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(Received for publication, January 4, 1996, and in revised form, April 4, 1996)
From the Vascular Biology Research Center and Division of
Hematology, Department of Internal Medicine, The University of Texas
Health Science Center at Houston, Houston, Texas 77030
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 ~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 ~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.
Volume 271, Number 24,
Issue of June 14, 1996
pp. 14631-14635
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
EVIDENCE FOR BIDOMAIN STRUCTURE AND SUCCESSFUL RECONSTITUTION OF
CATALYTIC ACTIVITY FROM TWO SEPARATE DOMAINS GENERATED BY A BACULOVIRUS
EXPRESSION SYSTEM
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