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  • Journal
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    ScienceDirect
    v. 247-251
    ScienceDirect
    v. 247
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    Stored offsite. Please request print.
    v. 247-251, 1980-82.
    SHELVED UNDER: Zentralblatt für Bakteriologie Originale Ser. "A".
    5
  • Article
    Njus D, Sehr PA, Radda GK, Ritchie GA, Seeley PJ.
    Biochemistry. 1978 Oct 03;17(20):4337-43.
    ATP hydrolysis and proton translocation in chromaffin granules were followed using 31P nuclear magnetic resonance. The intragranular pH affects the resonance frequency of the gamma-phosphate of granular ATP. By measuring frequency vs. pH in solutions which simulate the intragranular matrix, this may be calibrated to give quantitative pH measurements. The pH in the resting granule is 5.65 +/- 0.15. This drops by 0.4 to 0.5 pH unit when ATP is added externally and protons are actively pumped into the granules. Because of differences in the composition and pH of the internal and external solutions, the resonances of internal and external nucleotides and Pi can be distinguished. Consequently, ATP hydrolysis and changes in internal pH may be observed simultaneously and continuously in a single sample of chromaffin granules. From the measured buffering capacity of a reconstituted intragranular solution, pH changes were converted into an absolute number of protons translocated. The net proton flux (protons translocated/ATP hydrolyzed) was about 1.0 immediately after external ATP addition but fell toward zero as the pH gradient increased to a new steady state. These 31P NMR results agree with intragranular pH measurements determined from methylamine distribution and with H+/ATP stoichiometries calculated from pH changes observed in the external medium.
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