Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat β-cell tumour

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Abstract
Subcellular fractions were isolated from a rat .beta.-cell tumor [insulinoma] by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [.gamma.-32P]ATP, and labeling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 to 5 .mu.M. The enzyme had a Km (apparent) for ATP of 110 .mu.M (secretory granule) or 120 .mu.M (plasma membrane) and a Ka of Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 or 40 .mu.M-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 .mu.M. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. In the tumor .beta.-cell Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides may be present in the secretory granule and plasma membrane.

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