THE BOVINE PHOTORECEPTOR OUTER SEGMENT GUANYLATE-CYCLASE - PURIFICATION, KINETIC-PROPERTIES, AND MOLECULAR-SIZE

A simple protocol was developed to isolate the integral membrane guany late cyclase from bleached bovine photoreceptor outer segments. Hypoto nic and hypertonic washes strip the photoreceptor outer segment membra nes of peripheral proteins, The granylate cyclase activity is solubili zed by dodecyl-b-D-maltoside in a low salt concentration buffer. Phosp hatidylcholine, glycerol, and dithiothreitol are used to stabilize the activity during chromatography, GPT-affinity chromatography achieves a 250-fold increase in specific activity over that of membranes stripp ed of peripheral proteins. From 100 retinas, the protocol yields 100-1 40 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 Angstrom, whereas the purif ied protein forms homooligomers ranging from dimers to tetramers, Thes e properties are similar to those of ANP and guanylin receptors, indic ating that the photoreceptor protein shares characteristics of the mem brane receptor guanylate cyclase family. For the physiological substra te MgGTP, the K-m and V-max are 1.07 +/- 0.20 mM and 3262 +/- 514 nmol cGMP min(-1) mg(-1), respectively, generating a turnover rate of simi lar to 3.9 nmol cGMP s(-1) at physiological substrate concentrations, The relatively high K-m suggests that in vivo changes in GTP concentra tion might modulate the rate of cGMP synthesis. These properties indic ate that the photoreceptor membrane guanylate cyclase can sustain a ra te of cGMP synthesis comparable to the dark-adapted (basal) rate of cG MP degradation by the cGMP phosphodiesterase. (C) 1995 Academic Press, Inc.