Properties Of The Protein

The molecular weight of sulfhydryl oxidase determined with SDS-PAGE is 89 kDa (1). A similar value was obtained from light-scattering studies of a substantially purified preparation in 5 M guanidi-nium chloride (21, 22). Like other membrane proteins, preparations of the enzyme in the absence of dissociating agents are highly aggregated; thus, the size of the active protein as it exists in the membrane has not been established. The enzyme is present in skim milk in lipid vesicles > 0.3 pm in diameter as indicated by their exclusion from Nucleopore filters (21) and their limited penetration of 0.3-pm-diameter controlled-pore glass (3, 23). Active enzyme can be solubilized with nonionic detergents. Polyoxyethylene-9-lauryl ether (Cj2E9) and ft-octyl-D-glucoside are best suited for this purpose (2). More than 80% of the activity is solubilized in 1% Cj2E9; however, the complex is still quite large having limited ability to penetrate 7.5% polyacrylamide gels (2). Nevertheless, all of the solubilized enzyme was able to penetrate 0.1-pm-diameter controlled-pore glass (3). Dissociation of the aggregated enzyme with either nonionic detergent or 1 M guanidinium chloride results in increased activity (3, 21).

Analysis of a substantially purified form of the enzyme indicated that it contains 11% carbohydrate and 0.5 g-atoms of Fe/89,000. Moreover, treatment with EDTA caused removal of the Fe and complete loss of activity (1). Subsequent dialysis of the apopro-tein against dilute solutions of Fe2+ restored 70% of the original activity. However, treatment with other divalent metals did not restore any activity, with the exception of Cu2+ which yielded - 30% of the original activity (1). The active enzyme contains two DTNB-reactive sulfhydryl groups/89,000, whereas the denatured protein contains three reactive groups (1, 24). Kinetics of activity loss due to carboxymethylation with iodoacetate indicated that modification of one sulfhydryl group/89,000 caused complete inactivation (24).

That sulfhydryl oxidase activity and y-glutamyl-transferase activity arise from distinct proteins was shown by resolution of the proteins by transient cova-lent chromatography on cysteinylsuccinamidopropyl glass and by specific immunoprecipitation of sulfhy-dryl oxidase activity (25, 26). Furthermore, the activities of sulfhydryl oxidase, y-glutamyltransferase, xanthine oxidase, and alkaline phosphatase in deter-gent-solubilized skim milk membranes were resolved by isoelectric focusing (3).

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