STUDIES ON THE ANCHORAGE OF ATP-DIPHOSPHOHYDROLASE IN SYNAPTIC PLASMA-MEMBRANES FROM RAT-BRAIN

ATP diphosphohydrolases are described as ecto-enzymes in several tissu es. In the present study, synaptic plasma membrane (SPM) was exposed t o a series of agents used to distinguish between peripheral (hydrophil ic), G-PI-anchored and transmembrane-polypeptide-anchored membrane pro teins. These procedures included: (a) nondetergent extraction, (b) Tri ton X-114 phase partitioning, (c) phosphatidylinositol-specific phosph olipase C (PI-PLC) extraction and (d) protease incubation. In cases (a ): (c) and (d) the SPM was incubated with different agents and the ATP ase-ADPase activities and the protein concentration was determined in the original sample, in the pellet and in the supernatant obtained aft er 100,000g centrifugation. In procedure (b), the SPM was solubilized in 1% triton X-114 and submitted to phase separation onto a sucrose cu shion. The aqueous and detergent rich phases obtained by this treatmen t were assayed for ATPase-ADPase activities and protein determination. The results obtained suggest an intrinsic behaviour for ATP diphospho hydrolase since none of the nondetergent treatments was efficient in r emoving the enzyme from SPM. Moreover, ATPase and ADPase activities we re recovered predominantly (>50%) in the detergent-rich phase obtained by Triton X-114 partitioning. The enzyme was not released by PI-PLC o r proteases. These results indicate that the enzyme is not a GPI-ancho red protein, but is probably deeply anchored on the plasma membrane in agreement with the amino acid sequence of the enzyme recently publish ed. (C) 1998 Elsevier Science Ltd. All rights reserved.