H.Robert Horton

Sulfhydryl oxidase-catalyzed conversion of xanthine dehydrogenase to xanthine oxidase☆

Abstract Xanthine oxidase may be isolated from various mammalian tissues as one of two interconvertible forms, viz., a dehydrogenase (NAD+ dependent, form D) or an oxidase (O2 utilizing, form O). A crude preparation of rat liver xanthine dehydrogenase (form D) was treated with an immobilized preparation of crude bovine sulfhydryl oxidase. Comparison of the rates of conversion of xanthine dehydrogenase to the O form in the presence and absence of the immobilized enzyme indicated that sulfhydryl oxidase catalyzes such conversion. These results were substantiated in a more definitive study in which purified bovine milk xanthine oxidase, which had been converted to the D form by treatment with dithiothreitol, was incubated with purified bovine milk sulfhydryl oxidase. Comparison of measured rates of conversion (in the presence and absence of active sulfhydryl oxidase and in the presence of thermally denatured sulfhydryl oxidase) revealed that sulfhydryl oxidase enzymatically catalyzes the conversion of type D activity to type O activity in xanthine oxidase with the concomitant disappearance of its sulfhydryl groups. It is possible that the presence or absence of sulfhydryl oxidase in a given tissue may be an important factor in determining the form of xanthine-oxidizing activity found in that tissue.

Catalytic effect of sulfhydryl oxidase on the formation of three-dimensional structure in chymotrypsinogen A.

Abstract The effect of sulfhydryl oxidase on the rate of disulfide bond formation and polypeptide chain folding in reductively denatured chymotrypsinogen A has been investigated using an immobilized zymogen preparation and a cylindrical quartz flow-through fluorescence cell. Enzymatic oxidation of the 10 sulfhydryl groups in reduced chymotrypsinogen followed first order kinetics at pH 7.0 with an apparent first order rate constant governing sulfhydryl group disappearance of 4.2 × 10−2 min−1. This provides a t 1 2 of 16.3 min for the sulfhydryl oxidase-catalyzed oxidation, whereas 165 min are required for nonenzymatic aerobic oxidation of one-half the sulfhydryl groups. Refolding of the reductively denatured polypeptide chains, monitored by changes in protein fluorescence, did not follow first order kinetics characteristic of a simple two-state mechanism, nor did the return of trypsin activatability. It appears that at least one intermediate must exist in such refolding, in both the uncatalyzed and sulfhydryl oxidase-catalyzed processes. Estimation of the rate constants governing refolding, assuming a single intermediate between the denatured and native states, provided values of 3 × 10−2 min−1 and 7 × 10−3 min−1 for uncatalyzed autoxidation and 4 × 10−2 min−1 and 1.1 × 10−2 min−1 for the sulfhydryl oxidase-catalyzed transition. Thus, enzymic catalysis of disulfide bond formation can lead to apparent catalysis of protein refolding as monitored both by fluorescence and by acquisition of biological function.

Tissue distribution of mammalian sulfhydryl oxidase

Sulfhydryl oxidase activity is present in cow, goat, sow, human, and rat milks, and can also be measured in several rat tissues following homogenization in 1% polyoxyethylene-9-lauryl ether. These include lactating mammary tissue, kidney, and pancreas. Bovine kidney homogenates also exhibit sulfhydryl oxidase activity; however, no activity could be detected in rat thymus, brain, heart, liver, spleen, lung, or small intestinal tissue homogenates. Indirect immunofluorescent staining of tissue sections using rabbit antibodies directed against highly purified bovine milk sulfhydryl oxidase preparations revealed that the enzyme is closely associated with the plasma membrane of lactating cow and rat mammary tissues and the basal-lateral membrane of rat kidney cortex. In addition, the oxidase appears to be associated with endothelial cells lining the capillaries of rat kidney, heart, and small intestine, and centroacinar cells in pancreatic tissue slices also stain for sulfhydryl oxidase. In contrast, liver, brain, and thymus tissues do not exhibit fluorescent staining and appear to be devoid of sulfhydryl oxidase activity.

Resolution of renal sulfhydryl oxidase from γ-glutamyltransferase by covalent chromatography on cysteinylsuccinamidopropyl-glass☆

Sulfhydryl oxidase from bovine kidney cortex was purified 2500-fold by covalent chromatography using cysteinylsuccinamidopropyl-glass. GSH oxidation catalyzed by the resulting preparation was found to be totally enzymatic, as judged by the inability of the preparation to reduce nitro blue tetrazolium, and H2O2 was found to be a product, as had been previously observed with milk sulfhydryl oxidase. No GSH peroxidase activity could be detected, using either H2O2 or t-butylhydroperoxide. The chromatographically purified renal sulfhydryl oxidase was resolved from γ-glutamyltransferase as evidenced by a 12,000-fold increase in ratio of the two enzymatic activities over that exhibited by crude kidney homogenates, and antibodies raised against purified milk sulfhydryl oxidase cross-reacted with the kidney oxidase, but not the kidney transferase.

Air-reoxidation of reduced, denatured chymotrypsinogen A covalently attached to a solid matrix.

Bovine chymotrypsinogen A was covalently attached to porous succinylglass beads via the zymogens amino groups. The bound zymogen was completely reduced in 8 M urea, then allowed to reoxidize, and activated to chymotrypsin. Comparison of the ko (catalytic coefficient) of this preparation with that of a similar preparation which had never been exposed to reductant showed a 53% recovery of esterolytic activity towards N-benzoyl-L-tyrosine ethyl ester. Values of Km and Ko for non-reduced, matrix-bound zymogen, following its activation to chymotrypsin, were 12.8 × 10−5 M and 11.0 sec−1, respectively. Corresponding values for reduced, air-reoxidized preparations were 6.8 × 10−5 M and 3.1 sec−1.

Nonidentity of sulfhydryl oxidase and γ-glutamyltransferase in bovine milk

Abstract Sulfhydryl oxidase (glutathione-oxidizing activity) is closely associated with γ-glutamyltransferase (γ-glutamyl transpeptidase) in skim milk membranes. Similar close association of the two enzymatic activities in kidney membranes has led to the recent proposal that glutathione-oxidizing activity can be attributed to the action of γ-glutamyltransferase, itself, in generating cysteinylglycine which, in turn, catalyzes sulfhydryl group oxidation ( O. W. Griffith and S. S. Tate, 1980, J. Biol. Chem. 255 , 5011–5014 ). However, a previously published procedure for the isolation of highly purified sulfhydryl oxidase from skim milk membranes ( V. G. Janolino and H. E. Swaisgood, 1975, J. Biol. Chem. 250 , 2532–2538 ) leads to the effective separation of the two activities. Quantitative chromatographic analyses of GSH, GSSG, and Glu levels revealed that the highly purified sulfhydryl oxidase preparation catalyzes the direct oxidation of GSH to GSSG without detectable cleavage of the γ-glutamyl peptide bond. These results were confirmed by monitoring the time course of substrate disappearance and product formation using high-performance liquid chromatography. Conversely, a supernatant fraction enriched in γ-glutamyltransferase activity displayed no sulfhydryl group-oxidizing activity. 6-Diazo-5-oxo- l -norleucine selectively inhibited the transferase in crude preparations containing both sulfhydryl oxidase and γ-glutamyltransferase. It is concluded that sulfhydryl oxidase and γ-glutamyltransferase activities are distinct and separable.

[34] Covalently bound glutamate dehydrogenase for studies of subunit association and allosteric regulation

Publisher Summary This chapter presents studies of subunit association and allosteric regulation using covalently bound glutamate dehydrogenase. The technique of enzyme immobilization by covalent attachment to a surface poses a means for investigating various perturbations of the quaternary structure of oligomeric enzymes. Covalent attachment of a single protomer per molecule of oligomeric enzyme could allow experimental separation of subunits, and thus could provide a means for directly measuring the regain of secondary and tertiary structures within protomers in the absence of complicating reassociation reactions. The chapter presents the studies with two oligomeric dehydrogenases—glutamate dehydrogenase and lactate dehydrogenase. It is reported that immobilization of glutamate dehydrogenase allows examination of the effect of allosteric ligands independent of association-dissociation interactions.

[9] Covalent immobilization of proteins by techniques which permit subsequent release

Publisher Summary Characterization of immobilized species has been restricted to kinetic studies or to examination of fluorescent spectra because of the limitations imposed by the immobilization matrix. Among the approaches that have been used are derivatizations that permit cleavage of the immobilized species from the matrix with aqueous hydroxylamine and those that permit cleavage through the reduction of disulfide bridges. This chapter discusses thionyl chloride-activated succinamidopropyl-glass as an immobilization matrix. Succinamidopropyl-glass can be prepared from porous or nonporous glass beads by either an aqueous or a nonaqueous derivatization procedure. The aqueous procedure results in a surface that is more hydrated and contains more silane polymers than that produced by the nonaqueous method. Although both procedures yield surfaces that will covalently immobilize proteins (or other suitable nucleophiles) following activation with thionyl chloride, and that will subsequently release immobilized proteins upon treatment with hydroxylamine, the fact that subtle differences in the surfaces are obtained should be borne in mind in selecting the best method for a specific application.

Reversible allosteric modulation of activity of immobilized hepatic glutamate dehydrogenase

Summary Bovine liver glutamate dehydrogenase was covalently bound to porous succinamidopropyl-glass beads. Like native enzyme, the immobilized enzyme was subject to activation by ADP and inhibition by GTP, but such allosteric modulation was found to be fully reversible and not dependent on the association-dissociation proclivities of the “active monomeric” species.

Purification and properties of sulfhydryl oxidase from bovine pancreas

Immunofluorescent studies showed that antibodies prepared against bovine milk sulfhydryl oxidase reacted with acinar cells of porcine and bovine pancreas. A close inspection of the specific location within bovine pancreatic cells revealed that the zymogen granules, themselves, bound the fluorescent antibody. Bovine pancreatic tissue was homogenized in 0.3 M sucrose, then separated into the zymogen granule fraction by differential centrifugation. The intact zymogen granules were immunofluorescent positive when incubated with antibodies to bovine milk sulfhydryl oxidase, and glutathione-oxidizing activity was detected under standard assay conditions. Pancreatic sulfhydryl oxidase was purified from the zymogen fraction by precipitation with 50% saturated ammonium sulfate, followed by Sepharose CL-6B column chromatography. Active fractions were pooled and subjected to covalent affinity chromatography on cysteinylsuccinamidopropyl-glass using 2 mM glutathione as eluant at 37 degrees C. The specific activity of bovine pancreatic sulfhydryl oxidase thus isolated was 10-20 units/mg protein using 0.8 mM glutathione as substrate. Ouchterlony double-diffusion studies showed that antibody directed against the purified bovine milk enzyme reacted identically with pancreatic sulfhydryl oxidase. The antibody also immunoprecipitated glutathione-oxidizing activity from crude pancreatic homogenates. Western blotting analysis indicated a 90,000 Mr antigen-reactive band in both bovine milk and pancreatic fractions while sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-staining protein with an apparent Mr 300,000. Thus, we believe that sulfhydryl oxidase may exist in an aggregated molecular form. Bovine pancreatic sulfhydryl oxidase catalyzes the oxidation of low-molecular-weight thiols such as glutathione, N-acetyl-L-cysteine, and glycylglycyl-L-cysteine, as well as that of a high-molecular-weight protein substrate, reductively denatured pancreatic ribonuclease A.