Activation of the human P-glycoprotein ATPase by trypsin

The human MDR1 gene product, P-glycoprotein (Pgp), a tandemly duplicated mo lecule containing two putative ATP- and perhaps two drug-binding sites, is responsible for multidrug resistance in tumors. In this report, we characte rized the effects of trypsinization of Pgp on its ATPase function. Incubati on of Pgp-containing membranes with trypsin at a ratio of 1000: 1 (w/w) res ulted in a gradual increase in the basal- and the drug-stimulated ATPase ac tivities of Pgp in a time-dependent manner. The maximal basal-, verapamil-, and vinblastine-stimulated ATPase activities of the trypsinized Pgp were a pproximately 1.8-, 1.5-, and 1.75-fold higher than the activities of the na tive Pgp, respectively. Increased basal- and drug-stimulated ATPase activit ies of the Pgp were also observed when the ratio of membrane protein to try psin in the incubation mixtures was raised to 10:1 (w/w). Immunoblotting an alysis of Pgp tryptic digests using Pgp-specific NH(2)11, C219, and C494 an tibodies together revealed the degradation of full-length Pgp and formation of at least eight peptides migrating in the 36-60 kDa range. Immunoprecipi tation reactions using NH(2)11 and C494 antibodies have suggested that the peptides originating from the NH2 half of Pgp are in strong association wit h the COOH half of the peptide. These findings suggest that while Pgp fragm ents together exhibit the ATPase functional characteristics, Pgp possesses a cleavage activation site or region, and its cleavage leads to the activat ion of basal ATPase function of Pgp.