Phosphoinositide kinases in rat heart sarcolemma: biochemical properties and regulation by calcium.
Phosphatidylinositol (PtdIns) kinase and phosphatidylinositol 4-phosphate (PtdIns4P) kinase have been studied in a purified sarcolemmal fraction isolated from rat heart. Both enzymes were Mg(2+)-dependent and their activities were maximal at 2.5 mM Mg2+ and pH 7.5. Kinetic analysis of endogenous substrate phosphorylation by ATP showed that the apparent Km and Vmax values for PtdIns kinase were 292 +/- 17 microM and 1390 +/- 80 pmol.mg-1.min-1, respectively, while the apparent Km and Vmax values for PtdIns4P kinase were 398 +/- 25 microM and 382 +/- 24 pmol.mg-1.min-1. Under normal conditions, the activity of PtdIns4P kinase was lower than that of PtdIns kinase; however, the former activity increased several fold in the presence of PtdIns4P as an exogenous substrate. The enzymatic synthesis of intramembranal PtdIns4P and phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P2) was maximally enhanced by 0.1% Triton X-100 and inhibited by micromolar concentrations of Ca2+. Inhibition of PtdIns and PtdIns4P kinase showed IC50 values for Ca2+ of 20 and 6 microM, respectively, and was independent of either Ca(2+)-induced activation of phospholipase C and polyphosphoinositide monophosphoesterases or low ATP concentrations. The results indicate that purified rat heart sarcolemmal membranes contain a very active phosphoinositide phosphorylation system which is regulated by micromolar levels of Ca2+. The Ca2+ effect may contribute to the feedback inhibition of the receptor-activated formation of inositol 1,4,5-trisphosphate.
Mesaeli
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N
Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada.
Lamers
J M
JM
Panagia
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V
D019870:1-Phosphatidylinositol 4-Kinase; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D009355:Neomycin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases; D010770:Phosphotransferases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012508:Sarcolemma / Q000201:enzymology*
D015636:Magnesium Chloride; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D019870:1-Phosphatidylinositol 4-Kinase; D009355:Neomycin; D002118:Calcium