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) from nitroglycerin (NTG) in vascular smooth muscle cells has been characterized. The enzyme could be solubilized from vascular microsomes by several detergents, the most effective of which was 3-[(3-cholamidopropyl)-dimethylamino]-1-propanesulfonate (CHAPS). A partially purified enzyme preparation was obtained with CHAPS-solubilized vascular microsomes that were processed sequentially through an ion exchange column and a gel filtration column. The activity of this partially purified enzyme showed a dependence on substrate concentration, protein concentration and the duration of incubation. Enzyme activity was enhanced 2.7- to 4.2-fold by several thiols such as cysteine, N-acetylcysteine, reduced glutathione, and dithiothreitol. On the other hand, N-ethylmaleimide, iodoacetic acid, p-chloromercuric benzoic acid and 1-chloro-2,4-dinitrobenzene, reagents known to bind with the free sulfhydryl groups, inactivated the NO-generating activity from NTG. The enzyme activity could be reversibly bound to an organomercurial column. These results suggested 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 chromatographic steps including DEAE-Sephacel, Biogel A 1.5 m, hydroxylapatite and organomercurial columns, resulting in an increase in enzyme activity 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.
Seth
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Department of Pharmaceutics, School of Pharmacy, State University of New York at Buffalo 14260.
Fung
H L
HL
D000818:Animals; D002417:Cattle; D002793:Cholic Acids; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification*; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D005996:Nitroglycerin / Q000378:metabolism*
D002793:Cholic Acids; D003902:Detergents; D009569:Nitric Oxide; D003577:Cytochrome P-450 Enzyme System; D005996:Nitroglycerin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate