Characterization of Mg2+-ATPase activity in isolated B16 murine melanoma melanosomes

B16 murine melanoma melanosomes were purified using sucrose density gradient centrifugation. ATPase activity was evaluated in presence of specific ATPase inhibitors, and compared with melanosome ATP-driven proton translocating activity in the melanosome. Mg2+ dependent ATPase activity was greatly inhibited (82%) by the specific inhibitors of vaculor proton translocating ATPase; Cis-didimethylsulfoxide dichloroplatinum (II) at approximately 90 mu M and bafilomycin AI at two fold higher concentrations. Less inhibition, about 30 and 45% was obtained with N, N-1-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 mu M and 0.1-1.5 mM ranges, respectively. These drugs at similar concentrations also inhibited the proton pumping activity to the same extent as observed for ATPase activity and half-maximal inhibition of each activity was found at nearly similar concentrations. Carbonylcyanide p-trifluoromethoxyphenyl hydra zone (FCCP) prevented ATP from setting up a pH gradient across the melanosomal membrane but stimulated Mg2+ ATPase activity significantly. Replacement of 5 mM. Mg2+ with equimolar Ca2+ brought about a 60% inhibition in divalent cation -dependentATPase- activity, and an 85% inactivation of ATP-linked melanosomal H(+)pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+ ATPase activity was similar to that seen in a Mg2+ medium. In Ca2+ medium ATPase activity was inhibited by CDDP and stimulated by FCCP, however these effects were two to three fold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in 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 F- medium, and increased in the order of F- <So(4)(2-)< CL- = Br-. These results show that the ATPase activity may be related to the melanosomal proton pump.