Regulatory role of adenosine deaminase complexing protein (dipeptidyl peptidase IV=CD26) on the malignancy marker adenosine deaminase: Effect of membrane cholesterol and phase-transition

While adenosine deaminase (ADA) is an established malignancy marker and its clinical involvement in severe combined immunodeficiency (SCID) is underst ood, the biological significance of its binding to adenosine deaminase comp lexing protein (CP = DPPIV = CD26) remains enigmatic. The role of lipid-pro tein interactions in the modulation of ADA activity by membrane-bound CP wa s sought. ADA bound specifically to CP reconstituted in L-alpha-dimyristoyl phosphatidylcholine (DMPC) or asolectin vesicles. Without CP, the binding a nd specific activity of ADA were negligible. In the presence of CP, the spe cific activity recovered to values similar to those in buffer. The addition of cholesterol increased the fluorescence anisotropy of 1,6-diphenyl-1,3,5 -hexatriene (DPH)-labeled vesicles and resulted in a "bell-shaped" dependen ce of the specific activity of ADA on cholesterol concentration. Vesicles w ith small cholesterol ratios mimicked Rouse sarcoma virus-transformed chick embryo fibroblasts (CEF) in that both had reduced ADA activity and increas ed membrane fluidity. In contrast to continuous Arrhenius plots of ADA acti vity in solution, free or bound to CP, ADA bound to CP reconstituted in DMP C vesicles exhibited two breaks, around 25 and 13 degrees C, yielding three lints with similar apparent activation energies (E-a). Increased ADA activ ity of similar to 30% was observed at each of these discontinuities. A mode l in which active site accessibility is dependent on membrane fluidity led to a successful simulation of the phase transitions. This model could also account for ADA reduced activity associated with increased membrane fluidit y in transformed vs. normal fibroblasts. (C) 2000 Wiley-Liss, Inc.