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Mitochondria from pea leaves were purified by centrifugation on a self-generated Percoll gradient which contained a linear gradient of polyvinylpyrrolidone-25 (0-10010, w/v). The chlorophyll content of the purified mitochondria was less than 1 pg per mg protein. All substrates were rapidly oxidized by these mitochondria, the rate of glycine oxidation being between 200 and 300 nmol 0, min-' mg-I protein, depending on the age of the leaves used. These rates did not vary significantly over a period of 20 h, provided NAD+ was supplied exogenously, when the mitochondria were stored on ice. Respiratory control, ADP/O ratios and outer membrane integrity (always more than 95010) were also maintained during storage. The phospholipid composition of the membranes from the leaf mitochondria was virtually identical to that of mitochondria from non-photosynthetic tissues although their lipid to protein ratio was slightly lower. The polypeptide pattern of the membranes from green leaf mitochondria and those from etiolated leaves and hypocotyls were also similar, but marked differences were observed between the matrix proteins from the different tissues. In particular, intensely stained bands at 94, 51,41 and 15.5 kDa which were present in the matrix of green leaf mitochondria were missing or present in much smaller quantities in the non-photosynthetic tissues. This difference was correlated with the ability of the mitochondria to oxidize glycine, suggesting that the four polypeptides may be associated with the glycine decarboxylase complex.

Preincubation of potato (Solanum tuberosum) tuber mitochondria with 300 microM-H2O2 for 10 min nearly stopped the State 3 rate of citrate oxidation. Addition of isocitrate resulted in resumption of O2 uptake. The State 3 rates of succinate, external NADH and 2-oxoglutarate oxidation were unaffected by H2O2 over the dose range 50-500 microM. Preincubation of mitochondria with 300 microM-H2O2 for 5 min unmasked in the matrix space a paramagnetic signal with a peak at a g value of approx. 2.03. Aconitase was purified over 135-fold to a specific activity of 32 mumol/min per mg (with isocitrate as substrate) from the matrix of potato tuber mitochondria. The native enzyme was composed of a single polypeptide chain (molecular mass 90 kDa). Incubation of purified aconitase with small amounts of H2O2 caused the build up of a paramagnetic 3Fe cluster with a low-field maximum of g = 2.03 leading to a progressive inhibition of aconitase activity. The results show that aconitase present in the matrix space was the major intramitochondrial target for inactivation by H2O2.

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The subcellular distributions of folate and folate-synthesizing enzymes were investigated in pea leaves. It was observed that the mitochondrial folate pool (ϳ400 M) represented ϳ50% of the total pool. Furthermore, all the enzymes involved in tetrahydrofolate polyglutamate synthesis were present in the mitochondria. In marked contrast, we failed to detect any significant activity of these enzymes in chloroplasts, cytosol, and nuclei. The presence of the tetrahydrofolate synthesis pathway in mitochondria is apparently a general feature in plants since potato tuber mitochondria also contained a high folate concentration (ϳ200 M) and all the enzymes required for tetrahydrofolate polyglutamate synthesis.The specific activities of tetrahydrofolate-synthesizing enzymes were rather low (1.5-15 nmol h ؊1 mg ؊1 matrix protein), except for dihydrofolate reductase (180 -500 nmol h ؊1 mg ؊1 matrix protein). Dihydrofolate reductase was purified to homogeneity. The enzyme had a native molecular mass of ϳ140 kDa and was constituted of two identical 62-kDa subunits. Interestingly, this mitochondrial protein appeared to be a bifunctional enzyme, also supporting thymidylate synthesis. The cell distribution of thymidylate synthase was also investigated. No significant activity was observed in cell fractions other than mitochondria, indicating that plant cell mitochondria are also a major site for thymidylate synthesis.

High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane ('matrix extract') exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4 folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 x 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme ('P-protein'; 2 x 97 kDa), a carrier protein containing covalently bound lipoic acid ('H-protein'; 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity ('L-protein'; 2 x 61 kDa) and an H4 folate-dependent enzyme ('T-protein'; 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration on Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4 folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4 folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.

The glycine decarboxylase multienzyme complex comprises about one-third of the soluble protein of the matrix of pea (Pisum sativum) leaf mitochondria where it exists at a concentration of approximately 130 milligrams protein/milliliter. Under these conditions the complex is stable with an approximate subunit ratio of 2 P-protein dimers:27 H-protein monomers:9 T-protein mono glycine cleavage system or glycine synthase) has been reported from the mitochondria of a broad range of plant and animal tissues (8) as well as the cytosol of a number of bacteria (10, 1 1), it is found at its highest level in the mitochondria of C3 plant leaves. Indeed, glycine formed by the photorespiratory C-2 pathway is the predominant substrate oxidized by these mitochondria in illuminated leaf tissue (17).The concentration of the complex in plant tissue is controlled by light. In etiolated leaves or nongreen tissues the amount of enzyme activity is low (1, 3, 6, 22) and following illumination of etiolated leaves the amount ofenzyme activity increases about 10-fold (16,22). The increase in activity results from a de novo synthesis of new proteins and the increase in protein synthesis is largely regulated at the transcriptional level (9). The time course for the increase in mRNA concentrations for the specific component proteins of the enzyme complex closely parallels the increase in enzyme activity in greening pea tissues (9,12,22).The glycine decarboxylase complex consists of four different component proteins (2, 21). The 100 kD P-protein binds the PLP that forms the initial Schiff base with the a-amino group of glycine. The a-carboxyl of glycine is lost as CO2 and the remaining methylamine moiety is passed to the lipoamide cofactor of the 13.9 kD H-protein. The lipoamide-bound methylamine group is shuttled to the 45 kD T-protein where the methylene carbon is transferred to THF to produce methylene THF and the amino nitrogen is released as NH3. The last step of the reaction involves the oxidation of the resulting dihydrolipoamide of the H-protein by the 59 kD L-protein with the sequential reduction of FAD and NAD+.Although substantial progress has been made in understanding the enzymology and molecular biology of the glycine decarboxylase complex from leaf tissue, little is known about the physical structure of the complex. We describe here a series of experiments designed to study this multienzyme complex and some of the unusual enzymological consequences of the unique interactions between the subunits. MATERIALS AND METHODSPea (Pisum sativum) leaf mitochondria were isolated from young plants by differential centrifugation and purified on Percoll-polyvinylpyrrolidone gradients (5