Abstract
Guanylate cyclase-activating protein 1 (GCAP-1) is a Ca(2+)-sensing protein in vertebrate photoreceptor cells. It activates a membrane-bound guanylate cyclase. Three of four cysteines present in wild-type GCAP-1 were accessible to the thiol-modifying reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) in the presence of Ca(2+). Only Cys106 became exposed to the solvent after Ca(2+)-chelation. Since Cys106 is located in EF-hand 3, we could determine an apparent K(D) of 2.9 microM for Ca(2+) binding to this site with a fast off-rate (t approximately 2 ms). We conclude that the rapid dissociation of Ca(2+) from EF-hand 3 in GCAP-1 triggers activation of guanylate cyclase in rod cells.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Binding Sites
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Calcium / metabolism*
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Calcium-Binding Proteins / chemistry*
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Calcium-Binding Proteins / genetics
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Calcium-Binding Proteins / metabolism*
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Cattle
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Cysteine / metabolism*
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Disulfides
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Dithionitrobenzoic Acid / pharmacology
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EF Hand Motifs
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Egtazic Acid / pharmacology
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Enzyme Activation
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Guanylate Cyclase / metabolism*
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Guanylate Cyclase-Activating Proteins
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Mutation
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Nitrobenzoates / metabolism
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Rod Cell Outer Segment / enzymology
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Rod Cell Outer Segment / metabolism*
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Sulfhydryl Compounds / metabolism
Substances
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Calcium-Binding Proteins
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Disulfides
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Guanylate Cyclase-Activating Proteins
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Nitrobenzoates
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Sulfhydryl Compounds
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thionitrobenzoic acid
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Egtazic Acid
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Dithionitrobenzoic Acid
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Guanylate Cyclase
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Cysteine
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Calcium