Rabbit fast skeletal muscle phospholipase C Molecular weight determination by renaturation after polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate

[摘要]

Phosphoinositide specific phospholipase C from rabbit fast skeletal muscle has been enriched ca. 1,000-fold with a specific activity of 40 μmol × min⁻¹ × mg⁻¹. Following SDS-PAGE, renaturation of the enzyme protein in the presence of deoxycholate allowed the determination of an apparent molecular weight of 110 kDa. Gel-filtration of the native enzyme resulted in a very similar apparent molecular weight of 115 kDa, however, associated proteins of higher molecular weight were also found. Free Ca²⁺ concentrations needed for half-maximal activation of PtdIns(4,5)P₂, PtdIns4P and PtdIns hydrolysis are 6.3 μM, 85 μM and 1.8 mM, and the K_m values for these substrates 102, 340 and 937 μM, respectively.

[学科分类] 生物化学/生物物理

[关键词] Phospholipase C;Fast skeletal muscle;Molecular weight determination;Renaturation;Ins(1;4;5)P3;d-myo-inositol 1;4;5-triphosphate;PtdIns;1-(3-sn-phosphatidyl)-1d-myo-inositol;PtdIns(4)P;1-(3-sn-phosphatidyl)-1d-myo-inositol-4-phospahte;PtdIns(4;5)P2;1-(3-sn-phosphatidyl)-1d-myo-inositol-4;5-bisphosphate;PtdSer;(3-sn-phosphatidyl)2;serine;PtdFln;(3-sn-phosphatidyl)ethanolamine;DOC;deoxycholate;SDS;sodium dodecylsulfate