Binding kinetics of calbindin-D(28k) determined by flash photolysis of caged Ca(2+)
We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca(2+) binding to calbindin-D(28K). The experiments used saturated DM-nitrophen at a [Ca(2+)] of 1.5 microM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca(2+)] in the absence of calbindin-D(28K), and in its presence the decay of the flash-induced fluorescence was due solely to the Ca(2+) buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca(2+) buffer EGTA to confirm its validity. We provide evidence that calbindin-D(28K) binds Ca(2+) in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca(2+) with a k(on) comparable to that of EGTA (i.e., approximately 1 x 10(7) M(-1) s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca(2+) binding kinetics of Ca(2+) buffers, this method promises to be highly valuable for studying the Ca(2+) binding properties of other biologically important Ca(2+) binding proteins.
Nägerl
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Department of IDP Neuroscience, UCLA School of Medicine, Los Angeles, California 90095, USA. valentin@neuro.mpg.de
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Vergara
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D000085:Acetates / Q000494:pharmacology; D064026:Calbindins; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000737:chemistry; D004563:Electrochemistry; D005029:Ethylenediamines / Q000494:pharmacology; D005456:Fluorescent Dyes; D007700:Kinetics; D008956:Models, Chemical; D010782:Photolysis; D011485:Protein Binding; D064030:S100 Calcium Binding Protein G / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D000085:Acetates; D064026:Calbindins; D002614:Chelating Agents; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D064030:S100 Calcium Binding Protein G; C057014:DM-nitrophen; D004533:Egtazic Acid; D002118:Calcium