A P-31 NMR-STUDY OF MITOCHONDRIAL INORGANIC-PHOSPHATE VISIBILITY - EFFECTS OF CA2+, MN2+, AND THE PH GRADIENT

The effects of external pH, temperature, and Ca2+ and Mn2+ concentrations on the compartmentation and NMR visibility of inorganic phosphate (P(i)) were studied in isolated rat liver mitochondria respiring on succinate and glutamate. Mitochondrial matrix P(i) is totally visible by NMR at 8-degrees-C and at low external concentrations of P(i). However, when the external P(i) concentration is increased above 7 mM, the pH gradient decreases, the amount of matrix P(i) increases, and the fraction not observed by NMR increases. Raising the temperature to 25-degrees-C also decreases the pH gradient and the P(i) fraction observed by NMR. At physiologically relevant concentrations, Ca2+ and Mn2+ do not seem to play a major role in matrix P(i) NMR invisibility. For Ca2+ concentrations above 30 nmol/mg of protein, formation of insoluble complexes will cause loss of P(i) signal intensity. For Mn2+ concentrations above 2 nmol/mg of protein, the P(i) peak can be broadened sufficiently to preclude detection of a high-resolution signal. The results indicate that mitochondrial matrix P(i) should be mostly observable up to 25-degrees-C by high-resolution NMR. While the exact nature of the NMR-invisible phosphate in perfused or in vivo liver is yet to be determined, better success at detecting and resolving both P(i) pools by NMR is indicated at high field, low temperature, and optimized pulsing conditions.