Formation of Assimilatory Nitrate Reductase by in vitro Inter-Cistronic Complementation in Neurospora crassa

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 65; no. 1; pp. 137 - 144
• Main Authors: Nason, Alvin, Antoine, Alan D., Ketchum, Paul A., Frazier, William A., Lee, David K.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01.01.1970; National Acad Sciences
• Subjects: Biochemistry; Biological Sciences: Biochemistry; Cell-Free System; Centrifugation, Density Gradient; Complementation; Cytochromes; Enzyme Induction; Enzymes; Flavin-Adenine Dinucleotide; Genetic Complementation Test; Genetics, Microbial; Homogenization; Molecular weight; Mutation; NADP; Neurospora; Neurospora - enzymology; Neurospora crassa; Nitrates; Oxidoreductases - biosynthesis; Protein subunits; Pyridinium Compounds
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.65.1.137

In vitro complementation of the soluble assimilatory nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-nitrate reductase was attained by mixing cell-free preparations of certain Neurospora nitrate reductase mutants: induced nit-1 (uniquely possessing inducible NADPH-cytochrome c reductase) with (a) uninduced or induced nit-2 or nit-3, or (b) uninduced wild type. The complementing activity of induced nit-1 is soluble while that of nit-2, nit-3, and wild type is particulate but not of mitochondrial origin. All fractions are inactivated by heat or trypsin. The NADPH-nitrate reductase enzymes formed in the above three complementing mixtures are similar to the wild-type enzyme in sucrose density gradient profiles, molecular weight, substrate affinity, sensitivity to inhibitors and temperature, but show different ratios of associated enzyme activities. The data suggest that nitrate reductase consists of at least protein subunits: a nitrate-inductible subunit as reflected by inductible NADPH-cytochrome c reductase, and a constitutive protein which is activated (as indicated by the appearance of the flavine adenine dinucleotide, reduced form (FADH2)- and reduced methyl viologen-nitrate reductase activities) when it combines with the inductible subunit.