Glycine Metabolism and Oxalacetate Transport by Pea Leaf Mitochondria

Abstract

Isolated pea leaf mitochondria oxidatively decarboxylate added glycine. This decarboxylation could be linked to the respiratory chain (in which case it was coupled to three phosphorylations) or (21) with BSA as standard, and Chl was estimated by the method of Arnon (1). Mitochondrial protein was corrected for the contribution by broken thylakoids by assuming a thylakoid protein-to-Chl ratio of 7:1 (13). On this basis, thylakoids contributed 30 to 40% of the total protein in the mitochondrial fraction, and specific activities shown in figures and tables have been corrected for this contribution. 'Whole cell' rates of glycine decarboxylation, expressed as ,umol h-1 mg Chl' were estimated by taking into account the yield of isolated mitochondria and the total Chl extracted from the leaves (19). 02 consumption was measured using a Rank 02 electrode (Rank Bros., Cambridge, U. K.) in 2 to 3 ml of standard reaction medium (0.3 M sorbitol, 10 mm Tes buffer, 10 mm KH2PO4, 2 mm MgCl2, 0.1% BSA [pH 7.21) at 30 C. 02 in air-saturated medium was assumed to be 240 tLM.Enzyme assays were performed spectrophotometrically at room temperature. Mitochondria were disrupted either by incubating for 5 min with 0.5% digitonin (and then treating on a column of Sephadex G-25) (16) or by diluting with reaction medium and sonicating. Malate dehydrogenase was assayed according to Ochoa (26), NAD-malic enzyme according to Chapman and Hatch (6), and fumarase by the method of Huang and Beevers (18).Glycine decarboxylation via a reconstituted OAA-malate shuttle was measured as follows. Mitochondria (approximately 1 mg protein) were added to 0.9 ml standard reaction medium which also contained 5 ,UM antimycin A, 0.1 mm OAA, 4 mm NADP, and 2 to 3 units chicken-liver NADP-malic enzyme. The reaction was initiated by adding 10 mm glycine, and the reduction of NADP followed at 340 nm. NH3 release was measured simultaneously with 02 consumption in a sealed Perspex vessel into which were fitted an Orion ammonia electrode (connected to a Beckman pH meter) and a Clarke-type 02 electrode (Yellow-Springs, OH). The signals from both electrodes were monitored with a twin-channel Honeywell recorder. The capacity of the vessel was 9 ml. Mitochondria (0.5 ml, 3 to 7 mg protein) were added to 8 ml standard reaction 425 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from

Evidence for Metabolic Domains within the Matrix Compartment of Pea Leaf Mitochondria

“…This suggests that state 3 glycine oxidation is limited by the NADH reoxidation capacity of the respiratory chain. However, in these same mitochondrial preparations it could be shown that the state 3 rate of 02 uptake in the presence of glycine could be markedly increased by the addition of a second NAD-linked substrate such as malate (3,5). Thus, these data also support the hypothesis that the malate-oxidizing enzymes in leaf mitochondria have The concept of spatial organization of enzymes within the mitochondrial matrix is already well established for animal mitochondria (2,14,21,23).…”

“…OAA-supported glycine oxidation has been demonstrated many times with isolated leaf mitochondria (3,5,13,16,26). Indeed, a number of reports have demonstrated that OAAsupported rates of glycine oxidation are significantly higher than state 3 oxidation rates of glycine oxidation via the respiratory chain (3,16).…”

“…The differential distribution of glycine decarboxylase and malate oxidizing enzymes in pea leaf mitochondria was originally proposed by Dry and Wiskich (10) (3,5,16,19,26). This suggests that state 3 glycine oxidation is limited by the NADH reoxidation capacity of the respiratory chain.…”

Partial Purification and Reconstitution of the α-Ketoglutarate Carrier from Corn (Zea mays L.) Mitochondria

“…Furthermore, the inhibitor sensitivity of the reconstituted protein is similar to that of the mitochondrial a-ketoglutarate carrier (9,16,18,20). Among the inhibitors of the reconstited a-ketoglutarate exchange, the substrate analog phthalonate has been previously found to inhibit the a-ketoglutarate carrier in some plant mitochondria (20) but not in others (6,7). Because plant mitochondria contain a very active oxaloacetate carrier and this is very sensitive to phthalonate (1 1), the question arises as to whether or not the phthalonate-sensitive a-ketoglutarate/a-ketoglutarate exchange described in this paper is catalyzed by the oxaloacetate carrier.…”

Identification of AtNDI1, an Internal Non-Phosphorylating NAD(P)H Dehydrogenase in Arabidopsis Mitochondria

“…This, in turn, could stimulate transfer of reducing equivalents from the mitochondrial matrix to the cytosol, as proposed in Yarrowia lipolytica mitochondria (Kerscher et al, 2001). The increase in external NADH DH activity presented in this study could be due, therefore, to the action of a substrate shuttle across the inner mitochondrial membrane, such as the malate/oxaloacetate shuttle described in pea leaf mitochondria (Day and Wiskich, 1981).…”