Abstract
Rat mast cells were purified by a Percoll gradient, and the granules were isolated from the sonicated mast cells on a Percoll gradient. The granules were shown to contain a diphosphoinositide kinase which catalyzes the formation of triphosphoinositide (TPI) from diphosphoinositide (DPI). The enzyme requires ATP and Mg2+ for the activity. TPI formation is almost completely dependent on MgCl2 or MnCl2, and maximal response is observed at 20 mM or 1 mM, respectively. The Km for ATP is 3μM. TPI formation in the granules is dependent on the reaction time. The maximal response is seen at 23°C. NaCl, KCl, Na2HPO4 and KH2PO4 have no effect on the activity. One hundred μM adenosine, AMP, ADP, and 10μM cyclic AMP inhibit the kinase activity.
