Endothelial Nitric-oxide Synthase: EVIDENCE FOR BIDOMAIN STRUCTURE AND SUCCESSFUL RECONSTITUTION OF CATALYTIC ACTIVITY FROM TWO SEPARATE DOMAINS GENERATED BY A BACULOVIRUS EXPRESSION SYSTEM

Pei-Feng Chen,Ah-Lim Tsai,Vladimir Berka,Kenneth K Wu

doi:10.1074/jbc.271.24.14631

Pei-Feng Chen, Ah-Lim Tsai + Show 2 more

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https://doi.org/10.1074/jbc.271.24.14631

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Abstract

A baculovirus system was used to express the oxygenase and reductase domains of human endothelial nitric-oxide synthase (ecNOS) as distinct proteins. The oxygenase domain (residues 1-491) was expressed using a vector containing a His6 tag at the N terminus. The purified oxygenase domain had an apparent molecular mass of approximately 54 kDa, and retained the ability to bind L-arginine and form the ferrous CO complex. The purified reductase domain (residues 492-1244) had an apparent molecular mass of approximately 82 kDa and retained the ability to catalyze NADPH-dependent cytochrome c reduction, which was enhanced 10-fold by the presence of Ca2+/calmodulin. Both purified domains exhibited immunoreactivity to rabbit anti-ecNOS IgG. The NOS activity was successfully reconstituted by mixing the two domains. These results demonstrate for the first time that the two domains of ecNOS are catalytically intact and can be reconstituted in vitro.

Highlights

A baculovirus system was used to express the oxygenase and reductase domains of human endothelial nitric-oxide synthase as distinct proteins
We show for the first time that ecNOS has a bidomain structure and its catalytic activity can be reconstituted from these two separate domains
We generally obtained about 6 mg of purified oxygenase and 3 mg of purified reductase domains from 3 ϫ 109 sf9 cells. Both purified domains migrated as a single band on SDS-PAGE analysis with apparent molecular masses of ϳ54 and ϳ82 kDa for the oxygenase and reductase domains, respectively (Fig. 1A) and exhibited immunoreactivity with a polyclonal antibody raised against the purified recombinant ecNOS (Fig. 1B)

Summary

Introduction

A baculovirus system was used to express the oxygenase and reductase domains of human endothelial nitric-oxide synthase (ecNOS) as distinct proteins. The purified reductase domain (residues 492-1244) had an apparent molecular mass of ϳ82 kDa and retained the ability to catalyze NADPH-dependent cytochrome c reduction, which was enhanced 10-fold by the presence of Ca2؉/ calmodulin. Both purified domains exhibited immunoreactivity to rabbit anti-ecNOS IgG. There is a considerable sequence homology between ecNOS and other NOS isoforms, ecNOS contrasts from other NOSs in that it is mostly membrane-associated and may be structurally different [3, 26] It is, important to determine whether ecNOS has a bidomain structure and whether the enzyme activity can be reconstituted. We show for the first time that ecNOS has a bidomain structure and its catalytic activity can be reconstituted from these two separate domains

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