title: Proton pump-linked Mg2+-ATPase activity in isolated rat liver lysosomes.
abstract: ATPase activity in highly purified rat liver lysosome preparations was evaluated in the presence of other membrane cellular ATPase inhibitors, and compared with lysosome ATP-driven proton translocating activity. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 50% inhibition in divalent cation-dependent ATPase activity, and an 80% inactivation of ATP-linked lysosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+, ATPase activity was similar to that seen in an Mg2+ medium. Mg2+-dependent ATPase activity was greatly inhibited (from 70 to 80%) by the platinum complexes; cis-didimethylsulfoxide dichloroplatinum(II) (CDDP) at approximately 90 microM and cis-diaminedichloroplatinum(II) at twofold higher concentrations. Less inhibition, about 30 and 45%, was obtained with N,N'-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. The concentration dependence of inhibition by the above drugs was determined for both proton pumping and ATPase activities, and half-maximal inhibition concentration of each activity was found at nearly similar values. A micromolar concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented ATP from setting up a pH gradient across the lysosomal membranes, but stimulated Mg2+-ATPase activity significantly. ATPase activity in Ca2+ medium was also inhibited by CDDP and stimulated by FCCP, but both effects were two- to threefold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in a CDDP-supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton pump, was anion dependent. The lowest activity was recorded in a F-medium, and increased in the order of F- less than SO2-4 less than Cl- approximately equal to Br-. The CDDP-sensitive ATPase activity observed, supported by Mg2+ and less so by Ca2+, may be related to lysosome proton pump activity.
authors.affiliation: C.N.R. Center for the Study of Physiology of Mitochondria, University of Padova, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Dell'Antone
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D008099:Liver / Q000166:cytology* / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D009944:Organoplatinum Compounds / Q000494:pharmacology; D011522:Protons; D051381:Rats
chemical_list: D009944:Organoplatinum Compounds; D011522:Protons; C055423:didimethylsulfoxide dichloroplatinum(II); D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D008274:Magnesium; D005033:Ethylmaleimide; D002118:Calcium