Chicken Liver Xanthine Dehydrogenase Research Articles

The Structure of Chicken Liver Xanthine Dehydrogenase: cDNA CLONING AND THE DOMAIN STRUCTURE

The amino acid sequence of chicken liver xanthine dehydrogenase (EC 1.1.1.204) was determined by cDNA cloning and partial amino acid sequencing of the purified enzyme. The enzyme consisted of 1358 amino acids with calculated molecular mass of 149,633 Da. In order to compare the structure of the chicken and rat enzymes, limited proteolysis was performed with the purified chicken liver xanthine dehydrogenase. When the enzyme was digested with subtilisin, it was not converted from the NAD-dependent dehydrogenase type to the O2-dependent oxidase type, in contrast with the mammalian enzyme. However, the enzyme was cleaved mainly into three fragments in a manner similar to that for the rat enzyme at pH 8.2 (20, 37, and 84 kDa) and retaining a full complement of redox centers. The cleavage sites were identified by determination of amino-terminal sequences of the produced fragments. It was concluded that the 20-kDa fragment was amino-terminal, the 84-kDa fragment carboxyl-terminal, and the 37-kDa fragment an intermediate portion in the enzyme protein. On the other hand, when the enzyme was digested with the same protease at pH 10.5, the sample contained only the 20- and 84-kDa portions and lacked the 37-kDa portion. The resultant sample possessed xanthine dichlorophenol indophenol reductase activity, indicating that the molybdenum center remained intact. The absorption spectrum showed the sample was very similar to deflavo-enzyme. From these results and sequence analyses, the domain structure of the enzyme is discussed.

Paradoxical Stabilization of the Neutral Flavin Semiquinone of Xanthine Dehydrogenase at High pH

The pH dependence of the behavior of chicken liver xanthine dehydrogenase in the course of reductive titrations with sodium dithionite has been examined. Below pH 8.5, the behavior of xanthine dehydrogenase is similar to that of the much better understood milk xanthine oxidase, with the amount of neutral semiquinone accumulating transiently in the course of the titration increasing somewhat as the pH decreases. At pH 10, however, an anomalously large accumulation of the neutral semiquinone is observed by both UV/visible and EPR spectroscopy. Treatment of xanthine dehydrogenase with the thiol reagent iodoacetamide significantly diminishes the ability of the enzyme to stabilize the neutral flavin semiquinone at high pH. These data are consistent with the presence of a protein thiol in the immediate vicinity of the flavin, whose ionization above pH 8.5 results in thermodynamic stabilization of the neutral flavin semiquinone over the anionic form.

Studies on the induction and phosphorylation of xanthine dehydrogenase in cultured chick embryo hepatocytes.

Chick embryo hepatocytes, cultured in a chemically defined medium, were used to investigate hormonal requirements for xanthine-dehydrogenase induction and to determine whether the enzyme is phosphorylated. Triiodothyronine is found to be required to induce the synthesis of active enzyme. Inclusion of sodium tungstate in the medium resulted in the complete loss of enzyme activity but no decrease of immunochemically detectable levels of enzyme. Immunoprecipitated xanthine dehydrogenase from cell extracts migrates with enzyme purified from adult chicken liver on SDS/PAGE. Both the native 150-kDa subunit and the 130-kDa form of the enzyme is observed. N-terminal sequence analysis of the 150-kDa subunit shows the following; Ala-Pro-Pro-Glu-Thr-Gly-Asp-Glu-Leu-Val-Phe-Phe-Val-Asn-Gly-Lys-Lys-Val- Val which is similar to the published N-terminal sequences of rat, mouse and insect xanthine dehydrogenases. Autoradiography of denaturing gels of xanthine dehydrogenase isolated from 32P(i)-labeled hepatocytes demonstrates that the 150-kDa and the 130-kDa forms of the enzyme are phosphorylated. Chemical phosphate analysis of acid-precipitated, electrophoretically pure chicken liver xanthine dehydrogenase also shows the presence of covalently bound phosphate. Phosphoamino acid analysis of both 32P-labeled forms of the enzyme demonstrates the presence of phosphoserine. Thus, chicken liver xanthine dehydrogenase contains a phosphoserine residue as found previously in bovine milk xanthine oxidase [Davis, M. D., Edmondson, D. E. & Müller, F. (1984) Eur. J. Biochem. 145, 237-250].

Experimental Strategy to Study the pH Dependence of the Kinetic Behavior of Enzymes: Practical Application to Chicken Liver Xanthine Dehydrogenase

An experimental strategy has been developed to analyze the pH dependence of kinetic parameters in two-substrate enzyme kinetics based upon model discrimination, parameter refinement, and optimal discrete design. The experimental protocol proposed includes obtaining reproducible experimental results in different sessions, replicates of each experimental determination, and rejection of outliers. This strategy leads to an empirical kinetic equation that fits the experimental results. As a practical application of this strategy we studied the pH dependence of kinetic parameters of chicken liver xanthine dehydrogenase. We obtained an empirical rate law from the determination of the initial rates at different substrate concentrations and pH. Finally, we have proposed a plausible kinetic mechanism that may explain the experimental findings. This model is not the only one leading to the empirical equation, but it is based on previous experimental findings (Bruguera, P., López-Cabrera, A., and Canela, E. I., 1988, Biochem. J. 249, 171-178).

Kinetic comparison of reduction and intramolecular electron transfer in milk xanthine oxidase and chicken liver xanthine dehydrogenase by laser flash photolysis

A comparative study using laser flash photolysis of the kinetics of reduction and intramolecular electron transfer among the redox centers of chicken liver xanthine dehydrogenase and of bovine milk xanthine oxidase is described. The photogenerated reductant, 5-deazariboflavin semiquinone, reacts with the dehydrogenase (presumably at the Mo center) in a second-order manner, with a rate constant (k = 6 x 10(7) M-1 s-1) similar to that observed with the oxidase [k = 3 x 10(7) M-1 s-1; Bhattacharyya et al. (1983) Biochemistry 22, 5270-5279]. In the case of the dehydrogenase, neutral FAD radical formation is found to occur by intramolecular electron transfer (kobs = 1600 s-1), presumably from the Mo center, whereas with the oxidase the flavin radical forms via a bimolecular process involving direct reduction by the deazaflavin semiquinone (k = 2 x 10(8) M-1 s-1). Biphasic rates of Fe/S center reduction are observed with both enzymes, which are due to intramolecular electron transfer (kobs approximately 100 s-1 and kobs = 8-11 s-1). Intramolecular oxidation of the FAD radical in each enzyme occurs with a rate constant comparable to that of the rapid phase of Fe/S center reduction. The methylviologen radical, generated by the reaction of the oxidized viologen with 5-deazariboflavin semiquinone, reacts with both the dehydrogenase and the oxidase in a second-order manner (k = 7 x 10(5) M-1 s-1 and 4 x 10(6) M-1 s-1, respectively). Alkylation of the FAD centers results in substantial alterations in the kinetics of the reaction of the viologen radical with the oxidase but not with the dehydrogenase. These results suggest that the viologen radical reacts directly with the FAD center in the oxidase but not in the dehydrogenase, as is the case with the deazaflavin radical. The data support the conclusion that the environments of the FAD centers differ in the two enzymes, which is in accord with other studies addressing this problem from a different perspective [Massey et al. (1989) J. Biol. Chem. 264, 10567-10573]. In contrast, the rate constants for intramolecular electron transfer among the Mo, FAD, and Fe/S centers in the two enzymes (where they can be determined) are quite similar.

Kinetic isotope effect studies on milk xanthine oxidase and on chicken liver xanthine dehydrogenase

The effect of isotopic substitution of the 8-H of xanthine (with 2H and 3H) on the rate of oxidation by bovine xanthine oxidase and by chicken xanthine dehydrogenase has been measured. V/K isotope effects were determined from competition experiments. No difference in H/T(V/K) values was observed between xanthine oxidase (3.59 +/- 0.1) and xanthine dehydrogenase (3.60 +/- 0.09). Xanthine dehydrogenase exhibited a larger T/D(V/K) value (0.616 +/- 0.028) than that observed for xanthine oxidase (0.551 +/- 0.016). Observed H/T(V/K) values for either enzyme are less than those H/T(V/K) values calculated with D/T(V/K) data. These discrepancies are suggested to arise from the presence of a rate-limiting step(s) prior to the irreversible C-H bond cleavage step in the mechanistic pathways of both enzymes. These kinetic complexities preclude examination of whether tunneling contributes to the reaction coordinate for the H-transfer step in each enzyme. No observable exchange of tritium with solvent is observed during the anaerobic incubation of [8-3H]xanthine with either enzyme, which suggests the reverse commitment to catalysis (Cr) is essentially zero. With the assumption of adherence to reduced mass relationships, the intrinsic deuterium isotope effect (Dk) for xanthine oxidation is calculated to be 7.4 +/- 0.7 for xanthine oxidase and 4.2 +/- 0.2 for xanthine dehydrogenase. By use of these values and steady-state kinetic data, the minimal rate for the hydrogen-transfer step is calculated to be approximately 75-fold faster than kcat for xanthine oxidase and approximately 10-fold faster than kcat for xanthine dehydrogenase. This calculated rate is consistent with data obtained by rapid-quench experiments with XO. A stoichiometry of 1.0 +/- 0.3 mol of uric acid/mol of functional enzyme is formed within the mixing time of the instrument (5-10 ms). The kinetic isotope effect data also permitted the calculation of the Kd values [Klinman, J. P., & Mathews, R. G. (1985) J. Am. Chem. Soc. 107, 1058-1060] for substrate dissociation, including all reversible steps prior to C-H bond cleavage. Values calculated for each enzyme (Kd = 120 microM) were found to be identical within experimental uncertainty.

Differences in Protein Structure of Xanthine Dehydrogenase and Xanthine Oxidase Revealed by Reconstitution with Flavin Active Site Probes

The native flavin, FAD, was removed from chicken liver xanthine dehydrogenase and milk xanthine oxidase by incubation with CaCl2. The deflavoenzymes, still retaining their molybdopterin and iron-sulfur prosthetic groups, were reconstituted with a series of FAD derivatives containing chemically reactive or environmentally sensitive substituents in the isoalloxazine ring system. The reconstituted enzymes containing these artificial flavins were all catalytically active. With both the chicken liver dehydrogenase and the milk oxidase, the flavin 8-position was found to be freely accessible to solvent. The flavin 6-position was also freely accessible to solvent in milk xanthine oxidase, but was significantly less exposed to solvent in the chicken liver dehydrogenase. Pronounced differences in protein structure surrounding the bound flavin were indicated by the spectral properties of the two enzymes reconstituted with flavins containing ionizable -OH or -SH substituents at the flavin 6- or 8-positions. Milk xanthine oxidase either displayed no preference for binding of the neutral or anionic flavin (8-OH-FAD) or a slight preference for the anionic form of the flavin (6-hydroxy-FAD, 6-mercapto-FAD, and possibly 8-mercapto-FAD). On the other hand, the chicken liver dehydrogenase had a dramatic preference for binding the neutral (protonated) forms of all four flavins, perturbing the pK of the ionizable substituent greater than or equal to 4 pH units. These results imply the existence of a strong negative charge in the flavin binding site of the dehydrogenase, which is absent in the oxidase.

The Nicotinamide Adenine Dinucleotide-Binding Site of Chicken Liver Xanthine Dehydrogenase: Evidence for alteration of the redox potential of the flavin by NAD binding or modification of the NAD-binding site and isolation of a modified peptide

Abstract Affinity labeling of the NAD-binding site of chicken liver xanthine dehydrogenase by 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA) caused spectral perturbation around 450 nm in the same way as NAD. Reductive titration with xanthine of native xanthine dehydrogenase in the presence of NAD showed that redox potentials of the FAD/FADH. and FADH./FADH2 couples were shifted positive by NAD binding to the enzyme. The redox potentials of these couples were also shifted to some extent by modification of the NAD-binding site with 5'-FSBA. These results provide further evidence that binding of NAD to chicken liver xanthine dehydrogenase modulates the reactivity of the enzyme by shifting the redox potential of FAD. Proteolytic cleavage of the [14C]-5'-FSBA-modified enzyme yielded several domain peptides, only one of which contained radioactivity. The isolated radioactive peptide was further digested with Staphylococcus aureus protease and the 14C-labeled peptide was purified by two steps of high performance liquid chromatography. The amino acid sequence of the peptide was determined, and a reactive tyrosine residue was identified.

Reactivity of Chicken Liver Xanthine Dehydrogenase Containing Modified Flavins

Native FAD was removed from chicken liver xanthine dehydrogenase (XDH) and replaced with a number of artificial flavins of different redox potential. Dithionite titration of the 2-thio-FAD- or 4-thio-FAD (high potential)-containing enzymes showed that the first center to be reduced was the flavin. With native enzyme, iron-sulfur centers are the first to be reduced. With the low potential flavin, 6-OH-FAD, the enzyme-bound flavin was the last center to be reduced in reductive titration with xanthine. These shifts in the reduction profile support the hypothesis that the distribution of reducing equivalents in multi-center oxidation-reduction enzymes of this type is determined by the relative potentials of the centers. The reaction of molecular oxygen with fully reduced 2-thio-FAD XDH or 4-thio-FAD XDH resulted in 5 electron eq being released in a fast phase and one in a slow phase. Reduction of these enzymes by xanthine was limited at a rate comparable to that for the release of urate from native XDH. Xanthine/O2 turnover with these enzymes (and native XDH) resulted in approximately 40-50% of the xanthine reducing equivalents appearing as superoxide. Steady state turnover experiments involving all modified flavin-containing enzymes, as well as native enzyme, showed that shifting the flavin potential either positive or negative relative to FAD caused a decrease in catalytic activity in the xanthine/NAD reductase reaction. In the case of the xanthine/O2 reductase activity, there is no simple obvious relationship between the activity and the redox potential of the reconstituted flavin.

The reactivity of chicken liver xanthine dehydrogenase with molecular oxygen

The reaction of 6-electron reduced chicken liver xanthine dehydrogenase (XDH) with molecular oxygen was studied using both stopped flow and steady-state turnover techniques at pH 7.8, 4 degrees C. Oxidation of fully reduced XDH proceeded via four phases, three of which were detected with the stopped flow spectrophotometer. The fastest phase was second order in oxygen (1900 M-1 s-1), resulted in the appearance of flavin semiquinone and yielded no superoxide. The next phase was also second order in oxygen (260 M-1 s-1), involved the loss of flavin semiquinone and yielded, on average, 1 mol of superoxide/mol of XDH oxidized. The last 2 electron equivalents were located in the iron-sulfur centers. They were released one equivalent at a time in the form of superoxide. Steady-state kinetics were found to be critically dependent on temperature and oxygen concentration. When these factors were carefully controlled, both the xanthine-oxygen and NADH-oxygen reductase reactions gave linear Lineweaver-Burk plots. The xanthine-oxygen data yielded a turnover number of 43 min-1, which was 42% of that for xanthine-NAD turnover. During turnover, with xanthine and O2, 40-44% of the electron equivalents introduced by xanthine appeared as superoxide. Reduced pyridine nucleotides, NAD and 3-aminopyridine adenine dinucleotide, dramatically reduced the formation of superoxide at levels which did not seriously inhibit oxygen reactivity.

Rapid reaction studies on the reduction and oxidation of chicken liver xanthine dehydrogenase by the xanthine/urate and NAD/NADH couples.

Chicken liver xanthine dehydrogenase can be partially reduced by either xanthine or NADH. Reduction to approximately the 2-electron-reduced level occurs with NADH, and reduction beyond the 2-electron level occurs with xanthine. In both cases, the reaction is triphasic. The first and third phases are dependent on reductant concentration, whereas the second phase is not. Oxidation of fully (6-electron) reduced xanthine dehydrogenase by either urate or NAD is monophasic and dependent on the oxidant concentration. Oxidation stops at about the same level of reduction that was reached by the corresponding reductant. The position of this end point is sensitive to the potential of the reactants but is relatively insensitive to excess concentrations of oxidant or reductant. NADH binding to 2-electron-reduced enzyme is implicated in fixing the end point position in those reactions involving pyridine nucleotides, whereas urate binding is involved in fixing the end point of those reactions involving xanthine and urate.

53 LOCALIZATION OF XANTHINI DEHYDROGENASE IN LIVER

Xanthine dehydrogenase has been reported to be present in an endothelial cell in beef or rat liver and also in peroxisome of a hepatic cell. However, as the most enzyme was recovered in 100,000xg supernatant of rat or chicken liver homogenate, cellular and subcellular localization in liver are still obscure. We reexamined localization of xanthine dehydrogenase in the tissue of chicken liver by imrnunohistochemical method, or by the method of isolation of hepatic cells. Chicken liver xanthine dehydrogenase purified by the method of Nishino was used for the immunization of rabbit. The antisera obtained from the rabbit gave a distinct single precipitin line in agar against chicken liver xanthine dehydrogenase or crude extract. This antisera were used for immunohistochemical experiments. Chicken liver were perfused with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin and sectioned. They were stained with the indirect method using enzyme labeled antibody. The positive staining was observed in the cytoplasm of hepatocytes, Kupffer cells and endothelial cells, suggesting that these type cells contain xanthine dehydrogenase. After isolation of hepatocytes most of xanthine dehydrogenase was recovered in 100,000xg supernatant fraction from homogenate of hepatocytes and the specific activity of enzyme was similar to that in 100,000xg supernatant fraction from homogenate of liver.

Kinetic mechanism of chicken liver xanthine dehydrogenase.

The kinetic behaviour of chicken-liver xanthine dehydrogenase (xanthine/NAD+ oxidoreductase; EC 1.2.1.37) has been studied. Steady-state results, obtained from a wide range of concentrations of substrates and products, were fitted by rational functions of degree 1:1, 1:2, 2:2 and 3:3 with respect to substrates, and 0:1, 1:1, 0:2 and 1:2 with regard to products, using a non-linear regression program which guarantees the fit. The goodness of fit was improved using a computer program that combines model discrimination, parameter refinement and sequential experimental design. The AIC and F tests were also used for model discrimination. For comparative purposes, the xanthine/oxygen oxidoreductase reaction was also studied. From the functions which give the maximum improvement, the complete rate equation was deduced. The significance of the terms was stated by the above methods. It was concluded that xanthine dehydrogenase requires a minimum mechanism of degree 1:1 for xanthine, 2:2 for NAD+, 1:1 for uric acid and 1:2 for NADH in the xanthine/NAD+ oxidoreductase reaction. These are the minimum degrees required but a rate equation of higher degree is not excluded.

The influence of ammonium ions on the aggregation of chicken liver xanthine dehydrogenase

Conference Article| October 01 1987 The influence of ammonium ions on the aggregation of chicken liver xanthine dehydrogenase ANTONIO LOPEZ CABRERA; ANTONIO LOPEZ CABRERA 1Department of Biochemistry and Phisiology, University of Barcelona, Marti Franquès, 1, 08028 Barcelona, Catalunya, Spain Search for other works by this author on: This Site PubMed Google Scholar PAU BRUGUERA; PAU BRUGUERA 1Department of Biochemistry and Phisiology, University of Barcelona, Marti Franquès, 1, 08028 Barcelona, Catalunya, Spain Search for other works by this author on: This Site PubMed Google Scholar ENRIC I. CANELA ENRIC I. CANELA 1Department of Biochemistry and Phisiology, University of Barcelona, Marti Franquès, 1, 08028 Barcelona, Catalunya, Spain Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1987) 15 (5): 883–884. https://doi.org/10.1042/bst0150883 Article history Received: March 24 1987 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation ANTONIO LOPEZ CABRERA, PAU BRUGUERA, ENRIC I. CANELA; The influence of ammonium ions on the aggregation of chicken liver xanthine dehydrogenase. Biochem Soc Trans 1 October 1987; 15 (5): 883–884. doi: https://doi.org/10.1042/bst0150883 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: PAGE, polyacrylamide gel electrophoresis This content is only available as a PDF. © 1987 Biochemical Society1987 Article PDF first page preview Close Modal You do not currently have access to this content.

Evidence for the existence of a tyrosyl residue in the nicotinamide adenine dinucleotide binding site of chicken liver xanthine dehydrogenase.

Xanthine-NAD and NADH-methylene blue oxidoreductase activities of chicken liver xanthine dehydrogenase were inactivated by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA), an active site directed reagent for nucleotide binding sites. The inactivation reaction displayed pseudo-first-order kinetics. A double-reciprocal plot of inactivation velocity vs. 5'-FSBA concentration showed that 5'-FSBA and enzyme formed a complex prior to inactivation. NAD protected the enzyme from inactivation by 5'-FSBA in a competitive fashion. The modified enzyme had the same xanthine-dichlorophenolindophenol and xanthine-O2 oxidoreductase activities as the native enzyme, and on addition of xanthine to the modified enzyme, bleaching of the spectrum occurred in the visible region. The amount of radioactivity incorporated into the enzyme by incubation with [14C]-5'-FSBA was parallel to the loss of xanthine-NAD oxidoreductase activity, and the stoichiometry was 1 mol/mol of enzyme-bound FAD for complete inactivation. These results indicated that 5'-FSBA modified specifically the binding site for NAD of chicken liver xanthine dehydrogenase. The incorporated radioactivity was released slowly from 14C-labeled enzyme by incubation with dithiothreitol with concomitant restoration of catalytic activity. The modified residue responsible for inactivation was identified as a tyrosine.

The effects of pH on chicken liver xanthine dehydrogenase

The effects of pH on chicken liver xanthine dehydrogenase

Functional groups and quaternary structure of chicken liver xanthine dehydrogenase

Xanthine dehydrogenase from chicken liver is a dimeric enzyme, each hemimolecule containing one FAD and two Fe/S groups. Determination of sulfhydryl groups with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuribenzoic acid (PMB) showed a variable number of sulfhydryl groups depending onpH, ionic strength, and nature of the reaction medium and buffer. The number of disulfide bonds was determined with DTNB and reducing conditions. Amino groups were determined with 2,4,6,-trinitrobencensulfonic acid (TNBS). At constant temperature andpH the reaction of DTNB and TNBS with native xanthine dehydrogenase showed an exponential dependence on time. From the obtained parameters the number of available sulfhydryl and amino groups at infinite concentration of enzyme and the rate constant of the equation were determined. The absorption spectrum of the enzyme changed with time when a chaotropic agent (1 M sodium nitrate) was added to the medium. This difference was detected by measuring the absorbance in the range 450–550 nm. The absorption spectrum (between 350 and 600 nm) also changed when a denaturating agent (sodium dodecyl sulfate) was added. This modification increased with time and depended on the medium.

Subcellular localization of chicken liver xanthine dehydrogenase. A possible source of cytoplasmic reducing equivalents.

Classical fractionation studies showed that xanthine dehydrogenase (EC 1.2.1.37) was exclusively cytosolic in chicken liver. Fumarase (EC 4.2.1.2) and malate dehydrogenase (EC 1.1.1.37) were also found to have major cytosolic locations. These data indicate that urate synthesis in chicken liver produces substantial quantities of cytoplasmic NADH which may supply reducing equivalents of gluconeogenesis and other processes.

Evidence for the inorganic nature of the cyanolyzable sulfur of molybdenum hydroxylases.

Activation of the desulfo forms of milk xanthine oxidase, chicken liver xanthine dehydrogenase, and aldehyde oxidase with S2- is greatly facilitated in the presence of reducing agents. Upon anaerobic incubation with 1 mM S2- and 1 mM dithionite, desulfo xanthine oxidase and chicken liver xanthine dehydrogenase prepared by cyanide treatment of active enzymes, are activated to the specific activity predicted by their molybdenum content. Routine preparations containing desulfo molecules are also similarly activated to the extent predicted. Cyanide-inactivated chicken liver xanthine dehydrogenase was reconstituted with 35S2- in the presence of dithionite. 85% of enzyme-bound radioactivity was shown to be in the form of cyanolyzable sulfur, by comparison of enzyme activity, bound radioactivity, and 35SCN- yields from exposure of labeled enzyme to cyanide. This radiolabeled enzyme allowed the determination of the following. 1) The cyanolyzable sulfur is largely removed from the polypeptide by incubation at 37 degrees C for one hour in 1% sodium dodecyl sulfate, pH 7, or for 15 min in 6 M guanidinium chloride, pH 6.2. 2) The cyanolyzable sulfur is "acid labile." [35S]Methylene blue is formed in the theoretical quantity from oxidized or substrate-reduced enzyme under the standard conditions for labile sulfur analysis by the methylene blue method. These data strongly support the conclusion that the cyanolyzable sulfur is a terminal sulfur ligand of the Mo atom, and is not part of an organic moiety.

Comparison of the kinetic behaviour of xanthine oxidase and xanthine dehydrogenase

In the oxidation of hypoxanthine and xanthine by chicken liver xanthine dehydrogenase and bovine liver and milk xanthine oxidase, activation appears at high substrate concentrations. Hypoxanthine concentrations greater than 0.04 mM inhibit the bovine enzyme; concentrations greater than 0.09 mM inhibit the milk enzyme. On the other hand, uric acid is a mixed non-competitive inhibitor of bovine liver xanthine dehydrogenase. The phosphate (Na + or K +) and chloride (Na + or K +) ions do not affect bovine live xanthine oxidase activation, but modify the K and V values.