Alamethicin as a permeabilizing agent for measurements of Ca 2+-dependent ATPase activity in proteoliposomes, sealed membrane vesicles, and whole cells

Abstract

The channel-forming antibiotic peptide alamethicin was used in measurements of Ca-ATPase activity in sarcoplasmic reticulum (SR) vesicles, proteoliposomes containing Ca 2+-ATPase from SR, and native human platelets. Alamethicin was used as a permeabilizing agent providing for a free access of the whole cells or sealed vesicles interiors for ions, ATP, and other reactants. The experiments were carried out with the use of alamethicin preparations obtained in our laboratory and that purchased from the Upjohn Company (antibiotic U-22,324). A comparative study of the effects of Ca 2+-ionophore A23187 and alamethicin was performed on native SR vesicles containing Ca 2+-ATPase molecules with right orientation 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 Ca 2+ accumulation by the vesicles and therefore activates the Ca 2+-ATPase. Maximal specific activities of the Ca 2+-ATPase in native SR vesicles in the presence of either alamethicin, or A23187, or both of them, are equal in all cases to 20 activity units (μmol P i per min per mg protein). The operative range of alamethicin concentrations is 5–25 μg/ml and is a little wider than that for A23187. The ATPase activity of the SR vesicles treated with cholate reached 20 units in the presence of alamethicin while in the presence of A23187 it was only 10 units. These data suggest that alamethicin unlike A23187 allows ATP to reach the ATPase's active centers from the inside of the SR vesicles with ‘randomized’ membranes, the ATP transport through the membrane not being the rate-limiting stage of ATP hydrolysis. It was shown that diffusion flux of ATP through a BLM in the presence of alamethicin may reach 10% of the flux through the hole without the BLM. With the use of alamethicin it was 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 possible the use of alamethicin in accurate measurements of Ca 2+-ATPase activity in whole cells. A method was developed for determination of the Ca 2+-ATPase activity of whole platelets. The membrane-bound Ca 2+-ATPase activity of human platelets was found to be 90–100 nmol P i per min per mg protein.