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Rapid filtration measurements of Ca 2+ release from cisternal sarcoplasmic reticulum vesicles
Author(s) -
Sumbilla Carlota,
Inesi Giuseppe
Publication year - 1987
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(87)81292-9
Subject(s) - vesicle , endoplasmic reticulum , chemistry , biophysics , calcium , magnesium , liberation , filtration (mathematics) , molar concentration , chromatography , membrane , analytical chemistry (journal) , biochemistry , in vitro , biology , organic chemistry , statistics , mathematics
A radioactive tracer and rapid filtration method was applied to the study of Ca 2+ release from sarcoplasmic reticulum (SR) vesicles which were preloaded passively (equilibration with millimolar Ca 2+ ) or actively (in the presence of ATP or acetyl phosphate). The method allows complete substitution of the loading mixture with release medium in constant flow, and time resolution between 0.01 and 10.0 s. Net release can be clearly distinguished from isotope exchange. The latter is prominent in longitudinal SR vesicles. Net Ca 2+ release is observed only from cisternal SR vesicles, is Ca 2+ (micromolar) dependent, and is accelerated by inactive ATP analogues, or ATP itself, even in the presence of Mg 2+ . Net release has a strong pH dependence (between 6 and 7), and very little temperature dependence (consistent with a passive channel). In media of physiological significance (1 mM ATP, 1 mM magnesium, and free Ca 2+ in the micromolar range), net Ca 2+ release proceeds with a rate constant of approx. 100 s −1 .