Ca2+-controlled conformational states of the Ca2+ transport enzyme of sarcoplasmic reticulum.
The fluorescent reagent, S-mercuric N-dansyl-cysteine, reacts specifically with thiols of the purified Ca2+-ATPase of the sarcoplasmic reticulum, producing an increase of fluorescence of fluorescence intensity at 500 nm (lambda ex = 335 nm). The reaction is stoichiometric, and the increase of the fluorescence intensity is proportional to the number of blocked thiols. Twelve reactive thiols per 10(5) daltons of ATPase peptide fall into roughly three classes. Blocking of the most reactive thiol entails little inhibition of enzyme activity. Blocking of the five thiols reacting next (intermediate class) results in almost complete inhibition of both phosphorylated intermediate formation and ATP hydrolysis. The second order rate constants of the reaction of thiols have been determined by stopped flow studies. The most reactive thiol and the six least reactive thiols can each be treated as a single class with respect to the rate constant; five thiols of intermediate reactivity appear to have different rate constants (k2, k3, ..k6). Of these constants, k1, corresponding to the most reactive thiol, does not change with [Ca2+]. Upon increasing [Ca2+] from 10(-9) to 10(-5) M, k2 increase and k7-12 decreases; the changes roughly parallel the activation of ATPase activity and the Ca2+ binding to the high affinity alpha sites (Ikemoto, N. (1975) J. Biol. Chem. 250, 7219-7224). Upon further increase of [Ca2+] k2 decreases and k7-12 increase, in parallel with the inhibition of ATPase activity and with the Ca2+ binding to the low affinity gamma sites.
D000818:Animals; D001693:Biological Transport, Active; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D007700:Kinetics; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D000252:Calcium-Transporting ATPases