Abstract
The membrane-bound guanylyl cyclase in vertebrate photoreceptor cells is one of the key enzymes in visual transduction. It is highly sensitive to the free calcium concentration ([Ca2+]). The activation process is cooperative and mediated by a novel calcium-binding protein named GCAP (guanylyl cyclase-activating protein). We isolated GCAP from bovine rod outer segments, determined amino acid sequences of proteolytically obtained peptides, and cloned its gene. The Ca2+-bound form of native GCAP has an apparent molecular mass of 20.5 kDa and the Ca2+-free form of 25 kDa as determined by SDS-polyacrylamide gel electrophoresis. Recombinant GCAP was functionally expressed in Escherichia coli. Activation of guanylyl cyclase in vertebrate photoreceptor cells by native acylated GCAP was half-maximal at 100 nM free [Ca2+] with a Hill coefficient of 2.5. Activation by recombinant nonacylated GCAP showed a lower degree of cooperativity (n = 2.0), and half-maximal activation was shifted to 261 nM free [Ca2+]. Immunocytochemically we localized GCAP only in rod and cone cells of a bovine retina.
Highlights
The membrane-bound guanylyl cyclase in vertebrate photoreceptor cells is one of the key enzymes in visual transduction
An unexpected complexity has emerged from the cloning of a second photoreceptor-specific membrane guanylyl cyclase, retGC-2 [21]. (We refer to retGC activity, because we cannot distinguish between retGC-1 and retGC-2 activities in our rod outer segment (ROS) membrane preparations.) In order to gain further insight into the control of retGC activity, we screened cytoplasmic extracts of bovine ROS for guanylyl cyclase activating factors, isolated and cloned a protein (GCAP), studied its distribution in the vertebrate retina, and compared some of the molecular properties of two recombinant forms with the native form
Purification and Cloning of guanylyl cyclase-activating protein (GCAP)—An extract of illuminated bovine ROS contained a guanylyl cyclase-stimulating activity that could be further fractionated by column chromatography (Fig. 1A)
Summary
Purification of GCAP—ROSs were prepared from freshly obtained bovine eyes as described previously [22]. 12 ml of ROS (7–11 mg rhodopsin/ml) were adjusted to 100 mM NaCl, 1 mM MgCl2, 3 mM ATP, 1 mM dithiothreitol and bleached for 15 min at 37 °C. The concentrated extract was applied onto a gel filtration column (Superdex 75 16/60, Pharmacia Biotech Inc.), equilibrated with buffer A (50 mM NaCl, 20 mM Tris, pH 8.0, 1 mM dithiothreitol). GCAP was eluted by washing with 2–3 ml of 150 mM NaCl, 60 mM Tris-HCl, pH 8.0, 2.5 mM CaCl2 and diluted 1:3 with H20 prior to subsequent anion exchange chromatography on a MonoQ column. ROS membranes were diluted 5-fold with 5 mM Tris-HCl, pH 8.0 and centrifuged for 15 min (40,000 rpm, TLA45, 4 °C, Beckman). Fractions from column chromatography were added to washed ROS membranes and incubated at high and low Ca2ϩ for 5 min at 30 °C in a total volume of 50 l. Activity of retGC is V and Vmax, EC50 of Ca2ϩ-dependent retGC activation is K1⁄2, and Z is a constant taking into account that retGC activity is not zero at high free [Ca2ϩ]
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