CHARACTERIZATION OF AN ATP-STIMULATABLE CA2+-INDEPENDENT PHOSPHOLIPASE A(2) FROM CLONAL INSULIN-SECRETING HIT CELLS AND RAT PANCREATIC-ISLETS - A POSSIBLE MOLECULAR-COMPONENT OF THE BETA-CELL FUEL SENSOR

Isolated pancreatic islets from rats and humans express a plasmalogen-preferring ATP-stimulatable, Ca2+-independent phospholipase A(2) (ASCI-PLA(2)) enzyme which participates in the glucose-stimulated hydrolysis of arachidonate from membrane phospholipids and in insulin secretion. Here we report that clonal insulin-secreting HIT beta-cells contain substantial amounts of endogenous plasmalogens and express a similar ASCI-PLA(2) activity with the following properties: (1) Enzymatic activity as well as glucose-induced eicosanoid release and insulin secretion are inhibited by a mechanism-based suicide substrate directed towards ASCI-PLA(2). (2) HIT cell ASCI-PLA(2) is selectively activated and protected against thermal denaturation by ATP. (3) The magnitude of ASCI-PLA(2) activation by the nonhydrolyzable ATP analog AMP-PCP is similar to that by ATP. (4) The ATP concentrations required to activate ASCI-PLA(2) fall within physiologic ranges in the presence of Mg2+. (5) ADP induces a concentration-dependent attenuation of the activation of ASCI-PLA(2) by ATP. HIT cell ASCI-PLA(2) exhibited an apparent isoelectric point of 7.5 on chromatofocusing analysis and was quantitatively adsorbed to an ATP-agarose matrix and selectively desorbed from this column by ATP. Mono-Q anion-exchange analysis of the active ATP-agarose eluant yielded a peak of ASCI-PLA(2) activity associated with a single protein band with an apparent molecular mass of 40 kDa. Similar chromatographic behavior of the rat pancreatic islet ASCI-PLA(2) activity was observed during sequential ATP-agarose and Mono-Q anion-exchange steps. These results indicate that HIT cells express an ASCI-PLA(2) similar to the analogous islet enzyme and suggest that expression of this enzyme and of its preferred plasmalogen substrates may be a general property of insulin-secreting beta-cells.