Abstract
Abstract Addition of high levels of sodium molybdate (10-2 m, final concentration) to acid-treated purified bovine milk xanthine oxidase prior to incubation with an extract of the nitrate-induced nitrate reductase Neurospora crassa mutant nit-1 resulted in a significant enhancement of the subsequent in vitro assembly of wild type N. crassa NADPH-nitrate reductase (EC 1.6.6.2). Added molybdate had no effect on the wild type enzyme itself. The enhancement is highly specific for molybdenum, is independent of added phosphate, and also occurs with cell-free preparations of uninduced wild type and with other known acid-treated molybdenum enzymes including chicken liver xanthine dehydrogenase, rabbit liver aldehyde oxidase, bovine sulfite oxidase, and the nitrogenases of Azotobacter and Clostridium. Preincubation at 23° of acid-treated xanthine oxidase preparations resulted in a considerable loss of ability to form nitrate reductase and in a more striking enhancement by subsequently added molybdate. Prior addition of molybdate during the above 23° preincubation prevented in part the loss of the ability of the enzyme to form nitrate reductase, suggesting that the enhancement by molybdate is a restoration effect rather than a stimulatory one. Inclusion of molybdate in the buffer used for homogenization of the uninduced wild type also gave a significant increase in the subsequent in vitro assembly of the enzyme. Various forms and derivatives of molybdenum enhanced to varying degrees the ability of acid-treated xanthine oxidase to form nitrate reductase. Aqueous solutions of several complexes of Mo(V) with cysteine or glutathione under anaerobic conditions failed to substitute for the presumed molybdenum cofactor in the in vitro assembly of nitrate reductase. Sucrose density gradient profiles of NADPH-nitrate reductase formed in vitro by incubation of induced nit-1 extract with a 99Mo-labeled fraction of uninduced wild type (grown on 99MoO4=-containing medium) coincided with that of radioactivity. The addition of 99MoO4= to a cell-free preparation of unlabeled uninduced wild type or to acid-treated xanthine oxidase prior to its incubation with an extract of induced nit-1 gave similar results. Reciprocal experiments using an unlabeled fraction of uninduced wild type and an extract of nit-1 grown and induced in the presence of 99MoO4= showed no incorporation of radioactivity in the in vitro assembled enzyme. Nor was there in vitro exchange or incorporation of 99Mo into the already formed wild type enzyme. Comparable experiments with 55Fe showed that nit-1 and not uninduced wild type or xanthine oxidase is the source of iron in the in vitro assembly of Neurospora NADPH-nitrate reductase.
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