Abstract
Clathrin-coated vesicles contain a proton translocating ATPase which operates in parallel with a chloride transporter (Xie, X.-S., Stone, D.K., and Racker, E. (1983) J. Biol. Chem. 258, 14834-14838). The polypeptide antibiotic, duramycin, has a dual inhibitory effect on clathrin-coated vesicle acidification. Low amounts of duramycin (5 micrograms/100 micrograms of protein) inhibit by 50% the proton translocation facilitated by chloride translocation. Under these conditions duramycin inhibits also 36Cl uptake when driven by either the electrogenic proton pump or by inward directed K+ movement in the presence of valinomycin. Higher amounts of duramycin (20 micrograms/100 micrograms of protein) are needed to inhibit by 50% the proton pump itself, as evidenced by reduced proton translocation facilitated by an outward potassium movement in the presence of valinomycin. In addition, the amount of duramycin needed to inhibit the proton pump corresponded well with the amount needed to inhibit the ouabain-insensitive, N-ethylmaleimide-sensitive ATPase activity of clathrin-coated vesicles.
Highlights
From the Department of Biochemistry, Molecularand Cell Biology, Division of Biological Sciences, Cornell University, Zthoca, New York 14853
All other chemicals and enzymes were obtained from effect on clathrin-coated vesicle acidification
Amounts of duramycin(5 pg/100 pg of protein) inhibit Preparations-Clathrin-coated vesicles wereprepared from 20 boby 50%the proton translocationfacilitatedby chloride vine brains as described [9]
Summary
Aminco DW2 dual wavelength spectrophotometer as A absorbance ment in the presence of valinomycin. The amount of duramycin needed to inhibofitacidification are expressed in arbitrary optical density units asthe the proton pump corresponded well with the amount needed to inhibtiht e ouabain-insensitive,N-ethylmaleimide-sensitiveATPase activity of clathrin-coatedvesinitial slope of ATP-induced quenching of acridine orange. Acidification of bovine brain clathrin-coated vesicles occurs by means of a proton translocating ATPase which is readily distinguished from the mitochondrialproton pump by its insensitivity to oligomycin, azide, and efrapeptin [1, 2]. We have shown previously [3] that generaforma azacolutu, has antimicrobialactivityagainstGramtion of ApH by the proton pump of clathrin-coated vesicles positive bacilli, most notably Bacillus subtilis [5].It is mark- requires the presence of both an active chloride transporter edly water-soluble and has 19 amino acid residues, including and the presence of chloride or bromide outside the vesicle.
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