Rapid kinetic characterization of active transport and passive release of calcium in vesicular fragments of longitudinal and junctional sarcoplasmic reticulum.
1. Vesicular fragments of longitudinal and cisternal-junctional sarcoplasmic reticulum can be obtained, selectively, from skeletal muscle. 2. Longitudinal sarcoplasmic reticulum vesicles have a high content of Ca2+-ATPase, and provide the best system for characterization of the calcium pump. Equilibrium and transient state measurements provide equilibrium and kinetic constants for the partial reactions of the catalytic and transport cycle, including binding and translocation steps. Free energy estimates are then obtained based on an explicit mechanism, rather than simply on a transmembrane gradient (which is independent of the reaction mechanism). Kinetic evidence for a sequential pattern of calcium binding and translocation is presented. 3. The cisternal-junctional membrane contains (in addition to Ca2+-ATPase) calsequestrin and a large protein (MW greater than or equal to 350,000 Da) which corresponds to the junctional processes ("feet") and binds ryanodine. This protein is involved in rapid release of Ca2+ from loaded vesicles.
Inesi
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Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
D001693:Biological Transport, Active; D002118:Calcium / Q000493:pharmacokinetics*; D000252:Calcium-Transporting ATPases / Q000493:pharmacokinetics*; D005374:Filtration; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D000252:Calcium-Transporting ATPases; D002118:Calcium