Glutamic dehydrogenase of mung bean mitochondria.

Abstract

GLUTAMIC dehydrogenase has been demonstrated in several higher plants1 and is associated with mitochondria of pea2 and oat3. Mitochondria isolated from mung bean (Phaseolus aureus) seedlings4 were found capable of oxidizing glutamic acid (90 &µl. oxygen/hr./mgm. (nitrogen). The assay of glutamic dehydrogenase in intact mitochondria is limited by a permeability barrier to pyridine nucleotides. Reduced diphosphopyridine nucleotide was oxidized by mitochondria suspended in 0.2 M sucrose, 0.05 M phosphate buffer, pH 7.4 (change in optical density at 340 mµ of 0.1 in 4 min.) and the addition of 0.1 per cent (v/v) of a non-ionic detergent, O.P.C. 45 (Petrochemicals Ltd., London) increased the oxidation rate by 240 per cent. The suspension of mitochondria was immediately clarified by the addition of O.P.C. 45. To assay glutamic dehydrogenase, intact mitochondria were broken by exposure to 0.1 per cent O.P.C. 45 at 0°C. and immediately centrifuged for 30 min. at 20,000 g. The supernatant was assayed for glutamic dehydrogenase using the system : 0.02 M potassium glutamate, 0.05 M phosphate buffer, pH 7.4 and 0.0001 di- or tri-phosphopyridine nucleotide and measuring reduction of the nucleotides at 340 mµ. The glutamic dehydrogenase activity released by this method accounted for all the glutamic acid oxidase activity of the whole mitochondria. Freezing and thawing the mitochondria suspended in 0.1 M potassium bicarbonate5, released 61 per cent of total soluble glutamic dehydrogenase. Incubation of the potassium bicarbonate extract with 0.1 per cent O.P.C. 45 for 1 hr. at 0°C. was not found to inhibit the activity of glutamic dehydrogenase. Glutamic dehydrogenase reduced diphosphopyridine nucleotide four times as rapidly as it reduced triphosphopyridine nucleotide. Dual specificity may be due to the presence of a transhydrogenase which has been demonstrated in pea mitochondria2. Reduced diphosphopyridine nucleotide produced by glutamic dehydrogenase and glutamic acid was oxidised on the addition of α-ketoglutarate or ammonium chloride, indicating the reversibility of the system.