P-Glycoprotein (Pgp) was isolated from CHRC5 membranes by selective detergent extraction and further purified by lentil lectin affinity chromatography. The purified product displayed a very high basal ATPase activity (1.65 mumol/min per mg protein in the absence of added drugs or lipids) with an apparent Km for ATP of 0.4 mM. There was no evidence of cooperativity, suggesting that the two ATP sites operate independently of each other. Pgp ATPase activity was stimulated by verapamil, trifluoperazine and colchicine, and inhibited by daunomycin and vinblastine. All drugs and chemosensitizers acted as mixed activators or inhibitors, producing changes in both the Vmax of the ATPase and the Km for ATP. ADP competitively inhibited Pgp ATPase, with a Ki of 0.2 mM. The macrolide antibiotics bafilomycin A1, concanamycin A and concanamycin B, inhibited Pgp ATPase at concentrations of 0.1-10 microM, and at an inhibitor:protein stoichiometry of 0.65-1.0 mumol/mg protein, which is at the low end of the range characteristic of P-type ATPases. Pgp ATPase was relatively selective for adenine nucleotides. Several phospholipids stimulated Pgp ATPase activity in a dose-dependent manner, whereas others produced inhibition. Metabolic labelling showed that the endogenous phospholipids associated with purified Pgp consisted largely of phosphatidylethanolamine and phosphatidylserine, with only a small amount of phosphatidylcholine. 32P-Labelling studies indicated that purified Pgp was partially phosphorylated. It can be concluded that Pgp is a constitutively active, adenine nucleotide-specific ATPase whose catalytic activity can be modulated by both drugs and phospholipids.

The prokaryotic permeases are members of a superfamily of membrane transporters called traffic ATPases, which includes the medically important eukaryotic multidrug resistance (MDR) protein and cystic fibrosis transmembrane regulator (CFTR). Members of this superfamily have extensive sequence and structural similarity, in particular in an ATP-binding motif, and are believed to use ATP to energize translocation of substrates across biological membranes. The prokaryotic histidine permease is well-characterized and serves as a convenient model system. In this review, we highlight some of the biochemical and molecular biological approaches used to study the functional and architectural organization of this permease and relate the results of these approaches to what is known about other traffic ATPases. We have identified specific regions that we believe critical for the function of the histidine permease and propose that the corresponding regions in the eukaryotic traffic ATPases are also important for their function. In light of the fact that CFTR (and possibly the MDR protein) is an ion channel, we compare the properties of channels and transporters; in addition, we discuss the possibility that other members of the traffic ATPases may also have channel-like activity.

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