Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria.
Null point titration techniques have been developed for measurements of cytosolic free Mg2+ in isolated cells and matrix free Mg2+ in isolated mitochondria using antipyrylazo III as a spectrophotometric Mg2+ indicator. A cytosolic free Mg2+ of 0.37 +/- 0.02 mM was obtained with hepatocytes. This represented about 6% of the total cytosolic magnesium content (activity coefficient of 5.8 X 10(-2). Nondiffusable Mg2+-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 magnesium. The null point method gave a value of 0.35 +/- 0.01 mM for the mitochondrial matrix free Mg2+ concentration (activity coefficient of 8.8 X 10(-3). Nondiffusable Mg2+ 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 calcium. These data demonstrate the absence of a significant gradient of free Mg2+ between the cytosolic and mitochondrial compartments. They also demonstrate a high ligand binding capacity for magnesium in both compartments with relatively low affinity resulting in a constant value for free Mg2+ when total cell magnesium is constant. This maintains a ratio between free Mg2+ and free Ca2+ of about 2000 in the cytosol and 100 in the mitochondria. The high concentration and low affinity of Mg2+ binding sites results in rather large changes of free Mg2+ with small variations in total cell magnesium. This is apparent in hepatocytes isolated from streptozotocin diabetic rats which had a decreased total magnesium content and a cytosolic free Mg2+ of 0.16 +/- 0.02 mM.
Corkey
B E
BE
Duszynski
J
J
Rich
T L
TL
Matschinsky
B
B
Williamson
J R
JR
D000818:Animals; D001665:Binding Sites; D000001:Calcimycin / Q000494:pharmacology; D003201:Computers; D003600:Cytosol / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism; D009282:Naphthalenesulfonates / Q000378:metabolism; D009994:Osmolar Concentration; D051381:Rats
D009282:Naphthalenesulfonates; D000001:Calcimycin; C016238:antipyrylazo III; D008274:Magnesium