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

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119

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

Section: Role Of the H-protein In The Complexmentioning

confidence: 79%

“…H-protein activity was assayed by the method of Motokawa and Kikuchi (13,20 (18,19). Figure 1 shows the fractionation ofthe purified component enzymes and the concentrated matrix enzymes on identical sucrose density gradients.…”

Section: Methodsmentioning

confidence: 99%

Interaction between the Component Enzymes of the Glycine Decarboxylase Multienzyme Complex

Oliver

Neuburger²,

Bourguignon³

et al. 1990

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119

“…The four subunits of GDC have been isolated and characterized in detail from both plant and animal sources (2,7,8,10,19,23). Of the four proteins, the L protein, lipoamide dehydrogenase, is the only one known to be involved in other enzyme reactions, notably the a-keto acid dehydrogenase complexes, PDH, and 2-oxoglutarate dehydrogenase.…”

Section: Discussionmentioning

confidence: 99%

Changes to the Stoichiometry of Glycine Decarboxylase Subunits during Wheat (Triticum aestivum L.) and Pea (Pisum sativum L.) Leaf Development

Rogers¹,

Jordan²,

Rawsthorne³

et al. 1991

Changes In the levels of the four subunits of the mitochondrial enzyme glycine decarboxylase (EC 2.1.2.10) have been investigated during development in the 8 day old primary leaf of wheat (Triticum aestivum L.). Proteins were extracted from wheat leaf sections between the basal meristem and 8.5 centimeters. The individual glycine decarboxylase subunits were detected by Western blotting, using subunit-specific polyclonal antibodies, and quantified by laser densitometry. P, T, and H subunits showed similar developmental patterns along the leaf. All were below the level of detection up to 1.5 centimeters from the meristem, but then increased over the leaf length examined. In contrast, the increase in the L protein (lipoamide dehydrogenase) was more gradual, and levels in the youngest regions of the leaf were maintained at approximately 14% of those at 8.5 centimeters. In a complementary study, levels of the four subunits in light-grown leaf tissues were compared to those in etiolated leaves from wheat and pea (Pisum sativum L.), using the activity of the mitochondrial marker enzyme fumarase as the basis for comparison. For both wheat and pea, levels of P, T, and H proteins in etiolated tissues were between 25 and 30% of those in lightgrown tissue. However, in etiolated tissues L protein was present at levels of 60 to 70% of that in light-grown tissues. The results indicate that discrete mechanisms may control the synthesis of L, as compared to P, T, and H proteins.

“…INH, however, has limited usefulness because of the high concentrations (13) long incubation periods needed for inhibition. Cyanide has also been shown to inhibit glycine decarboxylase, probably by reacting with PLP (2,11), but this compound is pleiotropic in its effects. Glycine hydroxamate and aminoacetonitrile are structurally related to glycine and have been shown to inhibit glycine oxidation by cells (or leaf discs) and isolated mitochondria (6,16).…”

mentioning

confidence: 99%

“…Glycine hydroxamate and aminoacetonitrile are structurally related to glycine and have been shown to inhibit glycine oxidation by cells (or leaf discs) and isolated mitochondria (6,16). Because of the relative insensitivity of the isolated enzyme to aminoacetonitrile (11,18), it was suggested that this compound may not inhibit the glycine decarboxylase complex directly. Arsenite inhibits a wide range of thiol containing enzyme including the lipoamide-based complexes, pyruvate dehydrogenase and a-ketoglutarate dehydrogenase, and has been shown to inhibit isolated glycine decarboxylase (11) as well as glycine oxidation by mitochondria (17).…”

mentioning

confidence: 99%

Inhibition of Glycine Oxidation by Carboxymethoxylamine, Methoxylamine, and Acethydrazide

Sarojini¹,

Oliver²

1985

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ABSTRACICarboxymethoxylamine (amino-oxyacetate), methoxylamine, and acethydrazide are shown to be effective, although not completely specific, inhibitors of glycine oxidation by the isolated glycine decarboxylase multienzyme complex, mitochondria, protoplasts, and leaf discs from peas. The inhibition probably results from a reaction between these compounds and the pyridoxal 5-phosphate cofactor of the enzyme.The glycine decarboxylase multienzyme complex is a mitochondrial enzyme that is responsible for the photorespiratory oxidation of glycine. The enzyme complex is composed of at least four subunits: The P protein3 (PLP binding); the H protein (lipoamide containing); the T protein (THF binding); and the L protein (dihydrolipoamide dehydrogenase) according to the nomenclature of Kikuchi (5). The enzyme complex has been solubilized from pea leaf mitochondria in an active form (1 1) and purified to near homogeneity (12). The complex catalyzes the release of "'CO2 from [1-"'C]glycine dependent on the addition of PLP, THF, and NAD. It also catalyzes the exchange of "'CO2 with the carboxyl carbon of glycine and the synthesis of glycine from methylene THF, NADH, CO2, and NH4,. The isolated enzyme complex has a mol wt of about 290,000 and shows a strong dependence on the addition of DTT for full activity.Because of the unique role of this enzyme in photorespiration, substantial effort has been directed towards the identification of inhibitors of this enzyme. To date, three classes of inhibitors have been found: (a) compounds that react with PLP bound to the P protein, e.g. INH and cyanide; (b) compounds that react with the lipoamide cofactor of the H protein, like arsenite (1 1); and (c) structural analogs of glycine, such as aminoacetonitrile (16) and glycine hydroxamate (6). INH was the first reported inhibitor of glycine decarboxylase (10). It inhibits glycine oxidation by reacting with the PLP cofactor of the enzyme and is reasonably specific in that it shows little inhibition of photosynthesis under nonphotorespiratory conditions (13). INH, however, has limited usefulness because of the high concentrations (13) or '