Heme requirement for production of active endothelial nitric oxide synthase in baculovirus-infected insect cells.

Abstract:

: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.

authors

Seo HG,Fujii J,Soejima H,Niikawa N,Taniguchi N

doi

10.1006/bbrc.1995.1298

subject

Has Abstract

pub_date

1995-03-08 00:00:00

pages

10-8

issue

1

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(85)71298-3

journal_volume

208

pub_type

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