Purification and characterization of a human NO synthase.
A NO synthase (NOS, EC 1.14.23) was isolated from human cerebellum by two sequential chromatography steps, that is affinity chromatography on 2'5'ADP sepharose and size exclusion chromatography on Superose 6. Human NOS migrated as a single band of 160 kDa on SDS/PAGE. The enzyme was Ca2+/calmodulin-regulated and NADPH/tetrahydrobiopterin (BH4)-dependent, which are characteristics of a type I NOS previously isolated from rat cerebellum. Antisera raised against purified rat cerebellar NOS crossreacted specifically with a 160 kDa protein in crude supernatant fraction of human cerebellum and purified human NOS but not in crude supernatant fraction of the temporal lobe. These findings provide evidence that nitrinergic signal transduction through conversion of L-arginine to L-citrulline and NO does also occur in humans and NO may function as a neurotransmitter in the human central nervous system.
Schmidt
H H
HH
Northwestern University Medical School, Dept. of Pharmacology, Chicago, IL 60611.
Murad
F
F
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002531:Cerebellum / Q000201:enzymology*; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D019001:Nitric Oxide Synthase; D019335:Nitroarginine; D051381:Rats; D013702:Temporal Lobe / Q000201:enzymology*; D014268:Trifluoperazine / Q000494:pharmacology; D019323:omega-N-Methylarginine
D002147:Calmodulin; D007093:Imidazoles; D019335:Nitroarginine; D014268:Trifluoperazine; D019323:omega-N-Methylarginine; C031938:calmidazolium; D004533:Egtazic Acid; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases