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Substrate Regulation of Membrane Phosphorylation and of Ca2+ Transport in the Sarcoplasmic Reticulum

1973; Elsevier BV; Volume: 248; Issue: 10 Linguagem: Inglês

10.1016/s0021-9258(19)43982-3

1083-351X

Leopoldo de Meis, Maria Christina F. de Mello,

Pancreatic function and diabetes

Abstract Calcium uptake, Ca2+-dependent nucleoside triphosphatase activity, and Ca2+-dependent transfer of the γ-phosphate of nucleoside triphosphate to the membrane protein of isolated sarcoplasmic reticulum fragments were studied using different substrates. Ca2+ is a limiting factor for maximal membrane phosphorylation. The Ca2+ concentration required for half-maximal membrane phosphorylation varies with the temperature, the substrate used, and its concentration. Using ITP or GTP concentrations ranging from 10 µm to 60 µm at 0°, it is possible to measure the initial rate of membrane phosphorylation. With the use of ATP, this rate is very fast and cannot be measured by the techniques employed. The apparent Km for membrane phosphorylation at 0° was determined for GTP (0.45 µm) and ITP (0.31 µm). Through competitive experiments the apparent Km of other substrates were calculated. The values found were: acetyl phosphate, 3.8 x 10-4 m; UTP, 1.3 x 10-4 m; CTP, 4.0 x 10-5 m, and ATP, 2.4 x 10-8 m. The membrane phosphorylation reaction can be reversed by different nucleoside diphosphates. At 0° the ability of CDP, IDP, or GDP to reverse this reaction is 180 times lower than ADP. ITP, GTP, and ATP increase the rate of membrane dephosphorylation. Alkali ions inhibit the Ca2+ transport, substrate hydrolysis, and membrane phosphorylation of skeletal muscle sarcoplasmic reticulum vesicles. The inhibitory activity of Na+ and K+ varies with the substrate used and its concentration, the Ca2+ concentration and the temperature. A reaction sequence is proposed on the basis of the data presented.