Ramanadham S, Wolf M J, Ma Z, Li B, Wang J, Gross R W, Turk J
Division of Endocrinology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 1996 Apr 30;35(17):5464-71. doi: 10.1021/bi952652j.
Glucose-induced insulin secretion from pancreatic islets requires metabolism of glucose within islet beta-cells, and ATP has attracted interest as a messenger of glucose metabolism within beta-cells. Glucose-induced insulin secretion from islets and HIT insulinoma cells is accompanied by activation of an ATP-stimulatable Ca(2+)-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 beta-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 beta-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 beta-cell glucose sensor, which links glycolysis, phospholipid hydrolysis, and membrane electrochemical events involved in glucose-induced insulin secretion.
胰岛中葡萄糖诱导的胰岛素分泌需要胰岛β细胞内葡萄糖的代谢，而ATP作为β细胞内葡萄糖代谢的信使引起了人们的关注。胰岛和HIT胰岛素瘤细胞中葡萄糖诱导的胰岛素分泌伴随着一种ATP刺激的、不依赖钙离子的磷脂酶A2（ASCI-PLA2）的激活，该酶的催化活性存在于一种40 kDa的蛋白质中。最近发现心肌中一种类似的磷脂酶A2由一种40 kDa的催化蛋白与糖酵解酶磷酸果糖激酶（PFK）的一种同工型的四聚体组成的复合物构成。发现PFK同工型与心肌磷脂酶A2催化蛋白的结合赋予了该酶复合物ATP敏感性。在此我们证明，大多数HIT细胞和胰岛ASCI-PLA2催化活性从凝胶过滤柱上洗脱下来时，其洗脱区域对应于400 kDa，这表明40 kDa的β细胞ASCI-PLA2催化蛋白以更大分子量复合物的一部分形式存在。胰岛和HIT细胞的ASCI-PLA2活性被针对PFK的抗体免疫沉淀，免疫沉淀物中含有40 kDa和85 kDa的蛋白质，分别对应于磷脂酶A2催化蛋白和PFK单体的分子量。胰岛和HIT细胞的ASCI-PLA2活性被选择性地、可逆地吸附到含有固定化PFK的亲和基质上，但不吸附到含有固定化转铁蛋白或牛血清白蛋白的类似基质上。添加游离的PFK可阻止HIT细胞ASCI-PLA2活性与固定化PFK基质的结合，并促进先前结合到此类基质上的活性的解吸附。这些结果表明，β细胞ASCI-PLA2与心肌酶一样，以由催化蛋白和PFK样蛋白组成的复合物形式存在，并增加了ASCI-PLA2复合物可能代表β细胞葡萄糖传感器的一个组成部分的可能性，该传感器将糖酵解、磷脂水解以及与葡萄糖诱导的胰岛素分泌相关的膜电化学事件联系起来。
