Heme requirement for production of active endothelial nitric oxide synthase in baculovirus-infected insect cells.
We have cloned cDNAs encoding endothelial nitric oxide synthase (ecNOS) from a human fetal liver cDNA library. Overproduction of ecNOS in a baculovirus/insect cell expression system in conventional medium yielded a large amount of ecNOS protein localized in particulate components, but ecNOS activity was low. This activity was increased by addition of hemin to 2.5-fold. While a precursor for heme biosynthesis increased the activity, inhibitors of heme biosynthesis reduced the ecNOS activity to 50% without affecting the level of enzyme. After extraction of cells with 1% Triton X-100, ecNOS protein was purified by column chromatography. The resultant ecNOS required supplementation with cofactors for activity, but it did not require hemin. Binding of a protoporphyrin IX heme was confirmed by a pyridine hemochrome assay.
Seo
H G
HG
Department of Biochemistry, Osaka University Medical School, Japan.
Fujii
J
J
Soejima
H
H
Niikawa
N
N
Taniguchi
N
N
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D000622:Aminolevulinic Acid / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000201:enzymology*; D005415:Flavins / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006418:Heme / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase; D009573:Nitrites / Q000032:analysis; D010396:Penicillamine / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D017931:DNA Primers; D005415:Flavins; D009573:Nitrites; D011994:Recombinant Proteins; D006418:Heme; D000622:Aminolevulinic Acid; D004247:DNA; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D010396:Penicillamine; D007538:Isoniazid