A Chlamydomona~' reinhardtff molybdenum cofactor (MoCo).carrier protein (CP), capable of reconstituting nitrate redugtase activity with apoprotein from the Neurospora crassa mutant nit.l, was subjected to experiments of diffusion through a dialysis membrane and gel filtration. CP bonded firmly MoCo and did not release it efficiently unles-~ aponitrate reduetase was present in the incubation mixture, Stability of MoCo bound to CP against air and heat was very similar to that of fre¢-MoCo released from milk xanthine oxidase. Our data strongly suggest that MoCo is directly transferred from CP to aponitrate reductase to form an active enzyme, Molybdenum ¢ofactor; Molybdopterin; Nitrate reductase; Xanthine oxidase

Strain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild‐type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium‐grown cells from both the wild‐type and 21gr strains was small and blocked by sulphate 0·3 mM. However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild‐type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.

No abstract

Abstract. There were significant differences in the contents of molybdenum cofactor (Mo-co), both in a lowmolecular-mass form (free Mo-co) and in a protein-bound form, in seeds of seven Vicia faba genotypes. Lowmolecular-mass Mo-co species present in the extracts were detected by their ability to reactivate, through a dialysis membrane, aponitrate reductase from the Neurospora crassa nit-1 mutant. In extracts of all genotypes tested, the amount of Mo-co capable of directly reactivating nitrate reductase of the N. crassa nit-1 mutant was always much higher than that of low-molecular-mass Moco. These data cannot be explained by considering, as traditionally, that Mo-co detected directly, i.e. without any previous treatment for its release from Mo-coproteins, corresponds to free low-molecular mass Mo-co. A protein which bound Mo-co was purified to electrophoretic homogeneity. This protein consisted of a single 70-kDa polypeptide chain and carried a Mo-co that could be efficiently released when in contact with aponitrate reductase.