Hemoglobin Athens-Georgia has an increased affinity for oxygen, a normal heme-heme interaction and a normal Bohr effect. Hematological abnormalities are not associated with this variant.


Sodium trichloroacetate-induced helical conformation of poly(L-lysine). Sodium trichloroacetate which has been reported previously to be an effective denaturation reagent for proteins was applied to poly(L-lysine) and poly(L-glutamic acid) to see its effects on a coil-to-helix transition and on the chemical reactivities of the xi-amino group of poly(L-lysine). Addition of sodium trichloroacetate to poly(L-lysine) induced a helical conformation even at neutral pH where the xi-amino group of the polymer was protonated. On the other hand, little effect was observed on the coil-to-helix transition of poly(L-glutamic acid). The xi-amino group of poly(L-lysine) has an anomalously high reactivity with naphthoquinone 4,6-disulfonate carrying a negative charge. Sodium trichloroacetate inhibited the reaction of the xi-amino group with this reagent, while sodium trichloroacetate enhanced slightly the reaction of the xi-amino group of poly(L-lysine) with diazonium-1-H-tetrazole carrying a positive charge.


Lysyl oxidase: a pituitary hormone-dependent enzyme. Aldehyde-deficient non-crosslinked collagen obtained from lathyritic rats and collagen from penicillamine-treated rats, which is not deficient in aldehydes but the crosslinking of which is also inhibited, were implanted into the peritoneal cavity of hypophysectomized rats using the diffusion chamber technique. The enzyme lysyl oxidase which catalyses the aldehyde formation in certain lysyl residues of collagen and elastin was extracted from the skin of hypophysectomized rats. The activity of the enzyme was determined following its incubation with an L-[4,5-3H] lysine-labeled elastin substrate prepared from aortas of 17-day-old chick embryos. The result showed that the aldehyde deficient collagen did not crosslink while in the hypophysectomized animal indicating the lack of active lysyl oxidase in the rats. The enzyme activity in the skin of hypophysectomized animals was markedly reduced as compared with the controls indicating directly the dependance of lysyl oxidase activity on pituitary gland hormones.


Isolation and characterization of a hemagglutinin from Amphitrite ornata, a polychaetous annelid. Extracts of the marine polychaetous annelid, Amphitrite ornata, agglutinate rat, rabbit, chicken and human erythrocytes and in other work have been shown to inhibit the growth of Ehrlich ascites tumors in mice. Fractionation of extracts on Sephadex G-100 gave three active fractions with molecular weights of 30 000, 54 000 and 100 000. The 30 000 dalton fraction (B) was purified 72-fold by ammonium sulfate precipitation, gel filtration and preparative disc gel electrophoresis. The purified hemagglutinin, amphitritin, was homogenous on analytical disc gel electrophoresis at four different pH values and gave a sharp boundary in sedimentation velocity ultracentrifugation. The three fractions showed paralled specificity toward rat and chicken erythrocytes, the former giving the higher titer. The purified agglutinin was active toward human blood groups A, B and O and exhibited 4-fold higher activity toward group A. The hemagglutinin titer against rat red blood cells was lowered only by N-acetylgalactosamine, the terminal sugar residue of the group A determinant. None of the saccharides tested inhibited agglutination of chicken erythrocytes. Hemagglutinin activity was insensitive to dialysis or treatment with EDTA. The activity was not affected by digestion with trypsin or pronase, but was destroyed by phenol extraction. Analytical disc gel electrophoresis showed one protein band with high anodal mobility at pH 8.5, which was not affected by proteolytic enzymes but was removed by phenol. Activity was unaffected by heating at 70 degrees C for 30 min but was destroyed by similar treatemtn at 85 degrees C. Activity was at a maximum at pH 7-9 and decreased reversibly down to pH 4 at which point it was irreversibly inactivated. The higher molecular weight agglutinin (A1) could be dissociated to give amphitritin by treatment with 6M urea of precipitation in 55% (NH4)2SO4. This dissociation was not reversed by dialysis. Amphitritin is a glycoprotein with a molecular weight determined by gel filtration of 30 000 and by approach to equilibrium sedimentation of 32 000. Amino acid analysis showed a preponderance of aspartic and glutamic acids and relatively large amounts of glycine, proline, alanine, valine and cysteine. The carbohydrate moeity which represented 12.8% of the molecule, contained mannose, galactose, glucosamine and sialic acid. Amphitritin is the first hemagglutinin to be isolated from a polychaetous annelid.


Ceruloplasmin-anion interaction. A circular dichroism spectroscopic study. The effect of anion binding to ceruloplasmin has been studied using absorption and cirbular dichroism spectral data. At anion to ceruloplasmin molar ratios approaching infinite, OCN-, N3- and SCN- bind to ceruloplasmin giving rise to similar alterations in circular dichroism and absorption spectra. The positive bands at 610 and 520 nm in circular dichroism spectra disappear, a negative one apperars at 600 nm and the peak at 450 nm is only slightly modified. There is a new negative band at 410 nm well-defined in OCN- ceruloplasmin spectra. The decrease in absorption at 610 nm is ascribed to the disruption of one type I Cu-S(cysteine) bond owing presumably to the changes induced by anions in the protein secondary structure. The new band at 410 nm is assigned to a charge transfer transition from the ligand replacing cysteine at its binding site. Both absorption and circular dichroism spectra show isobestic points indicating that anion binding to the enzyme, disruption of one of the two type I Cu-S bonds and coordination of this Cu to another protein residue take place simultaneously.


Fluorescence and the structure of proteins. XXI. Fluorescence of aminotyrosyl residues in peptides and helical proteins. 1. Five peptides containing tyrosine were converted to the 3-aminotyrosyl peptides by nitration with tetranitromethane and subseuqent reduction of the nitro groups to amino groups. The fluorescence of these aminotyrosyl residues was found to be quite similar to that of 3-aminotyrosine and it is concluded that the fluorescence is not sensitive to incorporation of the amino acid into the peptide chain. 2. Fluorescence of 3-aminotyrosine derivatives was sensitive, however, to the nature of the solvent; as the dielectric constant decreased, fluorescence was enhanced ten fold and the emission maximum shifted from the 350-370 nm value in aqueous solution to 320 nm. It is predicted that similar differences might be expected for exposed and buried aminotyrosyl residues in a protein. 3. Exposed tyrosyl residues on the helical protein tropomyosin and a helical segment of paramyosin were aminated in part (39% and 34% of the total tyrosyl residues, respectively). The fluorescence of the aminated tyrosyl residues on these proteins was similar to that of the aminotyrosyl peptides in an aqueous medium. Although the fluorescence efficiency of an aminotyrosyl residue was much lower than that of a tyrosyl residue, it was easy to distinguish the fluorescence of the aminotyrosyl residues (350-355 nm) on the protein from that arising from unmodified tyrosyl residues (305 nm).


Fluorine nuclear magnetic resonance studies of trifluoroacetylinsulin derivatives. Effects of salts and denaturants. 19F nuclear magnetic resonance spectroscopy has been used to study the effects of salts and denaturants on the structure and aggregation properties of several trifluoroacetyl derivatives of insulin. This technique has been shown to be a powerful tool in the study of specific sites on the protein molecule. Circular dichroic and sedimentation velocity studies were also carried out to aid in the interpretation of the magnetic resonance data. At pH 6.8 Zn2+ had no effect on the 19F magnetic resonance spectrum, however, citrate and acetate ions significantly sharpened the signal from the trifluoroacetyl probe at the N-terminal end (glycine A-1) of the insulin A chain. No alterations were observed in the S20,W value of circular dichroic spectra, suggesting that the probe had gained a considerable degree of motional freedom without changes in aggregation or conformational properties. In the absence of perturbants the trifluoroacetyl group on glycine A-1 showed considerably more motional freedom than on phenylalanine B-1. Guanidine hydrochloride and sodium dodecyl sulfate were used to study the unfolding of several trifluoroacetylinsulin derivatives. The results suggested differential alterations in the environments of the probes located at glycine A-1, phenylalanine B-1, and lysine B-29 in the insulin molecule as the concentration of perturbant was increased.


Photosynthetic electron transport and electrochromic effects at sub-zero temperatures. Spinach chloroplasts, suspended in a liquid medium containing ethyleneglycol, showed reversible absorbance changes near 700 and 518 nm due to P-700 and "P-518" in the region from -35 to -50 degrees C upon illumination. The kinetics were the same at both wavelengths, provided absorbance changes due to Photosystem II were suppressed. At both wavelengths, the decay was slowed down considerably, not only by the System I electron acceptor methyl viologen, but also by silicomolybdate. The effect of the latter compound is probably not due to the oxidation of the reduced acceptor of Photosystem I by silicomolybdate, but to the enhanced accessibility of the acceptor to some other oxidant. In the presence of both an electron donor and acceptor for System I, a strong stimulation of the extent of the light-induced absorbance increase at 518 nm was observed. The most effective donor tested was reduced N-methylphenazonium methosulphate (PMS). The light-induced difference spectrum was similar to spectra obtained earlier at room temperature, and indicated electrochromic band shifts of chlorophylls a and b and carotenoid, due to a large potential over the thylakoid membrane, caused by sustained electron transport. It was estimated that steady-state potentials of up to nearly 500 mV were obtained in this way; the potentials reversed only slowly in the dark, indicating a low conductance of the membrane. This decay was accelerated by gramicidin D. The absorbance changes were linearly proportional to the membrane potential.


Redox potential of plastoquinone A in spinach chloroplasts. The redox potential of plastoquinone A in spinach chloroplasts was determined. The midpoint potential of the quinone is about +80 mV at pH 7.0 with an n value of 2. The pH-dependence of the potential is -30 mV per pH between pH 4.0 and 5.7, and -60 mV per pH between pH 5.7 and 8.0. The change of the slope at pH 5.7 is interpreted as the protonation of the oxidized plastoquinone A.


Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment. A comparative study is made, at 15 degrees C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O2 evolution was inhibited by low pH or by Tris-treatment. At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 mus. When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays (t 1/2 about 180 mus) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II (t 1/2 about 120 mus). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible. In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 mus.


Membrane-bound ATPase in chloroplasts of Euglena gracilis. Membrane-bound ATPase activities in chloroplasts of Euglena were examined. Ca2+- and Mg2+-dependent activities were relatively high in membrane preparations and could not be further activated by a number of procedures. The enzyme was found to be highly specific for purine nucleotides and was inhibited by the usual inhibitors of photophosphorylation. Km values of Ca2+ and Mg2+ ATPase for ATP were 2.5 and 2.1 mM, respectively. Both activities were competitively inhibited by ADP and inorganic phosphate. A relationship was found between Ca2+- or Mg2+-dependent ATPase activities and chloroplast completeness. The possibilities that these activities result from one enzyme depending on Ca2+ or Mg2+ or from two different enzymes are discussed.


Cytidine-5'-monophospho-N-acetylneuraminic acid galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosylceramide sialyltransferase in the neurohypophysis of the rabbit. 1. Cytidine-5'-monophospho-N-acetylneuraminic acid: (galactosyl-N-acetyl-galactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosylceramide sialyltransferase (CMP-NAcNeu: monosialoganglioside (GM1) sialyltransferase) activity was demonstrated in the neurohypophysis of the rabbit. 2. Optimum activity occurred at pH 6.5 and required the presence of exogenous galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosylceramide (GM1 ganglioside), detergent (Triton X-100), and divalent cation (Mn2+, Mg2+ or Ca2+). 3. The product of the reaction was characterized as N-acetylneuraminyl-galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosylceramide (GD1a) by ascending thin-layer chromatography. 4. Physiological stimulation of vasopressin secretion, by the substitution of 2.2% NaCl for drinking water for 14 days, had no effect on the enzyme activiity or the ganglioside content of the tissue.


The formation of cis-3-nonenal, trans-2-nonenal and hexanal from linoleic acid hydroperoxide isomers by a hydroperoxide cleavage enzyme system in cucumber (Cucumis sativus) fruits. 1. A particulate enzyme fraction and an acetone powder preparation from cucumber fruits cleaved 9- and 13-hydroperoxyoctadecadienoic acids to form volatile aldehydes and oxoacid fragments. 2. From the 9-hydroperoxide, the major volatile fragments were cis-3-nonenal and trans-2-nonenal using particulate enzyme and acetone powder preparations, respectively. 3. Hexanal was the only significant volatile fragment from the 13-hydroperoxide. 4. The particulate enzyme system was equally effective on both 9- and 13-hydroperoxide isomers and was fully active under anaerobic conditions and at pH 6.4. 5. An enzymic pathway for the biogenesis of hexanal, cis-3- and trans-2-nonenal (components of the characteristic flavour volatiles of cucumber) from linoleic acid is proposed. This involves the sequential activity of lipoxygenase, hydroperoxide cleavage and cis-3-: trans-2-enal isomerase enzymes.


Activity and properties of CTP: cholinephosphate cytidylyltransferase in adult and fetal rat lung. Cholinephosphate cytidylyltransferase (CTP : cholinephosphate cytidylyltransferase, EC 2.7.7.15) is located in both the microsomal and supernatant fractions of adult lung when the tissue is homogenized in 0.145 M NaCl. The activity is located predominantly in the supernatant fraction in fetal lung. Cholinephosphate cytidylyltransferase in the supernatant from fetal lung is stimulated 4- to 6-fold by the additions of total lung lipid. Serine phosphoglycerides and inositol phosphoglycerides specifically caused stimulation whereas choline phosphoglycerides and ethanolamine phosphoglycerides produced no stimulation. Lysophosphatidylcholine cause some stimulation, but only at high concentrations. A number of detergents were investigated. All produced inhibition except for the ampholytic detergent, miranol H2M which was not inhibitory. None of the detergents produced any stimulation of activity. Cytidylyltransferase activity in fetal lung when assayed in the absence of lipid is about 25% of the adult. The activity when assayed in the presence of lipid is equal or slightly higher than adult levels. The activity, measured without added phospholipid, increases 5- to 6-fold within 12 h after birth, to values higher than in the adult. The activity, measured in the presence of phospholipid, increased almost linearly from -2 day until +1 day. There is an inverse relationship between the concentration of phospholipid in the fetal lung supernatant and the degree of lipid stimulation. Chromatographic experiments with Biogel A 1.5 columns have shown that cytidylyltransferase can exist in two molecular sizes, a small molecular size that requires phospholipid for activity, and a larger molecular weight species which does not require the addition of phospholipid for activity. Fetal lung has a higher proportion of the low molecular weight form than adult lung. The small molecular weight species can be converted to the larger molecular weight form by the addition of phospholipids.


Effect of portacaval anastomosis on the activities of hepatic enzymes related to cholesterol and bile acid metabolism in rats. 1. The effect of a portacaval anastomosis on the activities of hepatic enzymes related to cholesterol metabolism was investigated in rats. 2. Portacaval anastomosis led to a fall in body weight and liver weight/body weight ratio, and to a rise in the activities of hydroxymethylglutaryl-CoA reductase and cholesterol 7alpha-hydroxylase per g of liver. The net effect was to maintain a normal activity of both enzymes per 100 g of rat. Diurnal rhythm in the activities of both enzymes was maintained after portacaval anastomosis. 3. The rate of excretion of total bile acids, per 100 g of rat, in bile fistula rats was not significantly decreased by portacaval anastomosis.


Coupling in secondary transport. Effect of electrical potentials on the kinetics of ion linked co-transport. In a previous paper kinetic equations of secondary active transport by cotransport have been derived. In the present paper these equations have been expanded by including the effect of an electrical potential difference in order to make them applicable to the more realistic systems of secondary active transport driven by the gradients of Na+ or H+. Thermodynamically an electrical potential difference is as a driving force fully exchangeable with an equivalent chemical potential difference. This is not necessarily so for the kinetics of co-transport. It is not always the same whether a given difference in electrochemical activity of the driver ion is mainly osmotic, i.e. due to difference in concentration, or electric, i.e. due to a difference in the electrochemical activity coefficient. In most cases a difference in concentration is more effective in driving co-transport than is an equivalent difference in electrical potential leading to the same difference in electrical activity. The effectiveness of the latter highly depends on the model, whether it is of the affinity type or of the velocity type, but also on whether the loaded or the unloaded carrier bears an electrical charge. With the same electrical potential difference co-transport is as a rule faster if the ternary complex rather than the empty carrier is charged. Also the "standard parameters", (see Glossary, page 62) Jmax and Km, of the overall transport respond differently to the introduction of an electrical potential difference, depending on the model. So an electrical potential difference will mostly affect Km if the loaded carrier is ionic, and mostly Jmax if the empty carrier is ionic, provided that the mobility of the loaded carrier is greater than that of the empty one. On the other hand, distinctive criteria between affinity type and velocity type models are partly affected by an electrical potential difference. If the translocation steps of loaded and unloaded carrier are no longer rate limiting for the overall transport, electrical effects on the transport rate are bound to vanish as does the activation by co-transport.


Tryptic- and chymotryptic-like proteinases in early and late preimplantation mouse blastocysts. Neutral tryptic- and chymotryptic-like enzyme activities have been identified in extracts of early and late mouse blastocyts. The enzyme activities are distinguishable my means of: (a) reactivity with specific naphthylamide derivatives; (b) reactivity with tosyl-L-lysine-chloromethyl ketone or L-tosylamido-2-phenylethyl-chloromethyl ketone, respectively; (c) electrophoretic mobilities; and (d) specific activity profiles of late vs. early blastocysts.


PHrmonal specificity of the melanotropin-sensitive adenylate cyclase of mouse melanoma and effect of cyclic AMP on the tyrosinase activity of mouse melanoma cells, in vitro. Transplantable mouse melanomas possess a melanotropin-sensitive adenylate cyclase system which is responsive to alpha-melanotropin, beta-melanotropin, adrenocorticotropin (ACTH) and prostaglandin E1. It was found that sensitivity to ACTH was not directed towards the ACTH activity but to the intrinsic melanotropin activity of the ACTH molecule. Therefore, the melanotropin-sensitive adenylate cyclase system is hormonally specific to the intrinsic melanotropin activity of peptide hormones and is unique in the melanoma tissue. The significance of the sensitivity to prostaglandin E1 is obscure at present. The melanotropin-sensitive adenylate cyclase requires the presence of Mg2+ or Mn2+, for its enzymic activity. Ca2+ inhibit the enzyme in the presence of a wide range of concentrations of Mg2+. The enzymic activity is ATP concentration-dependent and the saturation concentration appears to be 1 mM. The enzyme is very labile in the unfractionated tumor homogenates. A washed 11000 X g particulate fraction, representing about 30-60% of the total enzymic activity, was found to be more stable and could be stored at 5 degrees C for 2 h without appreciable loss of the activity. This fraction retained sensitivity to melanotropin, prostaglandin E1 and NaF. About 20% of the activity of the tumor homogenate could not be sedimented by centrifugation at 105000 X g for 60 min. This "soluble" fraction was not responsive to melanotropin, prostaglandin E1 and NaF and might be a degradative product produced by the fractionation. Cyclic AMP and alpha-melanotropin were able to increase the tyrosinase activity of isolated mouse melanoma-cells in vitro under the same conditions.


The effect of beta-adrenergic blocking agents on experimental porphyria induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in vivo and in vitro. The effect of DL-propranolol on 3',5'-diethoxycarbonyl-1,4-dihydrocollidine-induced experimental porphyria was studied. DL-Propranolol, a beta-adrenergic blocking agent with non-specific membrane effects, partially inhibited 3',5'-diethoxycarbonyl-1,4-dihydrocollidine-induced delta-aminolevulinate synthetase activity both in rats and in chick embryo liver cells in culture. In rats, DL-propranolol decreased urinary delta-aminolevulinate and porphobilinogen but no change occurred in the 24-h urinary excretion of total porphyrins and in the concentration of porphyrins in the liver. In cultured chick embryo liver cells treated with 3',5'-diethoxycarbonyl-1,4-dihydrocollidine, DL-propranolol decreased accumulation of porphyrins in the medium. D-Propranolol, oxprenolol and quinidine acted like DL-propranolol in chick embryo liver cells in culture treated with 3',5'-diethoxycarbonyl-1,4-dihydrocollidine. Pindolol, practolol and lidocaine had no effect. Phenobarbitone had a synergistic effect on the induction of delta-aminolevulinate synthetase by 3',5'-diethoxycarbonyl-1,4-dihydrocollidine in cultures of chick embryo liver cells. This induction was partially inhibited by propranolol. However, the increased accumulation of porphyrins in the medium caused by 3',5'-diethoxycarbonyl-1,4-dihydrocollidine was inhibited by the addition of phenobarbitone. This inhibited induction was further decreased by propranolol. Most of our results indicate that the drugs tested act mainly by their effects on membranes.


Studies on the production and assessment of experimental histidinemia in the rat. Intraperitoneal administration to rats of D- or DL-alpha-hydrazinoimidazolylpropionic acid was found to produce a substantial inactivation of hepatic histidine ammonia-lyase (EC 4.3.1.3) in vivo. Proportional to this loss in enzyme activity was an impairment of the ability of treated rats to oxidize L-[ring-2-14C] histidine to 14CO2. Rats in which hepatic histidine ammonia-lyase activity was either depressed by DL-hydrazinoimidazolylproprionic acid injection or elevated by feeding a high protein diet displayed proportionately altered rates of 3H2O release into plasma water following L-[3-3H] histidine administration. Plasma L-histidine clearance following loading with this amino acid was similarly affected by these treatments. Administration of DL-alphal-hydrazinoimisazolylproprionic acid to rats was also found to inactivate non-specifically pyridoxal 5-phosphate enzymes in vivo; pyridoxine injection was found to reverse the DL-alpha-hydrazinoimidazolylproprionic acid-induced inactivation of hepatic aspartate aminotransferase (EC 2.6.1.1) in vivo, but not that of hepatic histidine ammonia-lyase. These findings demonstrate that histidine ammonia-lyase is the rate-limiting factor in L-histidine degradation in the rat. The potential usefulness of DL-hydrazinoimidazolylproprionic acid in the production of an animal model for histidinemia (hereditary histidine ammonia-lyase deficiency) is discussed.


Photooxidation of methionine with immobilized methylene blue as photooxidizer. Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37 degrees C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84% acetic acid at 4 degrees C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94%, while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21%. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.