The divalent cation requirement of the mitochondrial glycerol-3-phosphate dehydrogenase

Abstract

Coupled respiration by blowfly mitochondria has been utilized to demonstrate an absolute divalent cation requirement for glycerol 3-phosphate respiration. With ADP, phosphate and EGTA, the respiration rate (state 3) decreases as a function of the amount of oxygen reduced, to approximately 15% of its maximum value, even at 40 mM dl-glycerol 3-phosphate; it can be increased to its maximum value by the addition of Ca 2+, Sr 2+ or Mn 2+. The decline in state 3 rate is not due to the removal of membrane-bound calcium into the matrix by the calcium carrier, since it occurs in the presence of ruthenium red. The effect is energy-dependent since the state 3 respiration does not decrease in the presence of uncouplers. The increase in respiration upon the addition of calcium is not due to the energy-dependent calcium transport since it is sensitive to oligomycin and insensitive to ruthenium red. The divalent cation effector site is located on the glycerol-3-phosphate dehydrogense, since state 3 (or state 4) pyruvate-proline respiration (NAD-linked) is not affected by EGTA. Yet the state 3 pyruvate-proline respiration removes calcium so effectively from the glycerol-3-phosphate dehydrogenase in the presence of EGTA, that added calcium stimulates glycerol 3-phosphate (26.4 mM) respiration about 22-fold. Since uncouplers stimulate the inhibited glycerol 3-phosphate respiration only to a very small extent, a calcium stimulation of the rate of phenazine methosulfate reduction by glycerol 3-phosphate (26.4 mM) which bypasses all phosphorylation sites, should be detectable. Only a 3-fold stimulation was observed. The present experiments suggest that upon complete removal of divalent cations from the dehydrogenase, glycerol 3-phosphate does not act as a homotropic effector in the coenzyme Q reductase reaction.