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Phosphoinositide kinases in rat heart sarcolemma: biochemical properties and regulation by calcium

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Abstract

Phosphatidylinositol (PtdIns) kinase and phosphatidylinositol 4-phosphate (PtdIns4P) kinase have been studied in a purified sarcolemmal fraction isolated from rat heart. Both enzymes were Mg2−-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 µM and 1390 ± 80 pmol · mg−1 · min−1, respectively, while the apparent Km and Vmax values for PtdIns4P kinase were 398 ± 25 µM 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 µM, respectively, and was independent of either Ca2+-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.

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Authors and Affiliations

  1. Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and Departments of Anatomy and Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada

    Nasrin Mesaeli & Vincenzo Panagia

  2. Department of Biochemistry, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, Rotterdam, The Netherlands

    Jos M. J. Lamers

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  1. Nasrin Mesaeli
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  3. Vincenzo Panagia
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Mesaeli, N., Lamers, J.M.J. & Panagia, V. Phosphoinositide kinases in rat heart sarcolemma: biochemical properties and regulation by calcium. Mol Cell Biochem 117, 181–189 (1992). https://doi.org/10.1007/BF00230758

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  • DOI: https://doi.org/10.1007/BF00230758

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