Various membrane ATPases have been tested for their sensitivity to baflomycin Al, a macrolide antibiotic. F1Fo ATPases from bacteria and mitochondria are not affected by this antibiotic. In contrast, ElE2 ATPases-e.g., the K+-dependent (Kdp) ATPase from Escherichia coli, the Na4+,K+ -ATPase from ox brain, and the Ca2+-ATPase from sarcoplasmic reticulum-are moderately sensitive to this inhibitor. Finally, membrane ATPases from Neurospora vacuoles, chromaffin granules, and plant vacuoles are extremely sensitive. and, in the case of the Na4 ,K4-ATPase, by ouabain (5-11).The vacuolar ATPases appear to hydrolyze ATP, generating a proton gradient that is used for acidification of compartments within cells (12,17,22). This group of ATPases has been distinguished from the other two by virtue of its inhibitor specificity. The vacuolar ATPases are not inhibited by azide, oligomycin, vanadate, or ouabain. Instead, inhibitors that have proved useful for these enzymes include (i) N,N'-dicyclohexylcarbodiimide, which also inhibits F1F0 and E1E2 ATPases, (ii) N-ethylmaleimide, which inhibits E1E2 enzymes (23) and at least one F1F0 ATPase (24), and (iii) NO3 -, which is effective only at millimolar concentrations or greater. Thus, no potent specific inhibitor of vacuolar ATPases has yet been identified.The bafilomycins A1, B1, C1, and D, macrolide antibiotics with a 16-membered lactone ring, were isolated from Streptomyces sp. (25). These compounds inhibited growth of Gram-positive bacteria and fungi in a disc diffusion assay. In addition, it has been reported that bafilomycin C1 inhibits the enzymatic activity of the Na4, 27). In this communication, we compare the effects of bafilomycin A1 on representative enzymes of the three classes of ATPases. The results show that bafilomycin A1 is useful for distinguishing among the different types of ATPases and that it is an extremely potent inhibitor of the vacuolar ATPases.

Various ATPases have been tested for their sensitivity to naturally occurring unusual macrolides and their chemically modified derivatives, which are structurally related to bafilomycin A1 (1), the first specific inhibitor of vacuolar ATPases. The structure-activity study showed that in general the concanamycins, 18-membered macrolides, are better and more specific inhibitors than the bafilomycins of this class of membrane-bound ATPases. The additional carbohydrate residue is not responsible for the improved activity. The importance of an intact hemiketal ring, which is part of an intramolecular hydrogen-bonding network, and the effects of the size of the macrolactone ring are discussed. The structurally related elaiophylin (13), a C2-symmetric macrodiolide antibiotic, proved to be inactive on vacuolar ATPases but still retained its inhibitory effect on P-type ATPases.

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Mutants of Escherichia coli lacking all of the known saturable K ؉ transport systems, "triple mutants," require elevated K ؉ concentrations for growth. K ؉ transport activity in such mutants, called TrkF activity, has low substrate specificity and a low rate that increases with increasing external pH. Attempts to isolate mutants requiring even higher concentrations of K ؉ failed, implying that either TrkF is essential or is composed of multiple minor K ؉ transport activities. Instead, we sought mutations that allowed triple mutants to grow at lower K ؉ concentrations. Mutations so identified include ones altering MscL, the large mechanosensitive channel, or Opp, the oligopeptide permease. However, a possible contribution of wild-type Opp and MscL to TrkF activity was not proven. In contrast, expression of wild-type ProP, TrkG, and TrkH proteins increased uptake when encoded on multicopy plasmids. In all of these situations, the driving force for K ؉ appeared to be the transmembrane electric potential, and in most cases substrate specificity was low; these are characteristics of TrkF activity. These results support the view that TrkF is composed of multiple, "aberrant" K ؉ transport activities, i.e., paths that, regardless of their physiological function, allow K ؉ to cross the cell membrane by a uniport process.