Characterization of Human Liver Inducible Nitric Oxide Synthase Expressed inEscherichia coli

Abstract

We have cloned the human liver inducible isoform of nitric oxide synthase (NOS) into anEscherichia coliexpression 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 ofl-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 bothl-Arg and H4B is highly active, with aVmaxof ∼800 nmol NO min−1mg−1and aKmforl-Arg of 22 μm. The cytochrome c reductase activity is 38,000 nmol·min−1mg−1. Similar values are obtained for the enzyme with and without the polyhistidine tail. Ethylene glycol bis(β-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.