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Polyphosphoinositide formation in isolated cardiac plasma membranes
C Kasinathan1, Z C Xu, M A Kirchberger
1Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, NY 10029.
Lipids
|September 1, 1989
Summary
This study characterized phosphatidylinositol kinase activity in canine heart plasma membranes. ADP and calcium did not significantly inhibit these enzymes, suggesting cardiac responses to hormones during hypoxia are largely unaffected.
Area of Science:
- Biochemistry
- Cellular Physiology
- Cardiovascular Research
Background:
- Phosphatidylinositol (PtdIns) and its phosphorylated derivatives are crucial signaling molecules in cellular processes.
- Understanding the regulation of PtdIns kinases is vital for comprehending cellular responses, particularly in the myocardium.
- Plasma membrane-associated lipid kinases play key roles in signal transduction pathways.
Purpose of the Study:
- To characterize phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase activities in canine left ventricle plasma membranes.
- To determine the sensitivity of these kinase activities to intracellular variables like calcium and ADP.
- To assess the potential impact of these enzymes' regulation on myocardial response to alpha-adrenergic hormones during hypoxia.
Main Methods:
- Isolation of plasma membranes from canine left ventricle.
- Assay of PtdIns and PtdIns4P kinase activities by measuring the formation of [32P]PtdIns4P and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PtdIns(4,5)P2) using [gamma-32P]ATP.
- Investigation of enzyme sensitivity to varying concentrations of Ca2+ and ADP, and determination of kinetic parameters (Km, Ki).
Main Results:
- PtdIns kinase activity produced [32P]PtdIns4P, while PtdIns4P kinase activity, requiring exogenous substrate, produced [32P]PtdIns(4,5)P2.
- Both kinase activities were insensitive to Ca2+ (0.1-30 microM).
- ADP showed competitive inhibition of PtdIns kinase with respect to ATP (Ki = 0.4 mM), but this inhibition is considered weak at physiological intracellular concentrations.
Conclusions:
- Canine left ventricle plasma membranes possess PtdIns and PtdIns4P kinase activities that are largely unaffected by physiological concentrations of Ca2+.
- ADP is a poor competitive inhibitor of PtdIns kinase under conditions relevant to normal or mildly hypoxic myocardium.
- The study suggests that myocardial responses to alpha-adrenergic hormones during mild hypoxia would likely be unimpaired by Ca2+ or ADP effects on these specific lipid kinases.