The bovine photoreceptor outer segment guanylate cyclase: purification, kinetic properties, and molecular size.
A simple protocol was developed to isolate the integral membrane guanylate cyclase from bleached bovine photoreceptor outer segments. Hypotonic and hypertonic washes strip the photoreceptor outer segment membranes of peripheral proteins. The guanylate cyclase activity is solubilized by dodecyl-b-D-maltoside in a low salt concentration buffer. Phosphatidylcholine, glycerol, and dithiothreitol are used to stabilize the activity during chromatography. GTP-affinity chromatography achieves a 250-fold increase in specific activity over that of membranes stripped of peripheral proteins. From 100 retinas, the protocol yields 100-140 mg of purified guanylate cyclase composed of a 115-kDa subunit. The molar ratio of the guanylate cyclase to rhodopsin is estimated to be 1:440. A significant portion of the freshly solubilized enzyme behaves as a monomer with a Stokes radius of 48.7 A, whereas the purified protein forms homooligomers ranging from dimers to tetramers. These properties are similar to those of ANP and guanylin receptors, indicating that the photoreceptor protein shares characteristics of the membrane receptor guanylate cyclase family. For the physiological substrate MgGTP, the Km and Vmax are 1.07 +/- 0.20 mM and 3262 +/- 514 nmol cGMP min-1 mg-1, respectively, generating a turnover rate of approximately 3.9 nmol cGMP s-1 at physiological substrate concentrations. The relatively high Km suggests that in vivo changes in GTP concentration might modulate the rate of cGMP synthesis. These properties indicate that the photoreceptor membrane guanylate cyclase can sustain a rate of cGMP synthesis comparable to the dark-adapted (basal) rate of cGMP degradation by the cGMP phosphodiesterase.
Committee on Neurobiology, University of Chicago, Illinois 60637, USA.
J G
JG
Aparicio
M L
ML
Applebury
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006162:Guanylate Cyclase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D012995:Solubility
D006162:Guanylate Cyclase