Nitric oxide synthase from rat colorectum: purification, peptide sequencing, partial PCR cloning, and immunohistochemistry.
Nitric oxide synthase (NOS) has been purified over 6,500-fold with a 3.4% yield from rat colorectum with 2',5'-ADP-Sepharose, DEAE cellulose, and gel filtration. The purified enzyme gave a single band corresponding to an apparent molecular mass of 160 Dka on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When assayed in the requisite presence of L-arginine, CaCl2, NADPH, calmodulin, tetrahydro-L-biopterin, and FAD, the purified enzyme exhibited a specific activity of 328 nmol/min/mg L-citrulline formed and an apparent Km for L-arginine of 2.9 microM. Amino acid sequencing of 12 peptides revealed identical sequences to that of the neuronal type enzyme except for two altered amino acid residues. When partial reverse transcription-polymerase chain reaction of RNA from rat colorectum and cerebellum was performed using primers designed according to the amino acid sequences determined, these amino acid changes were found in both cDNA fragments, indicating the identity of the colorectal enzyme to the cerebellar one. A polyclonal antibody raised against NOS purified from rat cerebellum cross-reacted with the NOS from colorectum but not that from IFN-gamma stimulated macrophage-derived cells, RAW 264.7. Immunohistochemical analysis of the colorectum using this specific antibody indicated that Auerbach's plexus is strongly immunoreactive, supporting the hypothesis that NO is an inhibitory transmitter for non-adrenergic and non-cholinergic nerves in the colorectum.
Seo
H G
HG
Department of Biochemistry, Osaka University Medical School.
Tatsumi
H
H
Fujii
J
J
Nishikawa
A
A
Suzuki
K
K
Kangawa
K
K
Taniguchi
N
N
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003106:Colon / Q000201:enzymology*; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D005260:Female; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019001:Nitric Oxide Synthase; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D012007:Rectum / Q000201:enzymology*; D014158:Transcription, Genetic
D018076:DNA, Complementary; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases