IN-VITRO AND IN-VIVO REGULATION OF ASSIMILATORY NITRITE REDUCTASE FROM CANDIDA-UTILIS

The nitrate assimilation pathway in Candida utilis, as in other assimi latory organisms, is mediated by two enzymes: nitrate reductase and ni trite reductase. Purified nitrite reductase has been shown to be a het erodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and N ADPH as electron donors. FAD, which is an essential coenzyme, stabilis ed the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thio l reagents. One cysteine was demonstrated to be essential for the enzy matic activity. In vitro, the enzyme was inactivated by ammonium salts , the end product of the path way, proving that the enzyme is assimila tory in function. In vivo, the enzyme was induced by nitrate and repre ssed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated to gether, which indicated that the primary level of regulation of this e nzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells g rown with the same salts as the source of nitrogen, the residual enzym atic activities were similar. Thus, a study of the in vitro inactivati on can give a clue to understanding the mechanism of in vivo regulatio n of nitrite reductase in Candida utilis.