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 yield ed 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 increase d the activity, inhibitors of heme biosynthesis reduced the ecNOS acti vity to 50% without affecting the level of enzyme. After extraction of cells with 1% Triton X-100, ecNOS protein was purified by column chro matography. The resultant ecNOS required supplementation with cofactor s for activity, but it did not require hemin. Binding of a protoporphy rin IX heme was confirmed by a pyridine hemochrome assay. (C) 1995 Aca demic Press, Inc.