The concentration and control of cytoplasmic free inorganic pyrophosphate in rat liver in vivo

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Abstract
The concentration of cytoplasmic free pyrophosphate was calculated in freeze-clamped livers of rats from the measured concentration of reactants and Keq. of the UDP-glucose pyrophosphorylase reaction (UDP–α-d-glucose 1-phosphate uridylyltransferase, EC 2.7.7.9). The Keq. of the UDP-glucose pyrophosphorylase reaction was redetermined at 38°C, pH7.0, I=0.25mol/l and free [Mg2+]=1mm, and was 4.55 in the direction of glucose 1-phosphate formation. The activity of UDP-glucose pyrophosphorylase in rat liver was between 46 and 58μmol of glucose 1-phosphate formed/min per g fresh wt. in the four dietary conditions studied. A fluorimetric assay with enzymic cycling was developed for the measurement of glucose 1-phosphate in HClO4 extracts of rat liver. The calculated free cytoplasmic PPi concentration in nmol/g fresh wt. of liver was 2.3±0.3 in starved, 3.8±0.4 in fed, 4.9±0.6 in meal-fed and 5.2±0.4 in sucrose-re-fed animals. These values agree well with recently determined direct measurements of total PPi in rat liver and suggest that there is not a large amount of bound or metabolically inert PPi in rat liver. The cytoplasmic [ATP]/[AMP][PPi] ratio is 103 times the cytoplasmic [ATP]/[ADP][Pi] ratio and varies differently with dietary state. The reaction PPi+H2O→2Pi catalysed by inorganic pyrophosphatase (EC 3.6.1.1) does not attain near-equilibrium in vivo. PPi should be considered as one of the group of small inorganic ions which is metabolically active and capable of exerting a controlling function in a number of important metabolic reactions.