Characterization of recombinant human endothelial nitric-oxide synthase purified from the yeast Pichia pastoris

Human endothelial nitric-oxide synthase (eNOS) was expressed in the methylo trophic yeast Pichia pastoris, making use of the highly inducible alcohol o xidase promoter. The recombinant protein constituted approximately 3% of to tal protein and was largely soluble (>75%). About 1 mg of purified eNOS was obtained from 100-ml yeast cell cultures by affinity chromatography of cru de cell supernatants. The purified enzyme had a V-max of 192 +/- 18 nmol of L-citrulline x mg(-1) x min(-1), had a K-m for L-arginine of 3.9 +/- 0.2 m u M, and showed an absolute requirement for tetrahydrobiopterin (H(4)biopte rin). NADPH oxidase activity was 136 +/- 9 and 342 +/- 24 nmol x mg(-1) x m in(-1) in the absence and presence of 0.1 mM L-arginine, respectively, and not affected by H(4)biopterin. The protein contained 0.56 +/- 0.06 equivale nts of FAD and 0.79 +/- 0.08 equivalents of FMN. On-line gel filtration/ind uctively coupled plasma mass spectrometry analysis confirmed that both iron (0.80 +/- 0.09 mol/subunit) and zinc (0.43 +/- 0.03 mol/subunit) were boun d to the enzyme. Graphite furnace-atomic absorption spectroscopy yielded a value for bound iron of 0.84 +/- 0.04 mol/subunit. The absorbance of the en zyme at 398 nm implied a heme content of 0.85 +/- 0.09 mol/subunit, and the high pressure liquid chromatography heme assay gave an estimate of 0.71 +/ - 0.02 mol heme/subunit. Gel permeation chromatography yielded one single p eak with a Stokes radius of 6.62 +/- 0.7 nm, indicating that the native pro tein is dimeric, Upon low temperature gel electrophoresis the untreated pro tein appeared mainly as a monomer (88 +/- 3%), but pretreatment with H(4)bi opterin and L-arginine led to a pronounced shift toward dimers (77 +/- 4%). Thus,in contrast to bovine eNOS (List,B.M., Klosch, B., Volker, C., Gorren , A C. F., Sessa, W. C., Werner, E. R, Kukovetz, W. R, Schmidt, a, and Maye r, B. (1997) Biochem J; 323, 159-165; Rodriguez-Crespo, L, Gerber, N, C., a nd Ortiz de Montellano, P.R (1996) J. Biol. Chem 271, 11462-11467), the hum an eNOS appears to be markedly stabilized by H(4)biopterin.