(Ca + Mg)-stimulated ATPase activity of a rat brain microsomal preparation. |
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Authors: | J D Robinson |
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Affiliation: | Department of Pharmacology, State University of New York, Upstate Medical Center, Syracuse, New York 13210 USA |
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Abstract: | ATPase activity of freshly prepared brain microsomes was stimulated 20% when 0.1 mm CaCl2 was added in the presence of a “saturating” concentration of MgCl2 (4 mm). This (Ca + Mg)-stimulated activity declined rapidly on storage. Treatment of the microsomes with 0.12% deoxycholate in 0.15 m KCl, followed by centrifugation and resuspension in sucrose, produced a preparation both stable on storage at ?15 °C and with an increased stimulation in the presence of CaCl2. SrCl2 was more effective than CaCl2, but BaCl2 was a poor activator. KCl and NaCl stimulated the (Ca + Mg)-ATPase activity by reducing substrate (ATP) inhibition. The Km for ATP was 0.1 mm, a third that of the Mg-ATPase. CTP, ITP, and GTP could not substitute for ATP, although they were fair substrates for the Mg-ATPase. The energy of activation of the (Ca + Mg)-ATPase was 21 kcal, nearly twice that of the Mg-ATPase. After sucrose density-gradient centrifugation of the microsomal preparation, the (Ca + Mg)-ATPase activity was distributed with the (Na + K)-ATPase and not with the mitochondrial marker succinic dehydrogenase. Studies with ouabain, oligomycin, and azide distinguished the (Ca + Mg)-stimulated ATPase from (Na + K)- and mitochondrial ATPases. Sensitivity to ruthenium red suggested a link to Ca transport, although the microsomal 45Ca accumulating system was much more sensitive to the inhibitor than was this ATPase activity. |
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