Phosphatidylinositol-4,5-bisphosphate phosphodiesterase and phosphomonoesterase activities of rat brain. Some properties and possible control mechanisms

Abstract

The phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] [and to a lesser extent, the phosphatidylinositol-4-phosphate (PtdIns4P)] phosphodiesterase and monoesterase activities of a rat brain supernatant were studied by using 32P-labeled substrates prepared from human red blood cells. PtdIns(4,5)P2 monoesterase is maximally stimulated by Mg2+, though some activity is detectable in Ca2+/EDTA (Mg2+-free) buffers. The phosphodiesterase is Ca2+-dependent, and in Ca2+/EDTA buffers with the pure lipid as substrate, shows maximal activity at 100 nM-Ca2+. If PtdIns(4,5)P2 is presented as a component of a lipid mixture of similar composition to that of the inner half of the lipid bilayer of a rat liver plasma membrane, the phosphodiesterase shows considerable activity at 1 .mu.M-Ca2+, and is maximal at 100 .mu.M-Ca2+. If it is assayed against the same substrate in Ca2+/EGTA [ethylene glycol bis-(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid] buffers with 3 mM-Mg2+ and 80 mM-KCl present (as an approximate parallel with the ionic environment in vivo), it shows no detectable activity below 100 .mu.M-Ca2+, and is maximal at 1 mM-Ca2+. The monoesterase can hydrolyze PtdIns(4,5)P2 in such a lipid mixture at all Ca2+ concentrations with 1 or 3 mM-Mg2+ present. PtdIns(4,5)P2 phosphodiesterase can be induced to attack its substrate under ionic conditions similar to those in vivo (0.1-1 .mu.M-Ca2+; 1 mM-Mg2+; 80 mM-KCl) by the conversion of its substrate into a non-bilayer configuration. If given such a substrate [by mixing PtdIns(4,5)P2 with an excess of phosphatidylethanolamine (PtdEtn)] it shows a shallow Ca2+-dependency curve from 0.1-100 .mu.M and then a steep rise to 1 mM-Ca2+. A perturbation in a membrane in vivo equivalent to a non-bilayer configuration would be sufficient to induce phosphodiesterase-catalyzed PtdIns(4,5)P2 breakdown. When given substrates mixed with excess PtdEtn at pH 7.25 (or 5.5), 1 .mu.M-Ca2+, 1 mM-Mg2+ and 80 mM-KCl, the rat brain supernatant phosphodiesterase activity hydrolyzed PtdIns(4,5)P 50- to 100-fold faster than it hydrolyzed phosphatidylinositol (PtdIns). If the supernatant was presented with such a non-bilayer mixture containing a 10-fold excess of PtdIns over PtdIns(4,5)P2, the latter phospholipid was still hydrolyzed by phosphodiesterasic cleavage at nearly 10 times the rate of the former. Receptor-stimulated phosphodiesterase cleavage of polyphosphoinositides is an early event in cell activation by many agonists. The properties of PtdIns(4,5)P2 phosphodiesterase in vitro suggest a change in the presentation of its substrate would be a sensitive and sufficient control on the enzyme''s activity in vivo.