title: Characterization of an ATP-stimulatable Ca(2+)-independent phospholipase A2 from clonal insulin-secreting HIT cells and rat pancreatic islets: a possible molecular component of the beta-cell fuel sensor.
abstract: Isolated pancreatic islets from rats and humans express a plasmalogen-preferring ATP-stimulatable, Ca(2+)-independent phospholipase A2 (ASCI-PLA2) 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-PLA2 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-PLA2. (2) HIT cell ASCI-PLA2 is selectively activated and protected against thermal denaturation by ATP. (3) The magnitude of ASCI-PLA2 activation by the nonhydrolyzable ATP analog AMP-PCP is similar to that by ATP. (4) The ATP concentrations required to activate ASCI-PLA2 fall within physiologic ranges in the presence of Mg2+. (5) ADP induces a concentration-dependent attenuation of the activation of ASCI-PLA2 by ATP. HIT cell ASCI-PLA2 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-PLA2 activity associated with a single protein band with an apparent molecular mass of 40 kDa. Similar chromatographic behavior of the rat pancreatic islet ASCI-PLA2 activity was observed during sequential ATP-agarose and Mono-Q anion-exchange steps. These results indicate that HIT cells express an ASCI-PLA2 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.
authors.affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: S
authors.initials: S
authors.lastname: Ramanadham
authors.forename: M J
authors.initials: MJ
authors.lastname: Wolf
authors.forename: P A
authors.initials: PA
authors.lastname: Jett
authors.forename: R W
authors.initials: RW
authors.lastname: Gross
authors.forename: J
authors.initials: J
authors.lastname: Turk
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D008647:Mesocricetus; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D010955:Plasmalogens / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
chemical_list: D010955:Plasmalogens; D000255:Adenosine Triphosphate; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium