A sup 31 P NMR study of mitochondrial inorganic phosphate visibility: Effects of Ca sup 2+ , Mn sup 2+ , and the pH gradient

• Published in: Biochemistry (Easton) Vol. 31:5
• Main Authors: Hutson, S.M., Williams, G.D., Berkich, D.A., LaNoue, K.F., Briggs, R.W.
• Format: Journal Article
• Language: English
• Published: United States 11.02.1992
• Subjects: 550201 - Biochemistry- Tracer Techniques; BASIC BIOLOGICAL SCIENCES; CELL CONSTITUENTS; ISOTOPES; LIGHT NUCLEI; MAGNETIC RESONANCE; MANGANESE COMPOUNDS; MITOCHONDRIA; NUCLEAR MAGNETIC RESONANCE; NUCLEI; ODD-EVEN NUCLEI; OXYGEN COMPOUNDS; PH VALUE; PHOSPHATES; PHOSPHORUS 31; PHOSPHORUS COMPOUNDS; PHOSPHORUS ISOTOPES; RESONANCE; STABLE ISOTOPES; TEMPERATURE DEPENDENCE; TRANSITION ELEMENT COMPOUNDS
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00120a007

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