Guanylate cyclase from bovine rod outer segments: solubilization, partial purification, and regulation by inorganic pyrophosphate.
In spite of its pivotal role in visual transduction, very little is known about guanylate cyclase of retinal photoreceptor cells. The enzyme has not yet been purified principally because of the difficulty in solubilizing it. We report here a simple method for solubilization of 67% of the cyclase activity from the retinal rod disk membranes (RDM). With Nonidet P-40 as detergent, the solubilization of cyclase is favored by a high concentration of KCl and exclusion of manganese. The solubilized and the residual insoluble enzymes are both highly unstable but could be partially stabilized by dithiothreitol. They were both insensitive to calcium, calmodulin, and atrial natriuretic factor. They also responded similarly to varying the manganese concentration in the assay. For the activity in both fractions, the Km for GTP was about 230 microM, Line-weaver-Burk plots showed that substrate binding was cooperative, and Hill plots suggested that there are two substrate binding sites. Cumulatively, these observations showed that while the entire activity could not be solubilized, the solubilized and the residual insoluble activities probably belonged to the same enzyme. Partial purification resolved the solubilized enzyme into two activities refered to as enzymes 1 and 2. Both had substrate saturation kinetics similar to the solubilized enzyme and were inhibited competitively by inorganic pyrophosphate, one of the products of the cyclase reaction. The Ki for PPi for enzyme 1 was 70-100 microM and 150-200 microM for enzyme 2. cGMP at concentrations up to 800 microM had no influence on the activity of either enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Hakki
S
S
Pennsylvania College of Optometry, Philadelphia, Pennsylvania 19141.
Sitaramayya
A
A
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D011756:Diphosphates / Q000378:metabolism; D004795:Enzyme Stability; D006162:Guanylate Cyclase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008566:Membranes / Q000201:enzymology; D010786:Photoreceptor Cells / Q000201:enzymology*; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology* / Q000648:ultrastructure; D012995:Solubility
D011756:Diphosphates; D008345:Manganese; D009320:Atrial Natriuretic Factor; D011755:Pyrophosphatases; D006162:Guanylate Cyclase; D002118:Calcium