A direct fluorescence study of the transient steps induced by calcium binding to sarcoplasmic reticulum ATPase

• Published in: The Journal of biological chemistry Vol. 255; no. 5; pp. 2072 - 2076
• Main Authors: Guillain, F, Gingold, M P, Büschlen, S, Champeil, P
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10-03-1980
• Subjects: Animals; Calcium - metabolism; Calcium-Transporting ATPases - metabolism; Kinetics; Magnesium - pharmacology; Mathematics; Muscles - enzymology; Protein Binding; Rabbits; Sarcoplasmic Reticulum - enzymology; Spectrometry, Fluorescence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)85995-1

The sarcoplasmic reticulum intrinsic fluorescence level was closely correlated with the ATPase functional state, from pH 5.5
to 8.5. The fluorescence signal was used in stopped flow measurements for direct study of transient pump kinetics after calcium
binding or removal. The signal change time course, which depends solely on the free calcium concentration in the observation
chamber, was analyzed as a single exponential. Rate constants (kobs) were relatively slow (5 to 20 s-1), indicating multistep
interaction between calcium and the transport protein. At pH 7 and 20 degrees C, and in the presence of 100 mM potassium and
1 to 20 mM MgCl2, kobs first decreased, and then increased as the calcium concentration rose. Similar experiments were performed
at pH 6. Data were analyzed according to a scheme in which sarcoplasmic reticulum . calcium complex formation is controlled
by a slow isomerization step occurring either before or after the rapid calcium binding to the high affinity site. The results
are discussed with reference to published rapid quenching experiments. Under our conditions, i.e. in the absence of a calcium
gradient across the membrane, the calcium pump cycle step in which reorientation of the calcium binding sites occurs cannot
be identified with the isomerization step mentioned above.