Evidence for Association of an ATP-Stimulatable Ca2+-Independent Phospholipase A2 from Pancreatic Islets and HIT Insulinoma Cells with a Phosphofructokinase-like Protein

Abstract

Glucose-induced insulin secretion from pancreatic islets requires metabolism of glucose within islet β-cells, and ATP has attracted interest as a messenger of glucose metabolism within β-cells. Glucose-induced insulin secretion from islets and HIT insulinoma cells is accompanied by activation of an ATP-stimulatable Ca²⁺-independent phospholipase A₂ (ASCI-PLA₂) enzyme, the catalytic activity of which resides in a 40 kDa protein. An analogous PLA₂ enzyme in myocardium was recently found to consist of a complex of a 40 kDa catalytic protein with a tetramer of an isoform of the glycolytic enzyme phosphofructokinase (PFK). Association of the PFK isoform with the myocardial PLA₂ catalytic protein was found to confer ATP sensitivity onto the enzyme complex. Here we demonstrate that the majority of HIT cell and islet ASCI-PLA₂ catalytic activity elutes from a gel filtration column in a region corresponding to 400 kDa, suggesting that the 40 kDa β-cell ASCI-PLA₂ catalytic protein exists as part of a larger molecular mass complex. Islet and HIT cell ASCI-PLA₂ activities were immunoprecipitated by antibodies directed against PFK, and the immunoprecipitates contained 40 and 85 kDa proteins which correspond to the molecular masses of the PLA₂ catalytic protein and of a PFK monomer, respectively. Islet and HIT cell ASCI-PLA₂ activities were selectively and reversibly adsorbed to affinity matrices containing immobilized PFK but not to similar matrices containing immobilized transferrin or bovine serum albumin. Addition of free PFK prevented binding of HIT cell ASCI-PLA₂ activity to immobilized PFK matrices and promoted desorption of activity previously bound to such matrices. These results suggest that β-cell ASCI-PLA₂, like the myocardial enzyme, exists as a complex comprised of a catalytic protein and a PFK-like protein and raise the possibility that the ASCI-PLA₂ complex may represent a component of the β-cell glucose sensor, which links glycolysis, phospholipid hydrolysis, and membrane electrochemical events involved in glucose-induced insulin secretion.