BIOCHEMICAL-CHARACTERIZATION OF A MEMBRANE-BOUND ENZYME RESPONSIBLE FOR GENERATING NITRIC-OXIDE FROM NITROGLYCERIN IN VASCULAR SMOOTH-MUSCLE CELLS

A membrane-bound enzyme responsible for generating nitric oxide (NO) f rom nitroglycerin (NTG) in vascular smooth muscle cells has been chara cterized. The enzyme could be solubilized from vascular microsomes by several detergents, the most effective of which was cholamidopropyl)-d imethylamino]-1-propanesulfonate (CHAPS). A partially purified enzyme preparation was obtained with CHAPS-solubilized vascular microsomes th at were processed sequentially through an ion exchange column and a ge l filtration column. The activity of this partially purified enzyme sh owed a dependence on substrate concentration, protein concentration an d the duration of incubation. Enzyme activity was enhanced 2.7- to 4.2 -fold by several thiols such as cysteine, N-acetylcysteine, reduced gl utathione, and dithiothretol. On the other hand, N-ethylmaleimide, iod oacetic acid, p-chloromercuric benzoic acid and 1-chloro-2,4-dinitrobe nzene, reagents known to bind with the free sulfhydryl groups, inactiv ated the NO-generating activity from NTG. The enzyme activity could be reversibly bound to an organomercurial column. These results suggeste d the presence of a free thiol group in the enzyme and that this thiol group was required for enzyme activity. The partially purified enzyme was active in the presence of 0.1% sodium dodecyl sulfate (SDS). The enzyme was purified to near homogeneity using several sequential chrom atographic steps including DEAE-Sephacel, Biogel A 1.5 m, hydroxylapat ite and organomercurial columns, resulting in an increase in enzyme ac tivity of about 94-fold. The subunit of this enzyme, as identified on an SDS-treated electrophoresis gel, had an apparent molecular size of 58 kDa.