Jg. Aparicio et Ml. Applebury, THE BOVINE PHOTORECEPTOR OUTER SEGMENT GUANYLATE-CYCLASE - PURIFICATION, KINETIC-PROPERTIES, AND MOLECULAR-SIZE, Protein expression and purification, 6(4), 1995, pp. 501-511
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.