Biomedical subjects
Publications and source records attributed to V A Pekkel'.
Methyl esters of aromatic a-amino acids, peptides with N-terminal tyrosine and C-terminal arginine, and amides of peptides with N-terminal aromatic amino acids all inhibit monoamine oxidases A and B from rat liver mitochondria with an IC50 of 0.2-3 mM.
The interaction between diamine oxidase (DAO) of human placenta and carboxyl-substituted lysines, including N-terminal lysine containing peptides, occurs at rather a high rate and is characterized by the following features. First, the enzyme catalyzes the oxidative deamination of one amino group in the N-terminal lysine at a rate which is inversely proportional to the peptide length. Second, the bound derivatives induce a noncompetitive reversible inhibition of DAO which is enhanced during their coincubation. The inhibiting capacity of this compound is directly proportional to the peptide length; therefore, the tripeptides with the N-terminal lysine can be effective inhibitors that are not practically deaminated in the presence of DAO. Third, the binding of carboxyl-substituted lysines to DAO as well as the inhibition reaction are reversible processes and, with some limitations, can be used for enzyme purification. An analysis of the total activity of DAO in the placenta before and after fractionation of tissue extracts on molecular sieves showed that part of the enzyme is in a blocked state in vivo which does not exclude the possibility that N-terminal lysine containing peptides are related to natural DAO inhibitors.
The phenomenology of inhibition of FAD-containing type A monoamine oxidase by clorgyline solutions containing negligibly small amounts of clorgyline that are insufficient for stoichiometric covalent blocking of a perceptible amount of the coenzyme was studied. The nature of this phenomenon consists in the fact that at monoamine oxidase concentrations of about 10(-8) M, more than 50% of the enzyme activity in inhibited by clorgyline (less than or equal to 10(-10) M), although is accordance with a well-defined mechanism after monoamine oxidase-catalyzed tautomerization clorgyline presumably interacts with FAD at a 1:1 stoichiometric ratio. This effect termed as secondary inhibition seems to be induced not by clorgyline proper, not by changes in the solvent induced by this compound. In other words, clorgyline may initiate the synthesis of a new hypothetical inhibitor (IIC) in aqueous media which causes a reversible inhibition of the same specific inhibitory site of the enzyme. This site is responsible for the initial binding of acetylene inhibitors and catalyzes the formation of their allenic derivatives.
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Human placenta was shown to contain practically all known types of aminooxidase, i.e., Membrane-bound and soluble monoamine oxidases A that predominantly oxidize serotonin (Km approximately 0.05 and 0.2 mM) and tyramine (Km approximately 0.03 and 0.085 mM), partly oxidize phenylethylamine (Km approximately 0.013 and 0.1 mM) and slightly oxidize benzylamine; Monoamine oxidase B and its intermediate form, B', with equal sensitivity towards the inhibitors, Lilly 51641 and deprenyl. The main substrates for these enzymes are phenylethylamine (Km = 0.011 mM for the membrane-bound and 0.019 mM for the soluble enzymes); Membrane-bound and soluble benzylamine oxidases that are stable to MAO inhibitors but are highly labile towards semicarbazide and aminoguanidine and that predominantly oxidize benzylamine. The Km value for the soluble enzyme is 0.19 mM, its specific activity is 0.058 nmol aldehyde/min/mg protein, which markedly exceeds that for serum benzylamine oxidase (i.e., 0.014 nmol/min/mg) and thus excludes its serum origin; Diamine oxidase that oxidizes putrescine (Km = 0.025 mM), histamine and cadaverine and only slightly oxidizes benzylamine. One characteristic feature of the placenta is the presence of soluble MAO as well as MAO incorporated into the endoplasmic reticulum membrane (microsomes). In all probability, these enzymes are precursors of the mitochondrial enzyme. The concentration of MAO A in the mitochondria is approximately 1.3%, that in microsomes--approximately 1%, kcat = 270 and 320 min-1, respectively.
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A procedure for isolation of adenylate deaminase from duck heart muscle has been developed. The method includes extraction of enzyme, chromatography on cellulose phosphate, fractionation by ammonium sulfate, chromatography on Sephadex G-25 and ion-exchange chromatography on DEAE-cellulose. The enzyme was purified approximately 4000-fold with a yield of 25%. Electrophoresis in polyacrylamide gel revealed that the enzyme contains no proteins other than adenylate deaminase. The enzyme has a UV absorption spectrum typical for proteins which contain no nucleic acid impurities. Using sievorptive chromatography, it was shown that the myocardial extract contains two adenylate deaminase forms, which are tetramers with mol. weights of 190 000 and 240 000. The molecular weights of the subunits are 47 000 and 63 000, respectively. In the oligomeric form the enzyme is only detected at high enzyme concentrations and in the presence of large amounts of substrate.
The activation of purified adenylate deaminase from the duck myocardium by K+ is accompanied by modification of the substrate specificity and by the appearance of the capacity to deaminate adenosine and adenine. Adenosine deaminase activity originates at the concentration of K+ of 0.15 M that possesses the most stimulating effect on adenylate deaminase activity; with the increase of potassium ions concentration adenosine deaminating activity is enhanced as well, with a parallel reduction of Hill's constant. The PH-dependence, mode of inhibition by phosphate ions and the effect of alkaline metals suggests that adenosine deamination is carried out by natural adenylate deaminase active centres when their conformation is changed under the activator action.
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Catalytic and physico-chemical properties of human placental monoamine oxidases (MAO) were studied. Both forms of the enzyme, membrane-bound and soluble, exhibited similar properties: optimal activity at pH8.3, equal rate of inhibition with selective MAO inhibitors, identical substrate specificity (the best substrate--serotonin). Some specific differences were found only for cytoplasmic (soluble) MAO: lower affinity for substrates as compared with membrane-bound enzymes, reversible interaction with an inhibitor Lilly 51641 even after long-term preincubation, whereas the mitochondrial MAO bound Lilly 51641 irreversibly. The property of cytoplasmic MAO to form aggregates during storage and/or in gel filtration and concentration did not affect the main catalytic properties of soluble enzyme. Analysis of isoenzyme spectra of membrane-bound and cytoplasmic MAO by means of selective inhibitors and electrophoresis showed that MAO of the two types--A and B were detected in all the subcellular fractions studied. Subunits of MAO of the A type had molecular masses 62, 61 and 61 kDa and of MAO of the B type--51, 55 and 55 kDa in mitochondria, microsomes and cytosol, respectively.
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Oxidation of p-nitro- and p-dimethylaminomethyl derivatives of benzylamine, catalyzed by amine oxidases from human placenta and blood serum, was studied. The amine oxidase activity was estimated by means of a spectrophotometric procedure involving measurement of aldehyde formed during the reaction after extraction with hexane. For extraction of benzaldehyde and p-nitrobenzaldehyde in the samples HCl was added up to the concentration of 0.17 M and for extraction of p-dimethylaminomethyl benzaldehyde--NaHCO3 up to the 0.5 M concentration. The reaction products were extracted with a yield of 94%, 83% and 78% respectively; molar extinction coefficients for aldehydes at the maximal absorption were equal to 13,080 (241 mn), 16,520 (258 nm), and 11,547 (248 nm), respectively. Analysis of the oxidative reactions using inhibitors Lilly 51,641, deprenyl, aminoguanidine and semicarbazide showed that monoamine oxidase of the A type (95%) and benzylamine oxidase (7%) catalyzed oxidation of 0.1 mM p-nitrobenzylamine in mitochondria and microsomes but oxidation of the substrate at 1 mM concentration was catalyzed by monoamine oxidase of the B type (20% in mitochondria and 35% in microsomes). In the soluble fraction oxidation of p-nitrobenzylamine was catalyzed mainly by diamine oxidase (55%); monoamine oxidase of the A type catalyzed oxidation of 30% of the amine, benzylamine oxidase-15%. The molecular activity of the mitochondrial monoamine oxidase of the A type with p-nitrobenzylamine as a substrate was equal to 61 nmol of the product per 1 mole of the enzyme per 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)
A modified radiometric procedure is developed for estimation of amine oxidase activity. The method avoids withdrawal of samples in order to measure the radioactivity of reaction products. The enzymatic reaction, extraction of the products and measurement of the radioactivity were carried out in the same scintillation vials. Although the experimental error was shown to be higher as compared with the conventional procedure it did not interfere in the practical use of the modified procedure up to 0.1 microCi concentration of substrate per a sample. The modified procedure exhibited high efficiency in estimation of the amine oxidase activity in soluble fraction of human placenta. The variation coefficient of the method was 5.6%.
48 women with normal (38-40 weeks) and premature (38-37 weeks) labor were examined. The rate of deamination of serotonin, tyramine, beta-phenylethylamine, putrescine, cadaverine and histamine in samples containing extracts of the control group placenta averaged 0.86; 0.62; 0.18; 0.145; 0.63; 0.12 (nmoles NH3 per I mg of protein within I min), respectively. In the placental extracts obtained after the premature labor the rate of deamination of the substrates studied was decreased and constituted 0.4; 0.23; 0.108; 0.105; 0.29 and 0.084 nmoles NH3, respectively. The decrease in the rate of deamination of the amines studied, exhibiting high biological activity, appears to be responsible for premature labor. In the control group a correlation was found between the rates of serotonin and tyramine deamination as well as of putrescine and cadaverine deamination. The rates of deamination of mono- and diamines did not correlate. Deamination of beta-phenylethylamine and histamine did not depend also on deamination of other substrates studied. The data obtained demonstrate the presence in placenta of at least two forms of the enzymes, deaminating monoamines (one form for serotonin and tyramine and the other form for beta-phenylethylamine) as well as two forms of diamine oxidase - one form deaminating cadaverine and putrescine and the second form - histamine.
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