• BMB Reports
• /
• 제29권4호
• /
• pp.335-342
• /
• 1996

Porcine liver cysteinesulfinic acid decarboxylase was purified approximately 460-fold by means of ammonium sulfate fractionation and sequential column chromatographic separation with Sephadex G-100, DEAE-cellulose and hydroxylapatite. The enzyme has a flat pH profile with maximum activity occurring between pH 6.0 and 7.6. Pyridoxal 5'-phosphate must be present in all buffers used for purification procedures in order to stabilize the enzyme. Addition of sulfhydryl reagents such as 2-mercaptoethanol are also necessary to maintain maximum enzyme activity throughout purification. The absorption spectrum shows that cysteinesulfinic acid decarboxylase is a pyridoxal 5' -phosphate-containing protein. The major absorption is at 280 nm with two smaller absorption regions, one at 425 nm which is ascribed to a Schiffs base between pyridoxal phosphate and protein, and another at 325 nm which is thought to be due to the interaction of 2-mercaptoethanol with the Schiffs base. A number of divalent cations tested did not affect enzyme activity with the exception of mercury, copper, and zinc which are inhibitory. The partially purified enzyme has an apparent $K_m$ of 0.94 mM for cysteinesulfinate. Cysteic acid is a competitive inhibitor of the enzyme with a $K_i$ of 1.32 mM. The molecular weight of the enzyme was estimated to be about 79,600 by using Sephadex G-200 column chromatography.