Purification of guanylyl cyclase from rod outer segments.
The particulate form of guanylyl cyclase from bovine rod outer segments has been solubilized and purified to near homogeneity by a combination of liquid chromatography and native gel electrophoresis. The procedure enriches enzyme activity 6700-fold from rod outer segment extracts to a final specific activity of 17.5 mumol/min per mg (when assayed with Mn-GTP as substrate). Purified preparations of guanylyl cyclase contain a single glycoprotein with an apparent molecular mass of 60,000 Da and a native isoelectric point of 7.6. Although crude or partially purified enzyme activity is modulated by sub-micromolar concentrations of Ca2+, the fully purified enzyme is insensitive to this cation. However, the purified enzyme remains sensitive to nitrovasodilators, being stimulated over 10-fold by sodium nitroprusside. These data suggest that retinal rods contain a unique isoform of guanylyl cyclase.
Department of Pharmacology, Northwestern University Medical School, Chicago, IL.
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Horio
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Murad
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000302:isolation & purification*; D007526:Isoelectric Point; D008562:Membrane Glycoproteins / Q000302:isolation & purification; D008970:Molecular Weight; D009599:Nitroprusside / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D008562:Membrane Glycoproteins; D009599:Nitroprusside; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D002118:Calcium