An isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isolated and purified from rabbit brain cytosol has previously been demonstrated to catalyze membrane fusion (Glaser and Gross, Biochemistry 33 (1994) 5805-5812; Glaser and Gross, Biochemistry 34 (1995) 12193-12203). Herein, we provide evidence suggesting that this GAPDH isoform can reconstitute in vitro protein-catalyzed fusion between naturally occurring subcellular membrane fractions involved in insulin exocytosis. Utilizing purified rat pancreatic β-cell plasma membranes and secretory granules, we show that a brain cytosolic factor catalyzed the rapid and efficient fusion of these two purified membrane fractions which could be inhibited by a monoclonal antibody directed against the brain isoform of GAPDH. Moreover, the brain cytosolic factor also catalyzed the fusion of reconstituted vesicles prepared from lipid extracts of islet plasma membranes and secretory granules. Although the brain cytosolic factor rapidly catalyzed membrane fusion between islet plasma membranes and secretory granules, it did not catalyze fusion between one secretory granule population with another. To identify the potential importance of brain cytosolic factor catalyzed membrane fusion in islet cells, we examined extracts of hamster insulinoma tumor cells (HIT cells) for fusion-catalyzing activity. A protein constituent was present in HIT cell cytosol which was immunologically similar to the rabbit brain GAPDH isoform. Although native HIT cell cytosol did not catalyze membrane fusion, removal of an endogenous protein inhibitor unmasked the presence of the protein which catalyzed membrane fusion activity and such fusion was ablated by a monoclonal antibody directed against the brain isoform of GAPDH. Collectively, these results suggest the possibility that an isoform of brain GAPDH, also evident in HIT cells, can catalyze fusion between the two naturally occurring subcellular membrane compartments involved in insulin secretion and suggest a novel paradigm potentially coupling glycolytic flux with insulin release. Copyright (C) 1998 Elsevier Science B.V.