Regulation of free and bound @entity1760 in @entity35 hepatocytes and XXXX .

@entity2383 @entity1025 @entity6 @entity35 @entity3839 @entity460 @entity141 @entity1760
Null point titration techniques have been developed for measurements of cytosolic free @entity1025 in isolated cells and matrix free @entity1025 in @entity2383 using antipyrylazo III as a spectrophotometric @entity1025 indicator. A cytosolic free @entity1025 of 0.37 +/- 0.02 mM was obtained with hepatocytes. This represented about 6% of the total cytosolic @entity1760 content (activity coefficient of 5.8 X 10(-2). Nondiffusable @entity1025 -binding sites in the cytosol were equal to 11.1 nmol/mg cell dry weight with an apparent dissociation constant of 0.71 mM and accounted for binding of 32% of the cytosolic @entity1760 . The null point method gave a value of 0.35 +/- 0.01 mM for the mitochondrial matrix free @entity1025 concentration (activity coefficient of 8.8 X 10(-3). Nondiffusable @entity1025 binding sites in the mitochondria were estimated at 25.7 nmol/mg mitochondrial protein with an apparent dissociation constant of 0.22 mM, compared with an apparent dissociation constant of 1.66 microM for bound @entity460 . These data demonstrate the absence of a significant gradient of free @entity1025 between the cytosolic and mitochondrial compartments. They also demonstrate a high ligand binding capacity for @entity1760 in both compartments with relatively low affinity resulting in a constant value for free @entity1025 when total cell @entity1760 is constant. This maintains a ratio between free @entity1025 and free @entity141 of about 2000 in the cytosol and 100 in the mitochondria. The high concentration and low affinity of @entity1025 binding sites results in rather large changes of free @entity1025 with small variations in total cell @entity1760 . This is apparent in hepatocytes isolated from @entity3839 @entity6 @entity35 which had a decreased total @entity1760 content and a cytosolic free @entity1025 of 0.16 +/- 0.02 mM.
