Simultaneous Oxidation of Glycine and Malate by Pea Leaf Mitochondria

Abstract

Mitochondria isolated from pea leaves (Pisum sativum L.) readily oxidized malate and glycine as substrates. The addition of glycine to mitochondria oxidizing malate in state 3 diminished the rate ofmalate oxidation.When glycine was added to mitochondria oxidizing malate in state 4, however, the rate of malate oxidation was either unaffected or stimulated. tricarboxylic acid cycle function.Several authors have suggested the total or partial inhibition of the tricarboxylic acid cycle in the light (1,2,7,10). Others have shown that the cycle continued in the light (3,8,15). The NADH produced by glycine oxidation can be used by malate dehydrogenase to reduce oxaloacetate to malate (17) and this malate can be linked by a shuttle system to the peroxisomal reduction of hydroxypyruvate to glycerate (12, 28).Isolated pea leaf mitochondria readily use both glycine and malate as substrates. In the experiments described, we added glycine to mitochondria metabolizing malate as a model reaction for determining the effect of photorespiratory glycine oxidation on tricarboxylic acid cycle function by these plants. MATERIALS AND METHODSDuring photosynthetic CO2 fixation by C-3 plants, a substantial amount of newly formed carbohydrate is oxidized back to CO2 by the process of photorespiration (29). The photorespiratory CO2 can arise either from the direct decarboxylation of glyoxylate (9, 19) or during the mitochondrial oxidation of glycine to NH3, C02, and methylene tetrahydrofolate by the enzyme glycine decarboxylase. In the latter case, NAD+ is the electron acceptor and it in turn is reoxidized by the mitochondrial electron transport chain (5, 17). Current data suggest that in soybean (20, 21) Mitochondria were isolated from 2-to 4-week-old greenhousegrown pea (Pisum sativum L.) seedlings. Younger plants could not be used because the high lipoxygenase activity precluded the isolation of well coupled mitochondria. After chilling for 1 h, the leaves and stems (approximately 300 g) were ground for 2 s in a 4-L Waring blender in 1 L of grinding medium (0.5 M sorbitol, 50 mM MOPS2-NaOH, pH 7.5, 5.0 mm EDTA, and 0.5% BSA). The inclusion of reducing agents, including 5 mm ascorbate, 2 mM DTT, 2 mm thioglycolate, or 10 mim cysteine, resulted in inconsistent coupling ratios and varying amounts of KCN-insensitive respiration and was avoided. The homogenate was filtered through two layers of Miracloth (Calbiochem) and the fitrate was centrifuged at 5,000g for 2 min.The mitochondria were then collected at 20,000g for 5 min. This pellet was resuspended in cold medium containing 0.5 M sorbitol, 20 mm MOPS-NaOH (pH 7.2), 0.1% BSA, and 2 mM glycine and centrifuged at l,000g for 10 min and then 7,000g for 10 min. The final pellet was resuspended in the same medium and either used directly or further purified on a Percoll (Pharmacia Fine Chemicals) gradient. In the latter case, 1 ml mitochondrial preparation was carefully layered atop a step gradient composed of (bottom to top) 2 ml each 40%1o, 25%, and 15% Percoll in 0.5 M sorbitol,...

Extraction and Partial Characterization of the Glycine Decarboxylase Multienzyme Complex from Pea Leaf Mitochondria

“…Arsenite can react with the lipoamide cofactor of the H protein and was also inhibitory. Arsenite inhibition of glycine oxidation by pea leaf mitochondria has been reported earlier (20). KCN, a mixed function inhibitor of glycine decarboxylase in spinach mitochondria (2,13), also inhibited the exchange reaction by the isolated enzyme.…”

NAD+-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

“…Etiolated seedlings were harvested after 7 to 10 d. Mitochondria were isolated by grinding and differential centrifugation as previously described (23 For further purification, the Matrix Gel Blue A pool was concentrated, filtered through a 0.45-,um filter, and applied to a 25-mL Superose 6 FPLC column previously equilibrated in 20 mm Mops, 50 mm NaCl, 5 mm j-mercaptoethanol, and 20% glycerol (pH 7.5) (all FPLC buffers were filtered and degassed before use). The column was eluted at a flow rate of 0.1 mL/min (0.7 MPa), and 0.5-mL fractions were collected and tested for activity.…”

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

“…malate or pyruvate, are less than the sum of the individual oxidation rates with these substrates (3,7,9,10,25), suggesting that the respective dehydrogenases compete for access to the respiratory chain. Under such conditions glycine is thought to be preferentially oxidized at the expense of the other TCA cycle substrates (3,7,9).…”