Functional characterization of a guanylyl cyclase-activating protein from vertebrate rods - Cloning, heterologous expression, and localization

被引:118
作者
Frins, S
Bonigk, W
Muller, F
Kellner, R
Koch, KW
机构
[1] FORSCHUNGSZENTRUM JULICH, FORSCHUNGSZENTRUM, INST BIOL INFORMAT VERARBEITUNG, D-52425 JULICH, GERMANY
[2] UNIV MAINZ, INST PHYSIOL CHEM & PATHOBIOCHEM, D-55099 MAINZ, GERMANY
关键词
D O I
10.1074/jbc.271.14.8022
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:8022 / 8027
页数:6
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