Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase.

• DOI: 10.1073/pnas.88.2.365
• Publication Date: 2006-05-31T11:47:53Z
• Authors: H H Schmidt; J S Pollock; M Nakane; L D Gorsky; U Förstermann; F Murad
• Keywords: Soluble guanylyl cyclase; Molecular mass

The soluble form of guanylyl cyclase-activating-factor (GAF) synthase from rat cerebellum was purified to homogeneity by sequential affinity chromatographic steps on adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin-agarose. Enzyme activity during purification bioassayed the L-arginine-, NADPH-, Ca2+/calmodulin-dependent formation a plasma membrane-permeable nitric oxide-like factor that stimulated cyclase in RFL-6 cells. With calmodulin NADPH as cofactors, GAF induced an increase 1.05 mumol cGMP per 10(6) cells 3 min mg protein. coproduct this signal-transduction pathway appeared be L-citrulline. catalyzed conversion 107 nmol L-arginine into L-citrulline Based these assays, represents approximately 10,076- 8925-fold with recoveries 16% 19%, respectively. Rechromatography enzyme Mono P (isoelectric point = 6.1 +/- 0.3), Q, Superose 12 or 6 resulted no further specific activity. A Stokes radius 7.9 0.3 nm sedimentation coefficient s20,w 7.8 0.2 S were used calculate molecular mass about 279 25 kDa for native enzyme. SDS/PAGE revealed single protein band 155 kDa. These data suggest is homodimer 155-kDa subunits dependent upon presence calmodulin.