Characterization of rat neuronal nitric oxide synthase expressed in Saccharomyces cerevisiae.
A cDNA encoding rat neuronal nitric oxide synthase (nNOS) was cloned into the yeast expression vector pMA56 to generate pA379. Transformation of Saccharomyces cerevisiae strain BJ2168 with this plasmid resulted in the synthesis of nNOS at levels of 0.5-1.0 mg/liter. The protein is localized in the cytosol and is catalytically active as determined by the conversion of [3H]-L-arginine to [3H]-L-citrulline and NO. The enzyme was purified by calmodulin-Sepharose affinity chromatography and its catalytic activity was found to be both calcium and calmodulin dependent. Overexpression of nNOS in S. cerevisiae and purification of the recombinant protein will facilitate detailed characterization of this important enzyme.
Black
S M
SM
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Ortiz de Montellano
P R
PR
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D007700:Kinetics; D008969:Molecular Sequence Data; D019331:NG-Nitroarginine Methyl Ester; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D019331:NG-Nitroarginine Methyl Ester