In vitro assembly of Neurospora assimilatory nitrate reductase from protein subunits of a Neurospora mutant and the xanthine oxidizing or aldehyde oxidase systems of higher animals.
In vitro assembly or complementation of a hybrid assimilatory nitrate reductase was attained by mixing a preparation of nitrate-induced N. crassa mutant nit-1 specifically with acid-treated (pH 2.5) bovine milk or intestinal xanthine oxidase, rabbit liver aldehyde oxidase, or chicken liver xanthine dehydrogenase. The complementation reaction specifically required induced nit-1, the only nitrate reductase mutant of Neurospora that lacked xanthine dehydrogenase and was unable to use hypoxathine or nitrate as a sole nitrogen source. The complementing activities of the above acid-treated enzymes correspond to their xanthine or aldehyde oxidizing activity profiles on sucrose density gradients. The resulting soluble, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductases are the same as the Neurospora wild type enzyme in sucrose density gradient profile, molecular weight, substrate affinities, and sensitivity to inhibitors and temperature. By analogy to a similar in vitro complementation of nitrate reductase in mixtures of induced nit-1 and individual nonalleic Neurospora mutants, or uninduced wild type, the complemented nitrate apparently consists of an inducible protein subunit (possessing inducible NADPH-cytochrome c reductase) furnished by nit-1 and a subunit from the acid-treated xanthine or aldehyde oxidizing system which can substitute for the constitutive component furnished by the other mutants or uninduced wild type. The data suggest that Neurospora nitrate reductase and the xanthine oxidizing system and aldehyde oxidase of animals, all of which are molybdenum-containing enzymes catalyzing the reduction of nitrate to nitrite, share a highly similar protein subunit.
D000447:Aldehydes; D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002645:Chickens; D005182:Flavin-Adenine Dinucleotide; D005816:Genetic Complementation Test; D005827:Genetics, Microbial; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007422:Intestines / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008892:Milk / Q000201:enzymology; D009154:Mutation; D009249:NADP; D009491:Neurospora / Q000201:enzymology* / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000032:analysis / Q000378:metabolism*
D000447:Aldehydes; D009566:Nitrates; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D010088:Oxidoreductases; D014969:Xanthine Oxidase