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PubMed1978

[Presence of SH-groups and histidine in the active site of Chlorella glutamine synthetase].

The presence of two cysteine residues per each six monomers comprising the oligomer of Chlorella glutamine synthetase (E.C.6.3.1.2) is demonstrated using homogenous enzyme preparation. p-Chloromercuribenzoate (p-CMB) is found to inhibit glutamine synthetase activity, the degree of inhibition depending on the inhibitor concentration. The following enzyme reactivation by dithiotreitol (10(-2) M) was observed only when the enzyme was inactivated with 10(-5) M p-CMB under 15 min. preincubation. Preincubation of the enzyme with 10(-4) M p-CMB for 45 min. did not result in its reactivation. Gel filtration of glutamine synthetase treated with 10(-4) M p-CMB has revealed the dissociation of the enzyme into inactive monomers. Incubation of glutamine synthetase with p-CMB at various pH values, incubation after pre-treatment with urea and experiments with HgCl2 indicate the presence of free and masked inside the globula SH-groups in the enzyme molecule. Competitive character of the enzyme inhibition with p-CMB with respect to ATP indicates that SH-groups of the active site participate in the ATP binding, probably, as Mg-ATP or Mn-ATP complexes. Data on the estimation of ionization constant of glutamate-binding group and experiments on the effect of histidine photooxidation on the enzyme activity indicate the presence of histidine residue in the enzyme active site, which participates in glutamate binding.

Adenosine Triphosphate↗

PubMed1977

Interactions of platinum complexes with the essential and nonessential sulfhydryl groups of thymidylate synthetate.

Thymidylate synthetase (methylenetetrahydrofolate:deoxyuridylate C-methyltransferase) from Lactobacillus casei was progressively inactivated when incubated at 25 degrees C, pH 6.8, in the presence of trans-Pt(NH3)2Cl2. The inhibition appeared to be irreversible, and the rate ofa ctivity loss was dependent on the inhibitor concentration. The corresponding cis isomer was incapable of inhibiting the enzyme under the same conditions. The presence of 2-mercaptoethanol protected the enzyme from inhibition, but did not reactivate enzyme preparations which had been inhibited prior to the addition of the thiol. The interactions of cis- and trans-Pt(NH3)2Cl2 with the enzyme's sulfhydryl (-SH) groups were inferred from the results of spectrophotometric titrations of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate. The results suggested that the cis isomer reacted with an average of 1.3 of the enzyme's 4-SH groups and that these were not essential for catalysis. The trans isomer reacted with a total of approximately 2.5 -SH groups, 1.2 of which are essential for catalysis. Neither the trans isomer nor a combination of both isomers was able to react with 1.2 of the 4 -SH groups. Further evidence that the Pt complexes are interacting with enzyme's -SH groups was obtained by reversibly blocking the -SH groups of thymidylate synthetase, and demonstrating the resistance of these preparations to inhibition by the trans Pt complex. Possible explanations for the preferential inhibition of thymidylate synthetase by only one of the two geometric isomers of Pt(NH3)2Cl2 are considered.

Binding Sites↗

PubMedDate not supplied

Studies on the Zn2+/Co2+ exchange with acylamino acid amidohydrolase from pig kidney.

The kinetics of inactivation of the Zn2+-metalloenzyme acyl-amino acid amido hydrolase by chelating ligands were studied. The rate of inactivation by 1,10-phenanthroline is enhanced by histidine and inhibited in the presence of phenyl-alanine. Removal of the metal ion increases the heat stability and decreases the pH stability of the enzyme. Reactivation of the inactive metal free enzyme is possible with Zn2+ and Co2+. Titration of the metal free protein with a free enzyme is possible with Zn2+ and Co2+. Titration of the metal free protein with a Co2+/nitrilotriacetate metal buffer revealed a dissociation constant of about 10(-7) M for the Co2+ enzyme (Zn2+ enzyme = 10(-10) M). The Co2+ substituted enzyme is less stable than the Zn2+ enzyme. Histidine and phenylalanine protect the Co2+ enzyme against inactivation by 1,10-phenanthroline.

Amidohydrolases↗

PubMed1950

Enzyme kinetics and the rate of biological processes.

It is found empirically that a simple modification of the usual theoretical kinetic formulation (in which a transformation in the temperature scale is made) describes the temperature dependence of a wide variety of biochemical processes with a greater accuracy than hitherto achieved. Used in conjunction with the formulation of the theory of absolute reaction rates this empirical relation facilitates the determination of the thermodynamic functions. The results of applying these relations to biochemical processes support the contention that in the lower temperature range of enzyme activity a thermodynamic equilibrium exists between catalytically active and inactive forms of the enzyme. It is suggested that at low temperatures the formation of intramolecular hydrogen bridges converts reactive enzyme particles to a catalytically inactive condition, in which the active centers either lose their specific configuration or are no longer exposed to the substrate. Upon the basis of this interpretation, values of the entropy changes that are calculated theoretically are found to be in agreement with those calculated from the experimental data. The reactive configuration of the enzyme is apparently possessed in only a relatively narrow temperature band, being lost at both high and low temperatures. The kinetics of biological processes appear to differ only quantitatively from those of in vitro enzyme-catalyzed reactions. In both cases the non-linearity of the Arrhenius plots appears to be due to the fact that in the lower temperature range of enzyme activity a series of reactions are involved in the formation of the activated complex. These include reactions which lead to the formation of the catalytically reactive form of the enzyme followed by that which leads to the formation of the activated complex. The conversion of enzymes to the catalytically inactive form is essentially completed at temperatures of -10-0 degrees C. in living systems, whereas in in vitro experiments with purified enzyme preparations this condition is not attained until temperatures 30-60 degrees C. lower have been reached.

Biological Phenomena↗

PubMed1988

In vivo protection against soman toxicity by known inhibitors of acetylcholine synthesis in vitro.

Soman inhibits the enzyme acetylcholinesterase, essentially irreversibly, producing an accumulation of acetylcholine (ACh) which is responsible for many of its toxic effects. Current approaches to treatment include: (1) atropine, a muscarinic receptor blocker; (2) pyridine-2-aldoxime methylchloride (2-PAM), an enzyme reactivator; and (3) carbamate protection of the enzyme. However, no fully satisfactory regimen has been found, primarily because of the rapid aging process. In this study, compounds known to inhibit ACh synthesis in vitro were evaluated in combination with atropine and 2-PAM so as to assess their potential utility in protection against soman toxicity in rats. Acetylsecohemicholinium (100 micrograms/kg, i.c.v.t., 30 min prior to soman), an inhibitor of high affinity choline uptake (HAChU) and cholineacetyltransferase (ChAT) activity in vitro, enhanced the protective effects of atropine and 2-PAM, reducing the mortality within the first 2 hr following soman. N-Hydroxyethylnaphthylvinylpyridine (NHENVP), a quaternary ChAT inhibitor (1.7 mumol/kg, i.m.), significantly reduced the overall percent mortality due to soman from 80% to 20%. The compound was most effective when administered 2-3 min prior to soman and was effective only by the intramuscular route. N-Allyl-3-quinuclidinol, a potent HAChU inhibitor (1 mumol/kg, i.m.) was the most effective quinuclidine analog evaluated, also reducing the percent mortality for a 24-hr period. Unlike NHENVP, it was most effective when given 30-60 min prior to soman. It is suggested from the data that compounds that disrupt presynaptic ACh synthesis in vitro may prove effective in treating organophosphate poisoning. The results demonstrate interesting differences among the compounds studied and provide insight for the design of protectants against soman toxicity. These findings further underscore the need to examine the structure activity and pharmacokinetic properties of these compounds, i.e. comparison of routes of administration, dose-response relationships, and time to effect.

Acetylcholine↗

PubMed1990

The mechanism of action of the nitrosourea anti-tumor drugs on thioredoxin reductase, glutathione reductase and ribonucleotide reductase.

The nitrosoureas BCNU, CCNU, ACNU, and Fotemustine covalently deactivate thioredoxin reductase, glutathione reductase and ribonucleotide reductase by alkylating their thiolate active sites. Since thioredoxin reductase and glutathione reductase function as alternative electron donors in the biosynthesis of deoxyribonucleotides, catalyzed by ribonucleotide reductase, the inhibition of these electron transfer systems by the nitrosoureas could determine the cytostatic property of this homologous series of drugs. A detailed study of the kinetics and mechanism for the inhibition of purified thioredoxin reductases from human metastatic melanotic and amelanotic melanomas by the nitrosoureas showed significantly different inhibitor constants. This difference is due to the regulation of these proteins by calcium. Calcium protects thioredoxin reductase from deactivation by the nitrosoureas. In addition, it has been shown that reduced thioredoxin displaces the nitrosourea-inhibitor complex from the active site of thioredoxin reductase to fully reactivate enzyme purified from human metastatic amelanotic melanoma. It has been possible to label the active sites of thioredoxin reductase and glutathione reductase by using chloro[14C]ethyl Fotemustine, resulting in the alkylation of the thiolate active sites to produce chloro[14C]ethyl ether-enzyme inhibitor complexes. These complexes can be reactivated via reduced thioredoxin and reduced glutathione, respectively, by a beta-elimination reaction yielding [14C]ethylene and chloride ions as reaction products.

Antineoplastic Agents↗

PubMed1990

Leukotriene A4 hydrolase: an epoxide hydrolase with peptidase activity.

Purified leukotriene A4 hydrolase from human leukocytes is shown to exhibit peptidase activity towards the synthetic substrates alanine-4-nitroanilide and leucine-4-nitroanilide. The enzymatic activity is abolished after heat treatment (70 degrees C, 30 min). At 37 degrees C these substrates are hydrolyzed at a rate of 380 and 130 nmol/mg/min, respectively, and there is no enzyme inhibition during catalysis. Apo-leukotriene A4 hydrolase, obtained by removal of the intrinsic zinc atom, exhibits only a low peptidase activity which can be restored by the addition of stoichiometric amounts of zinc. Reconstitution of the apoenzyme with cobalt results in a peptidase activity which exceeds that of enzyme reactivated with zinc. Preincubation of the native enzyme with leukotriene A4 reduces the peptidase activity. Semipurified preparations of bovine intestinal aminopeptidase and porcine kidney aminopeptidase do not hydrolyze leukotriene A4 into leukotriene B4.

Aminopeptidases↗

PubMedDate not supplied

Comparison of a commercial hexadimethrine bromide method and low-ionic-strength solution for antibody detection with special reference to anti-K.

The sensitivities of manual low-ionic hexadimethrine bromide (Polybrene, LIP) and low-ionic Polybrene indirect antiglobulin tests (LIPAT) were compared with those of a manual low-ionic-strength indirect antiglobulin test (LISS) by using a commercial Polybrene kit. One hundred antibodies were coded, titrated, and tested in parallel. LIP did not detect 36 antibodies: 31 anti-K, two anti-E, two anti-Fya, and one anti-Jka. LIPAT did not detect seven anti-K, two anti-E, and two anti-Jka. The combination of LIP and LIPAT did not detect two anti-E that were reactive only in a two-stage enzyme test and seven anti-K that had titers of 2 or lower by LISS. LISS detected all antibodies except for the two enzyme-reactive anti-E. There were no significant differences in the titers of 63 percent of the antibodies studied. For 54 percent of the antibodies in the Kell system, LISS produced significantly higher titers; for 25 percent of antibodies in the Rh system, LIP did so. The poor sensitivity of the Polybrene kit for anti-K makes it unsuitable as a primary method for antibody screening.

Coombs Test↗

PubMed1980

Purification and some properties of human heart arginase.

Arginase from extracts of human heart was purified about 1500-fold. In polyacrylamide-gel electrophoresis the enzyme migrated to the cathode at pH 5.5, showing very low mobility at pH 8.9. Molecular weight determined by gel filtration was 120 000. The Km for L-arginine was 5 mM. L-Ornithine and L-lysine were competitive inhibitors. The enzyme was completely inactivated by treatment with EDTA, and dissociated into subunits with mol.wt. of about 30 000. Addition of Mn2+ ions to the inactive subunits resulted in reappearance of the enzyme activity; the molecular weight of the reactivated enzyme corresponded to that of the native form.

Amino Acids↗

PubMedDate not supplied

Biotinylated isocoumarins, new inhibitors and reagents for detection, localization, and isolation of serine proteases.

Eight new biotinylated, mechanism-based isocoumarin serine protease inhibitors have been designed and synthesized to detect, localize, and isolate serine proteases. Isocoumarins that contain a 4-chloro group, a biotinylated substituent at the 7-position, and different 3-alkoxy groups are inhibitors of various serine proteases including human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), trypsin, human recombinant granzyme A, chymotrypsin, and cathepsin G. Insertion of spacers between the isocoumarin moiety and the biotin moiety enhanced enzyme inhibitory potency and may also promote binding of the enzyme-inhibitor complex to avidin. The 3-alkoxy groups conferred selectivity toward different serine proteases with chymotrypsin being inhibited effectively by compounds with 3-phenylethoxy groups while derivatives with 3-methoxy, ethoxy, or propoxy groups were potent inhibitors of HLE and moderate inhibitors of PPE. Full enzymatic activity was regained after the immediate addition of hydroxylamine to the inactivated chymotrypsin and PPE derivatives, which indicated that a simple acyl enzyme derivative is formed initially in the inhibition reaction. Egg avidin did not effect the rate of spontaneous enzyme reactivation rate while streptavidin accelerated the reactivation reaction. PPE inhibited by 7-[[6-(biotinylamino)caproyl]amino]-4-chloro-3- ethoxyisocoumarin (BIC 5) or 7-[[6-[[6-(biotinylamino)caproyl]amino] caproyl]amino]-4-chloro-3-methoxyisocoumarin (BIC 7) was bound to immobilized avidin columns. Most of inhibited PPE could be eluted from the monomeric or tetrameric avidin columns but only a portion (40-70%) of the enzyme was active due to the partial formation of a stable alkylated enzyme derivative during the isolation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Photoreactivation of superoxide dismutase by intensive red (laser) light.

The effects of helium-neon laser (HNL) on activity, absorption spectra, and ESR signals of superoxide dismutase (SOD; E Cul.15.1.1) from bovine erythrocytes in acid medium were investigated. It was found that incubation during 2 hours at pH 5.9 led to inactivation of the enzyme. The subsequent illumination of SOD by HNL brought about the enzyme reactivation. Both absorption and ESR spectra were changed after incubation at pH 5.9 and restored after laser irradiation. In a model system, copper-histidine complex, absorption maximum was shifted from 632-633 nm at pH 5.8 to 639-640 nm at pH 8.5-9.0. The similar shift of the maximum was observed after illumination by HNL at pH 5.8. It may be postulated that the photoreactivation of SOD consists essentially in deprotonation of His-61 residue in the enzyme active site and subsequent recovery of imidasol bridge between copper and zinc which had been destroyed at low pH. Since many other enzymes possess similar histidine-copper structures in their active sites, one may expect diverse effects of red (laser) light on the enzyme activity. Heme-containing enzyme, catalase was also found to be photoreactivated by HNL after inactivation at pH 6.0.

Animals↗

PubMed1981

Reactivity of sulphydryl groups of cytosolic and mitochondrial bovine aspartate aminotransferases.

Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs2 while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.

Animals↗

PubMed1979

Mechanism of inhibition of malate dehydrogenase by thyroxine derivatives and reactivation by antibodies: homogeneous enzyme immunoassay for thryroxine.

Pig heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) is about 90% inhibited upon labelling an average of two amino groups per subunit with an active ester of thyroxine. Inhibition is probably associated primarily with thyroxine binding to one specific group which is normally unreactive but becomes activated upon noncovalent binding of thyroxine derivatives to the enzyme. Enzyme inhibition is due to a decrease in the rate of association of NAD. Antibodies to thyroxine induce a slow conformational change with partial reversal of inhibition of more heavily labelled conjugates. The antibody-induced activation is not cooperative and does not require bivalent association of the antibody. Activation can be blocked by the presence of free thyroxine and is the basis for a clinically useful assay for serum thyroxine.

Animals↗

PubMed1984

Inhibitory characteristics of 3,5-dibromo-1-acetoxy-4-oxo-2,5-cyclohexadien-1-acetonitrile, a semisynthetic derivative of aeroplysinin-1 from sponges (Aplysinidae), on Na+ - K+-ATPase.

3,5-Dibromo-1-acetoxy-4-oxo-2,5-cyclohexadien-1-acetonitrile (dienone A) inhibited Na+ - K+-ATPase with a half-maximal inhibition concentration (I50) equal to 2.9 X 10(-6)M. Inhibition was time- and pH-dependent and complete after 20-30 min preincubation within a range of pH from 7.0 to 9.0. Kinetic evaluation of the cationic substrate activation of Na+ - K+-ATPase indicated mixed type inhibition with regard to Na+ and K+ and competitive inhibition with regard to ATP activation of the enzyme. The presence of Mg2+ caused an increased inhibition. Also, K+-p-nitrophenyl phosphatase activity was altered by dienone A and mixed type inhibition with regard to p-nitrophenyl phosphate and K+ was demonstrated. Inhibition was partially restored by repeated washing. Preincubation with sulfhydryl reagents protected the enzyme from inhibition. A significant linear correlation between reactive enzyme sulfhydryl contents [SH] and Na+ - K+-ATPase activity in the presence of varying concentrations of dienone A was observed. One of the factors causing cytotoxic activity of this compound might be its interaction with some thiol groups of the membrane-bound Na+ - K+-ATPase.

4-Nitrophenylphosphatase↗

PubMed1983

Modification of arginines in D-beta-hydroxybutyrate dehydrogenase.

D-beta-Hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate: NAD+ oxidoreductase, EC 1.1.1.30) is a lipid-dependent enzyme which has an absolute and specific requirement for phosphatidylcholine for function. Chemical derivatization studies using 1,2-cyclohexanedione, an arginine-specific reagent, have been carried out on the purified enzyme devoid of lipid as well as on the enzyme reactivated with phospholipid. Cyclohexanedione inactivated the active enzyme-phospholipid complex and the lipid-free enzyme was rendered inactivatable by phospholipid. From kinetic studies and by direct chemical derivatization studies with [1-14C]cyclohexanedione, we find that incorporation of a single cyclohexanedione molecule per enzyme monomer resulted in complete loss of enzymic activity. The presence of NADH or NAD+, cofactors for the enzyme, offered no protection for the rate of inactivation. The substrates beta-hydroxybutyrate and acetoacetate with or without coenzyme gave little or no protection. However, 2-methyl malonate, a competitive inhibitor for beta-hydroxybutyrate, strongly protected against inactivation. These studies indicate that: (1) a single arginine serves a vital role and is essential for function; (2) the arginine is located in the proximity of the substrate binding site.

Animals↗

PubMed2000

Kinetics of ampicillin synthesis catalyzed by penicillin acylase from E. coli in homogeneous and heterogeneous systems. Quantitative characterization of nucleophile reactivity and mathematical modeling of the process.

Kinetic regularities of the enzymatic acyl group transfer reactions have been studied using ampicillin synthesis catalyzed by E. coli penicillin acylase as an example. It was shown that ampicillin synthesis proceeds through the formation of an acylenzyme-nucleophile complex capable of undergoing hydrolysis. The relative nucleophile reactivity of 6-aminopenicillanic acid (6-APA) is a complex parameter dependent on the nucleophile concentration. The kinetic analysis showed that the maximum yield of antibiotic being synthesized depended only on the nucleophile reactivity of 6-APA, the ratio between the enzyme reactivities with respect to the target product and acyl donor, and the initial concentrations of reagents. The parameters characterizing the nucleophile reactivity of 6-APA have been determined. The algorithm of modeling the enzymatic synthesis has been elaborated. The proposed algorithm allows the kinetics of the process not only in homogeneous, but also in heterogeneous ("aqueous solution-precipitate") systems to be quantitatively predicted and described based on experimental values of parameters of the reaction. It was shown that in heterogeneous "aqueous solution-precipitate" systems PA-catalyzed ampicillin synthesis proceeds much more efficiently compared to the homogeneous solution.

Ampicillin↗

PubMed1985

Fatal infantile cytochrome c oxidase deficiency: decrease of immunologically detectable enzyme in muscle.

A 2-month-old boy had progressive generalized weakness, hypotonia, and respiratory insufficiency requiring assisted ventilation. At age 3 1/2 months, he started having seizures and recurrent pulmonary infections; he died at age 7 months. Serum lactate was chronically elevated, but there was no aminoaciduria. Histochemical and ultrastructural studies of muscle biopsies at ages 2 and 3 months showed excessive mitochondria, lipid, and glycogen; a third biopsy at 6 months showed marked increase in perimysial fibrous and fat tissue. Cytochrome c oxidase activity was 7% of normal in the first biopsy and undetectable in the others. Cytochrome spectra of mitochondria isolated from postmortem muscle showed complete lack of cytochrome aa3. Antibodies were obtained against cytochrome c oxidase purified from normal human heart. Immunotitration and enzyme-linked immunosorbent assay (ELISA) showed decreased immunologically reactive enzyme protein in the patient's muscle, but SDS-PAGE electrophoresis of immunoprecipitates of muscle mitochondrial extracts showed the presence of all cytochrome c oxidase subunits. These data suggest that decreased synthesis of one or more subunits may result in markedly decreased concentration of electrophoretically normal complex IV in skeletal muscle.

Cytochrome-c Oxidase Deficiency↗

PubMed1987

Mitochondrial encephalomyopathy and partial cytochrome c oxidase deficiency.

A 52-year-old man had slowly progressive weakness and wasting of limb-muscles, sensorineural hearing loss, and complex partial seizures. CT showed cerebral atrophy, but he was not demented. Muscle biopsy showed ragged-red fibers and decreased histochemical stain for cytochrome c oxidase. Biochemical studies showed decreased cytochrome c oxidase activity in crude muscle extracts and in isolated mitochondria (44 and 30% of normal), while other mitochondrial enzymes were normal. A comparable decrease of immunologically reactive enzyme protein was shown by immunotitration with antibodies against human heart cytochrome c oxidase. Partial defects of cytochrome c oxidase may cause adult-onset, slowly progressive mitochondrial encephalomyopathies.

Atrophy↗