ALAMETHICIN AS A PERMEABILIZING AGENT FOR MEASUREMENTS OF CA2-DEPENDENT ATPASE ACTIVITY IN PROTEOLIPOSOMES, SEALED MEMBRANE-VESICLES, AND WHOLE CELLS()

The channel-forming antibiotic peptide alamethicin was used in measure ments of Ca-ATPase activity in sarcoplasmic reticulum (SR) vesicles, p roteoliposomes containing Ca2+-ATPase from SR, and native human platel ets. Alamethicin was used as a permeabilizing agent providing for a fr ee access of the whole cells or sealed vesicles interiors for ions, AT P, and other reactants. The experiments were carried out with the use of alamethicin preparations obtained in our laboratory and that purcha sed from the Upjohn Company (antibiotic U-22,324). A comparative study of the effects of Ca2+-ionophore A23187 and alamethicin was performed on native SR vesicles containing Ca2+-ATPase molecules with right ori entation and SR vesicles treated with cholate in order to randomize Ca 2+-ATPase molecules orientation in the membrane. It was found out that alamethicin, like A-23187, prevents the ATP-dependent Ca2+ accumulati on by the vesicles and therefore activates the Ca2+-ATPase. Maximal sp ecific activities of the Ca2+-ATPase in native SR vesicles in the pres ence of either alamethicin, or A23187, or both of them, are equal in a ll cases to 20 activity units (mumol P(i) per min per mg protein). The operative range of alamethicin concentrations is 5-25 mug/ml and is a little wider than that for A23187. The ATPase activity of the SR vesi cles treated with cholate reached 20 units in the presence of alamethi cin while in the presence of A23187 it was only 10 units. These data s uggest that alamethicin unlike A23187 allows ATP to reach the ATPase's active centers from the inside of the SR vesicles with 'randomized' m embranes, the ATP transport through the membrane not being the rate-li miting stage of ATP hydrolysis. It was shown that diffusion flux of AT P through a BLM in the presence of alamethicin may reach 10% of the fl ux through the hole without the BLM. With the use of alamethicin it wa s found out that the quality of randomization of the ATPase molecules orientation in the membrane depends on the proteoliposome preparation technique. The ATP transport through the alamethicin pores makes possi ble the use of alamethicin in accurate measurements of Ca2+-ATPase act ivity in whole cells. A method was developed for determination of the Ca2+-ATPase activity of whole platelets. The membrane-bound Ca2+-ATPas e activity of human platelets was found to be 90-100 nmol P(i) per min per mg protein.