A comparative study of calcium transients by isotopic tracer, metallochromic indicator, and intrinsic fluorescence in sarcoplasmic reticulum ATPase.
Comparative measurements were carried out in order to evaluate the significance of intrinsic fluorescence transients with respect to various steps of the catalytic and transport cycle of sarcoplasmic reticulum ATPase. The enzyme can acquire three levels of intrinsic fluorescence. Level 1 (lowest fluorescence) is observed in the absence of Ca2+ and ATP. Level 2 (highest fluorescence) is induced by Ca2+ through a sequential mechanism including two binding steps interspaced by an isomerization step. This transition occurs more rapidly in the presence of ATP and produces enzyme activation. Level 3 (slightly higher fluorescence than level 1) is observed immediately upon ATP binding (or phosphorylation with Pi) in the absence of Ca2+. When ATP is added to the enzyme X calcium complex, the enzyme is rapidly phosphorylated and the bound calcium is translocated to a position which is protected from La3+ added to the medium. This initial phenomenon is followed by a slow isomerization of the phosphoenzyme which is revealed by a decrease of fluorescence intensity and produces calcium release inside the vesicles before hydrolytic cleavage of the phosphoenzyme. A reaction cycle is considered and subjected to analysis, based on three main enzyme states: E, in the absence of Ca2+; E', in the presence of Ca2+; and *E, subsequent to phosphorylation.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001150:Arsenazo III / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007811:Lanthanum / Q000378:metabolism; D008433:Mathematics; D008956:Models, Chemical; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D001150:Arsenazo III; D004533:Egtazic Acid; D007811:Lanthanum; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D002118:Calcium