Characterization of human liver inducible nitric oxide synthase expressed in Escherichia coli.
We have cloned the human liver inducible isoform of nitric oxide synthase (NOS) into an Escherichia coli expression vector and have expressed and purified the enzyme. The protein has been expressed with and without a polyhistidine tail. In both cases, expression of functional protein requires coexpression with calmodulin and inclusion of tetrahydrobiopterin (H4B) in the purification buffers. Unlike the constitutive isoforms of NOS, this isoform is unstable in the absence of L-arginine (L-Arg) and H4B toward loss of the heme group and the formation of a low-spin species spectroscopically distinct from that of the cofactor-bound protein. The enzyme purified in the presence of both L-Arg and H4B is highly active, with a Vmax of approximately 800 nmol NO min(-1) mg(-1) and a Km for L-Arg of 22 microM. The cytochrome c reductase activity is 38,000 nmol x min(-1) mg(-1). Similar values are obtained for the enzyme with and without the polyhistidine tail. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid does not inhibit the activity of the protein, nor is the activity of the enzyme increased by the addition of exogenous calmodulin and/or Ca2+. These findings contrast with an earlier report, based on experiments with extracts of COS-1 cells expressing the recombinant enzyme, that the enzyme responds to changes in the Ca2+ concentration. The human hepatic isoform is similar in its properties to the inducible NOS isoform purified from macrophages.
Gerber
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NC
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Nishida
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CR
Ortiz de Montellano
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PR
D000818:Animals; D001120:Arginine / Q000378:metabolism; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods; D003574:Cytochrome c Group / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004926:Escherichia coli; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D013053:Spectrophotometry
D002147:Calmodulin; D003574:Cytochrome c Group; D007527:Isoenzymes; D011994:Recombinant Proteins; D004533:Egtazic Acid; D001120:Arginine; D019001:Nitric Oxide Synthase; D002118:Calcium