Turkey liver xanthine dehydrogenase. Relation between nicotinamide-adenine dinucleotide oxidoreductase activity and the content of functional enzyme.

The turkey liver xanthine dehydrogenase-catalysed oxidation of NADH by Methylene Blue, by ferricyanide or by O2 is not dependent on the integrity of the active-centre persulphide groups. By contrast, the NADH-dichlorophenol-indophenol oxidoreductase and NADH-trinitrobenzenesulphonate oxidoreductase activities are directly proportional to the content of functional enzyme. These findings help to identify the sites of egress of electrons to oxidizing substrates.     

Turkey liver xanthine dehydrogenase. Reactivation of the cyanide-inactivated enzyme by sulphide and by selenide

1. The glycogen present in the liver of rat foetuses was labelled by injecting a trace amount of [6-(3)H]glucose into the mother at 19.5 days of gestation. The radioactivity incorporated in the glycogen 4h after the administration of the label was still present 38h later. A large proportion of this radioactivity was on the outer chains of the polysaccharide. These results indicate that there is normally almost no glycogen degradation in the foetal liver. In contrast, glycogen breakdown occurs very rapidly in the livers of foetuses whose mother is anaesthetized. 2. Glycogen synthetase is present in the liver at day 16 of gestation at a concentration as high as 30% of that in the adult, but essentially as an inactive (b) enzyme. The appearance of synthetase phosphatase between days 18 and 19 corresponds to that of synthetase a and to the beginning of glycogen synthesis. From day 19 to 21.5 the amount of synthetase a present in the foetal liver is just sufficient to account for the actual rate of glycogen deposition. 3. The content of total phosphorylase in the foetal liver increases continuously from day 16 to birth. However, a precise measurement of the a and b forms of the enzyme in the liver of non-anaesthetized foetuses is not possible. Taking the rate of glycogenolysis as an appropriate index of phosphorylase activity, we conclude that this enzyme is almost entirely in the inactive form in the foetal liver under normal conditions. 4. The accumulation of glycogen in the liver during late pregnancy may therefore be explained by a relatively slow rate of synthesis and a nearly total absence of degradation.     

Histidine residues and the enzyme activity of pig heart supernatant malate dehydrogenase (Short Communication)

1. Supernatant pig heart malate dehydrogenase is completely inhibited by reaction with diethyl pyrocarbonate at pH6.5, when 0.58+/-0.1 residue of ethoxycarbonylhistidine is formed per NADH-binding site. 2. Oxaloacetate and hydroxymalonate protect the enzyme from inhibition in the absence of coenzyme. 3. Limited ethoxycarbonylation does not alter the binding of NADH to the enzyme but prevents the enzyme-NADH complex from interacting with hydroxymalonate in a ternary complex.     

Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)]

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.     

Direct measurement of proton binding to the active ternary complex of pig heart lactate dehydrogenase (Short Communication)

With the use of a glass electrode it is shown that one proton is taken up from the solvent when o-nitrophenylpyruvate forms a ternary complex with lactate dehydrogenase and NADH at pH8 and before the complex has rearranged to give products.     

Aging effects on the liver aldolase of rabbits (Short Communication)

A chemical method is used to determine the amount of aldolase sequence in rabbit livers. It is demonstrated that the aldolase sequences of the livers of young rabbits are more efficient catalytically than are those from the livers of old rabbits. The presence of altered residues in the aldolase sequences of old animals may account for these observations.     

Purification of xanthine dehydrogenase from rat liver: a rapid procedure with high enzyme yields

Xanthine dehydrogenase (EC 1.2.1.37) was purified approximately 1000-fold from liver homogenates of adult male Sprague-Dawley rats. Enzyme recovery was good (greater than 20% of the starting activity was obtained), and the homogeneously pure enzyme had a molecular mass of approximately 300,000 Da. The purified protein exhibited a specific activity of 2470 units/mg protein and spectral properties identical to those of the best preparations of this enzyme reported by other investigators. Routine preparations of this enzyme also possess higher dehydrogenase:oxidase ratios (typically between 5 and 6) than do other xanthine dehydrogenase preparations so far reported in the literature. Maximum dehydrogenase:oxidase ratios, greater than 10, could be obtained from this procedure if only peak dehydrogenase fractions from the chromatography columns were saved. The present small-scale purification method, which can be completed in 48-60 h, utilizes ammonium sulfate fractionation, Sephadex G-200 column chromatography, Blue Dextran-Sepharose column chromatography, and preparative gel electrophoresis.     

Structural and functional peculiarities of alpha-ketoglutarate dehydrogenase with non-interacting active sites

The properties of alpha-ketoglutarate dehydrogenase with non-interacting active sites were investigated. The substrate and coenzyme saturation curves are found to be hyperbolic, which is consistent with the absence of cooperativity between the active sites of the enzyme. The peculiarities of KGD of this form, determining its functional properties, were revealed. Thus, 6 cysteine residues of the enzyme possess different properties in comparison with the form of the enzyme with interacting active sites. 3 Sulfhydryl groups of the "non-cooperative" enzyme form were rapidly oxidized in the process of the enzyme isolation and storage; thereafter they could not be reduced by dithiols. Three other cysteine residues are probably involved in the formation of disulfide bonds. Two of them are supposed to form intramolecular disulfide, whereas the third one is thought to be modified by some low molecular weight disulfide. The reduction of these sulfhydryl groups by dithiols is shown to be accompanied by the appearance of the kinetic cooperativity with respect to the substrate. It is suggested that the thiol/disulfide exchange in vivo can regulate a reversible conversion of the "non-cooperative" KGD form into one with interacting sites.     

Purification of rat liver S-adenosyl-l-methionine decarboxylase (Short Communication)

The amount of S-adenosyl-l-methionine decarboxylase present in rat liver was enhanced 17-fold by administration of methylglyoxal bis(guanylhydrazone),* a specific inhibitor of the enzyme. The enzyme was purified 1400-fold in 50% yield from such liver extracts by chromatography on columns of the inhibitor bound to Sepharose. The purified enzyme had no spermidine synthetase activity.     

Nonlinear kinetics of the xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase

The xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase has been shown to give nonlinear kinetics of the type which has been identified as substrate activation. When a very wide range of substrate (pteridine) concentrations were studied, it was found that a downward deflection in reciprocal plots (substrate activation) occurs in the high region and an upward deflection in the very low region. When product (isoxanthopterin) was included in reaction mixtures, the upward deflection was enhanced and shifted to higher substrate concentration ranges. In addition, reciprocal plots with a second substrate (oxygen) and a product (isoxanthopterin) were nonlinear.     

Kinetic equivalence of the active sites of alcohol dehydrogenase from horse liver.

The reduction, catalysed by liver alcohol dehydrogenase, of benzaldehyde in the presence and absence of pyrazole, and the oxidation of benzyl alcohol and cyclohexanol in the presence of isobutyramide, has been measured by the stopped-flow technique. In performing these experiments particular care was taken to purify the enzyme, coenzymes, substrates and inhibitors, and to minimise as much as possible the effects of a blank substrate reaction. The calculation of the amount of substrate converted to product during the various phases of the transient process was based on the absorption coefficients for the enzyme-coenzyme and enzyme-coenzyme-inhibitor complexes determined in the absence of substrate. The results show that the two active sites of liver alcohol dehydrogenase are kinetically equivalent and that the enzyme does not exhibit half-of-the-sites reactivity.     

Distribution of reducing equivalents on xanthine oxidase molecules and the rates of the intramolecular electron-transfer reactions (Short Communication)

When xanthine oxidase turns over 1-methylxanthine aerobically at pH8.2, the time-sequence in development of its electron-paramagnetic-resonance signals is not primarily due to slow intramolecular reactions among its centres. It derives instead from gross differences in electron distribution within enzyme molecules reduced by the substrate in comparison with those that have subsequently been partly reoxidized by one-electron reaction with oxygen.     

The presence of desulfo xanthine dehydrogenase in purified and crude enzyme preparations from rat liver

Crude and purified xanthine dehydrogenase preparations from rat liver were examined for the existence of a naturally occurring inactive form. Reduction of the purified enzyme by xanthine under anaerobic conditions proceeded in two phases. The enzyme was inactivated by cyanide, which caused the release of a sulfur atom from the molybdenum center as thiocyanate. The amount of thiocyanate released was almost in parallel with the initial specific activity. The active and inactive enzymes could be resolved by affinity chromatography on Sepharose 4B/folate gel. These results provided evidence that the purified enzyme preparation from rat liver contained an inactive form. A method for the determination of the active and inactive enzymes in crude enzyme preparations from rat liver was devised based on the fact that only active enzyme could react with [14C]allopurinol and both active and inactive enzymes could be immunoprecipitated quantitatively by excess specific antibody to xanthine dehydrogenase. The amount of [14C]alloxanthine (derived from [14C]allopurinol) bound to the active sulfo enzyme in crude rat liver extracts was about 0.5 mol/mol of FAD. As this content is closely similar to that in the purified enzyme, these results suggest the existence of an inactive desulfo form in vivo.     

Proteolytic conversion of xanthine dehydrogenase from the NAD-dependent type to the O2-dependent type. Amino acid sequence of rat liver xanthine dehydrogenase and identification of the cleavage sites of the enzyme protein during irreversible conversion by trypsin

The primary structure of rat liver xanthine dehydrogenase (EC 1.1.1.204) was determined by sequence analysis of cDNA and purified enzyme. The enzyme consists of 1,319 amino acid residues with a calculated molecular mass of 145,034 Da, including initiation methionine, and is homologous to the previously reported Drosophila melanogaster enzyme (Lee, C. S., Curtis, D., McCarron, M., Love, C., Gray, M., Bender, W., and Chovnick, A. (1987) Genetics 116, 55-66; Keith, T. P., Riley, M. A., Kreitman, M., Lewontin, R. C., Curtis, D., and Chambers, G. (1987) Genetics 116, 67-73) with an identity of 52%. The enzyme exists originally as the NAD-dependent type in a freshly prepared sample. When the purified NAD-dependent type enzyme was digested with trypsin, it cleaved into three fragments with molecular masses of 20, 40, and 85 kDa and was irreversibly converted to the O2-dependent type. Comparison of the amino-terminal sequences of the three peptide fragments with the cDNA-deduced sequence reveals that the 20-, 40-, and 85-kDa peptide fragments correspond residues to 1-184, 185-539, and 540-1319 of the enzyme, respectively. Comparison of the 5'-p-fluorosulfonylbenzoyladenosine-labeled peptide sequence of the chicken enzyme (Nishino, T., and Nishino, T. (1989) J. Biol. Chem. 264, 5468-5473) reveals that the NAD binding site is associated with the 40-kDa fragment portion of the enzyme. Hydropathy analysis around the cysteine residues suggests that the 2Fe/2S sites are associated with the 20-kDa fragment portion of the enzyme.     

Induction of cystathionase in human foetal liver (Short Communication)

Incubation of liver explants from second-trimester human foetuses with dexamethasone, glucagon or dibutyryl cyclic AMP (plus theophylline) increased the activity of liver cystathionase from unmeasurable or trace values to adult values. Simultaneous incubation with cycloheximide or actinomycin D inhibited this effect.