Variation of the polypeptide composition of mitochondria isolated from different potato tissues

The protein contents of mitochondria from different potato (Solanum tuberosum L.) tissues (tubers, dark-grown shoots, and green leaves) grown in a greenhouse or in vitro were compared by two-dimensional polyacrylamide gel electrophoresis. Two different methods were used: using the method that gave the highest resolution, an average number of 360 polypeptides was revealed on the mitochondrial patterns after silver staining. The mitochondrial protein patterns of etiolated tissues (tubers, dark-grown shoots) are roughly similar but distinct from those of green leaves. The four subunits of the glycine decarboxylase complex (involved in photorespiration) and a few other polypeptides are very abundant in green tissues, compared with nonphotosynthetic tissues. Conversely, some other polypeptides that are abundant in tubers and dark-grown shoots are hardly detectable in green leaf mitochondria. A rabbit antiserum was raised against a 40 kilodalton polypeptide that is among the most characteristic of these nonphotosynthetic tissue-specific polypeptides, and the N-terminal sequence of this polypeptide was determined. No effect of in vitro culture was observed on the protein composition of mitochondria isolated from differentiated tissues. However, the protein patterns of callus and cell suspension mitochondria are distinct from those of any differentiated tissues, although their basic pattern is clearly mitochondrial.     

Purification and primary amino acid sequence of the L subunit of glycine decarboxylase. Evidence for a single lipoamide dehydrogenase in plant mitochondria.

In order to purify the lipoamide dehydrogenase associated with the glycine decarboxylase complex of pea leaf mitochondria, the activity of free lipoamide dehydrogenase has been separated from those of the pyruvate and 2-oxoglutarate dehydrogenase complexes under conditions in which the glycine decarboxylase dissociates into its component subunits. This free lipoamide dehydrogenase which is normally associated with the glycine decarboxylase complex has been further purified and the N-terminal amino acid sequence determined. Positive cDNA clones isolated from both a pea leaf and embryo lambda gt11 expression library using an antibody raised against the purified lipoamide dehydrogenase proved to be the product of a single gene. The amino acid sequence deduced from the open reading frame included a sequence matching that determined directly from the N terminus of the mature protein. The deduced amino acid sequence shows good homology to the sequence of lipoamide dehydrogenase associated with the pyruvate dehydrogenase complex from Escherichia coli, yeast, and humans. The corresponding mRNA is strongly light-induced both in etiolated pea seedlings and in the leaves of mature plants following a period of darkness. The evidence suggests that the mitochondrial enzyme complexes: pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and glycine decarboxylase all use the same lipoamide dehydrogenase subunit.     

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

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.     

Subcellular distribution, multiple forms and development of glutamate-pyruvate (glyoxylate) aminotransferase in plant tissues

According to a sucrose density gradient analysis of cell organelles from homogenates of green leaves of rye, wheat and pea seedlings glutamate-pyruvate aminotransferase was predominantly localized in the leaf microbodies (peroxisomes; 90%) and to a minor extent in the mitochondria (10%) but completely absent from chloroplasts. In etiolated rye leaves the distribution of the enzyme was similar. In other non-green tissues glutamate-pyruvate aminotransferase was predominantly associated with the mitochondria but also present in the microbodies of dark-grown pea roots and in the glyoxysomes of Ricinus endosperm. In the microbodies isolated from potato tubers the enzyme was not detectable. Glutamate-pyruvate aminotransferase activity was not associated with the proplastid fractions of the non-green tissues. The distribution of glutamate-oxaloacetate aminotransferase was different from that of glutamate-pyruvate aminotransferase. Glutamate-oxaloacetate aminotransferase was found in chloroplasts, proplastids, mitochondria, microbodies and in the supernatant. Evidence is presented that glutamate-pyruvate and glutamate-glyoxylate aminotransferase activities were catalyzed by the same enzyme. Both activities showed the same organelle distribution on sucrose gradients and both were eluted at the same salt concentration from DEAE-cellulose. By chromatography of preparations from rye leaf extracts on DEAE-cellulose two forms of glutamate-pyruvate (glyoxylate) aminotransferase were separated. The major fraction eluting at a low salt concentration was identified as peroxisomal form and the minor fraction eluting at a higher salt concentration was identified as a mitochondrial form. Both the glutamate-glyoxylate and the glutamate-pyruvate aminotransferase activities of the peroxisomal as well as of the mitochondrial forms of the enzyme were strongly (about 80%) inhibited by the presence of 10 mM glycidate, previously described as an inhibitor of glutamate-glyoxylate aminotransferase in tobacco tissue. Pig heart glutamate-pyruvate aminotransferase exhibited no glutamate-glyoxylate aminotransferase activity and was only slightly inhibited by glycidate. The development of glutamate-pyruvate aminotransferase activity in the leaves of rye seedlings was strongly increased in the light, relative to dark-grown seedlings, and very similar to that of catalase activity while the development of glutamate-oxaloacetate aminotransferase was, in close coincidence with the behavior of leaf growth, only slightly enhanced by light. It is discussed that in green leaves an extrachloroplastic synthesis of alanine is of considerable advantage for the metabolic flow during photosynthesis.     

Extraction and partial characterization of the glycine decarboxylase multienzyme complex from pea leaf mitochondria

Glycine decarboxylase has been successfully solubilized from pea (Pisum sativum) leaf mitochondria as an acetone powder. The enzyme was dependent on added dithiothreitol and pyridoxal phosphate for maximal activity. The enzyme preparation could catalyze the exchange of CO₂ into the carboxyl carbon of glycine, the reverse of the glycine decarboxylase reaction by converting serine, NH₄⁺, and CO₂ into glycine, and ¹⁴CO₂ release from [1-¹⁴C]glycine. The half-maximal concentrations for the glycine-bicarbonate exchange reaction were 1.7 millimolar glycine, 16 millimolar NaH¹⁴CO₂, and 0.006 millimolar pyridoxal phosphate. The enzyme (glycine-bicarbonate exchange reaction) was active in the assay conditions for 1 hour and could be stored for over 1 month. The enzymic mechanism appeared similar to that reported for the enzyme from animals and bacteria but some quantitative differences were noted. These included the tenacity of binding to the mitochondrial membrane, the concentration of pyridoxal phosphate needed for maximum activity, the requirement for dithiothreitol for maximum activity, and the total amount of activity present. Now that this enzyme has been solubilized, a more detailed understanding of this important step in photorespiration should be possible.     

Mitochondria from leaf mesophyll cells of C(4) plants are deficient in the H protein of glycine decarboxylase complex

Mesophyll mitochondria from green leaves of the C(4) plants Zea mays (NADP-ME-type), Panicum miliaceum (NAD-ME-type) and Panicum maximum (PEP-CK-type) oxidized NADH, malate and succinate at relatively high rates with respiratory control, but glycine was not oxidized. Among the mitochondrial proteins involved in glycine oxidation, the L, P and T proteins of glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) were present, while the H protein of GDC was undetectable. In contrast, mesophyll mitochondria from etiolated leaves of Z. mays oxidized glycine at a slow rate and with no respiratory control, and contained the H protein as well as the other GDC proteins and SHMT. The T and P proteins and SHMT were present in the mitochondria from etiolated leaves at significantly higher levels than in those from green leaves of Z. mays. The content of the L protein was almost identical in all three C(4) plants examined and close to the value obtained for mesophyll mitochondria from the C(3) plant Pisum sativum, whereas the other GDC proteins and SHMT were less abundant than the L protein. We discuss possible reasons for the H protein's absence in mesophyll mitochondria of C(4) plants, as well as the role(s) the other GDC components could play in its absence.     

Light-dependent and tissue-specific expression of the H-protein of the glycine decarboxylase complex.

Glycine decarboxylase is a mitochondrial enzyme complex, which is the site of photorespiratory CO2 and NH3 release. Although the proteins that constitute the complex are located within the mitochondria, because of their intimate association with photosynthesis their expression is controlled by light. Comparisons of the kinetics of mRNA accumulation between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and the H-protein of glycine decarboxylase during the greening of etiolated Arabidopsis thaliana suggest that their expression is controlled in parallel. A genomic clone for the H-protein (gdcH) was isolated from Arabidopsis and sequenced. The upstream region from -856 to +62 was fused to the beta-glucuronidase (GUS) reporter gene, and this construct was transformed into tobacco. This 5' upstream regulatory region appears to control GUS expression in a manner very similar to that of the endogenous H-protein gene. Constructs with deletions in the 5' upstream region were transformed into tobacco. These deletions revealed that light-dependent and tissue-specific expression was largely controlled by a 259-bp region between -376 and -117 bp. This region contains several putative GT boxes with the GGTTAA consensus core sequence. Once these strong light-dependent elements were removed, a second level of control was revealed. In constructs in which the gdcH 5' regulatory region was shortened to -117 bp or less, there was more GUS activity in the roots than in the leaves, and in dark-grown plants than in light-grown plants. This suggests that more proximal control elements may be responsible for the constitutive low levels of gene expression noted in all nonphotosynthetic tissues.     

Oxaloacetate translocator in plant mitochondria

(1) Mitochondria were prepared from leaves of spinach, green and etiolated seedlings and roots of pea, potato tuber and rat liver and heart. In the case of leaf mitochondria, an improved isolation procedure resulted in high respiratory rates (460–510 nmol/mg protein per min) and good respiratory control ratio (6.8–9.8) with glycine as substrate. (2) In these mitochondria oxaloacetate transport was studied either by following the inhibitory effect of oxaloacetate on the respiration of NADH-linked substrates or by determining the consumption of [4-¹⁴C]oxaloacetate. (3) Studies of the competition by other carboxylates and effect of inhibitors on the oxaloacetate transport demonstrate that mitochondria from spinach leaves, green pea seedlings, etiolated pea seedlings and pea roots contain a specific translocator for oxaloacetate with a very high affinity to its substrate (K_m = 3–7 μM) and an even higher sensitivity to its competitive inhibitor phthalonate (K_i = 3–5 μM). The V_max values ranged from 150 to 180 nmol/mg protein per min for mitochondria from etiolated pea seedlings and pea roots and from 550 to 570 nmol/mg protein per min for mitochondria from spinach leaves and green pea seedlings. In mitochondria from potato tuber, the K_m was about one order of magnitude higher (V_max = 450 nmol/mg protein per min). In mitochondria from rat liver and rat heart, a specific translocator for oxaloacetate was not found. (4) The oxaloacetate translocator enables the functioning of a malate-oxaloacetate shuttle for the transfer of reducing equivalents across the inner mitochondrial membrane. (5) This malate-oxaloacetate shuttle appears to play a role in the photorespiratory cycle in catalyzing the transfer of reducing equivalents generated in the mitochondria during glycine oxydation to the peroxysomal compartment for the reduction of β-hydroxypyruvate. (6) Interaction between the mitochondrial and the chloroplastic malate oxaloacetate shuttles would make it possible for surplus-reducing equivalents, generated by photosynthetic electron transport, to be oxidized by mitochondrial electron transport.     

Developmental Regulation of Respiratory Activity in Pea Leaves

The developmental pattern of mitochondrial respiratory activity in pea (Pisum sativum) leaves has been investigated in an attempt to determine changes in mitochondrial function as plant cells mature. NADH and succinate dehydrogenase and cytochrome c oxidase activities remained relatively constant during cell maturation (from d 0 to d 14). Alternative oxidase and glycine decarboxylase activity, however, were low in young leaf tissue (d 0-6) but increased substantially as the tissue matured (d 7-14) and gained photorespiratory activity. Western blot analysis of the alternative oxidase protein revealed that it was primarily in an oxidized state in young leaves (d 0-6) but switched dramatically to the reduced form of the protein as the pea cells matured (d 7-14). The switch to the reduced form of the protein correlated with an increase in alternative oxidase activity. Results are discussed in terms of the changing function of plant mitochondria during leaf development.     

Cloning and characterization of the P subunit of glycine decarboxylase from pea (Pisum sativum).

A pea leaf cDNA library constructed in lambda gt11 was screened with an antibody raised to the P subunit of glycine decarboxylase. One of the positive clones isolated was sequenced and shown to contain an open reading frame, which encoded the entire P subunit polypeptide. Aligning the deduced amino acid sequence with the amino acid sequence determined directly from the NH2 terminus of the mature P subunit shows the presence of a putative 86 amino acid leader sequence, presumably required for import into the mitochondria, and gives a Mr of the mature protein of 105,000. Comparison of this deduced amino acid sequence with the sequence of a pyridoxal phosphate-containing peptide isolated from the P subunit of chicken liver glycine decarboxylase shows remarkable conservation. The P subunit, however, shows little sequence homology with other published amino acid decarboxylases. Expression of the P subunit mRNA shows a pattern very similar to that of the corresponding polypeptide: it is strongly light induced and is expressed at a much higher level in leaves than in other tissues. Southern blot analysis suggests that the P subunit is encoded by a small multigene family.     

Evidence for the presence of ferritin in plant mitochondria.

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI-TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.     

Glycine oxidation in mitochondria isolated from light grown and etiolated plant tissue

Mitochondria were isolated from light grown and dark grown monocotyledonous (wheat- Triticum aestivum and barley- Hordeum vulgare ) and dicotyledonous (pea- Pisum sativum ) plants and their capacity to oxidize glycine was measured. In all of the studied plant species the rate of mitochondrial glycine oxidation was high in light grown leaves. Glycine oxidation in mitochondria from etiolated leaves was also very substantial; the rate of glycine oxidation relative to the oxidation of other substrates was about half as compared to green tissue. In etiolated non-photosynthetic tissues the relative glycine oxidation was only ca 20% of that measured in green leaves. The effect of light on the development of glycine oxidation capacity was studied using etiolated barley which was transferred to light for 6 to 24 h. During this time the rate of glycine oxidation as compared to the oxidation of NADH and malate increased, approaching the ratio observed in light grown leaves. It is concluded that the synthesis of proteins involved in glycine oxidation is regulated both in a light dependent and in a tissue specific manner. Monocotyledonous plants should be very useful for further studies of this aspect due to the relatively small developmental difference between etiolated and light grown leaf tissue.     

The organisation and expression of the genes encoding the mitochondrial glycine decarboxylase complex and serine hydroxymethyltransferase in pea (Pisum sativum)

Summary Restriction fragment length polymorphisms have been used to determine the chromosomal location of the genes encoding the glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) of pea leaf mitochondria. The genes encoding the H subunit of GDC and the genes encoding SHMT both show linkage to the classical group I marker i. In addition, the genes for the P protein of GDC show linkage to the classic group I marker a. The genes for the L and T proteins of GDC are linked to one another and are probably situated on the satellite of chromosome 7. The mRNAs encoding the five polypeptides that make up GDC and SHMT are strongly induced when dark-grown etiolated pea seedlings are placed in the light. Similarly, when mature plants are placed in the dark for 48 h, the levels of both GDC protein and SHMT mRNAs decline dramatically and then are induced strongly when these plants are returned to the light. During both treatments a similar pattern of mRNA induction is observed, with the mRNA encoding the P protein of GDC being the most rapidly induced and the mRNA for the H protein the slowest. Whereas during the greening of etiolated seedlings the polypeptides of GDC and SHMT show patterns of accumulation similar to those of the corresponding mRNAs, very little change in the level of the polypeptides is seen when mature plants are placed in the dark and then re-exposed to the light.     

Purification and characterization of the multiple forms of aldehyde reductase in ox kidney.

A mutant of Arabidopsis thaliana (L.) Heyn. (a small plant in the crucifer family) that lacks glycine decarboxylase activity owing to a recessive nuclear mutation has been isolated on the basis of a growth requirement for high concentrations of atmospheric CO2. Mitochondria isolated from leaves of the mutant did not exhibit glycine-dependent O2 consumption, did not release 14CO2 from [14C]glycine, and did not catalyse the glycine-bicarbonate exchange reaction that is considered to be the first partial reaction associated with glycine cleavage. Photosynthesis in the mutant was decreased after illumination under atmospheric conditions that promote partitioning of carbon into intermediates of the photorespiratory pathway, but was not impaired under non-photorespiratory conditions. Thus glycine decarboxylase activity is not required for any essential function unrelated to photorespiration. The photosynthetic response of the mutant in photorespiratory conditions is probably caused by an increased rate of glyoxylate oxidation, which results from the sequestering of all readily transferable amino groups in a metabolically inactive glycine pool, and by a depletion of intermediates from the photosynthesis cycle. The rate of release of 14CO2 from exogenously applied [14C]glycollate was 14-fold lower in the mutant than in the wild type, suggesting that glycine decarboxylation is the only significant source of photorespiratory CO2.     

Inhibition of the glycine decarboxylase multienzyme complex by the host-selective toxin victorin.

Victoria blight of oats is caused by the fungus Cochliobolus victoriae. This fungus is pathogenic due to its ability to produce the host-selective toxin victorin. We previously identified a 100-kD protein that binds victorin in vivo only in susceptible genotypes and a 15-kD protein that binds victorin in vivo in both susceptible and resistant genotypes. Recently, we determined that the oat 100-kD victorin binding protein is the P protein of the glycine decarboxylase complex (GDC). In this study, we examined the effect of victorin on glycine decarboxylase activity (GDA). Victorin was a potent in vivo inhibitor of GDA. Leaf slices pretreated for 2 hr with victorin displayed an effective concentration for 50% inhibition (EC50) of 81 pM for GDA. Victorin inhibited the glycine-bicarbonate exchange reaction in vitro with an EC50 of 23 microM. We also identified a 15-kD mitochondrial protein that bound victorin in a ligand-specific manner. Based on amino acid sequence analysis, we concluded that the 15-kD mitochondrial protein is the H protein component of the GDC. Thus, victorin specifically binds to two components of the GDC. GDA in resistant tissue treated with 100 micrograms/mL victorin for 5 hr was inhibited 26%, presumably as a consequence of the interaction of victorin with the H protein. Victorin had no detectable effect on GDA in isolated mitochondria, apparently due to the inability of isolated mitochondria to import victorin. These results suggest that the interaction of victorin with the GDC is central to victorin's mode of action.     

Comparative aspects of aminooxyacetate inhibition of glycine oxidation and aminotransferase activity of pea leaf mitochondria

Aminooxyacetate (AOA) was found to inhibit glycine oxidation by pea leaf mitochondria at micromolar levels. The inhibition resulted from an inhibition of both glycine decarboxylase and serine hydroxymethyltransferase (SHMT) activity. Aspartate: 2-oxoglutarate aminotransferase (AsAT) and alanine: 2-oxoglutarate aminotransferase activities of pea leaf mitochondria were also very sensitive to AOA inhibition. Inhibition of both glycine oxidation and aminotransferase activity was likely competitive with respect to the amino group substrate, but also displayed a time-dependent increase in inhibition at constant AOA concentration. In the case of glycine oxidation, this time-dependent component may be related to the rate of penetration of AOA across the inner mitochondrial membrane. Furthermore, the AOA-inhibition of glycine oxidation could be reversed by pyridoxal 5-phosphate (PLP), whereas AOA-inhibited aminotransferase activity was not reversed. The results indicate that the pyridoxal 5-phosphate antagonist, AOA, results in varying types of inhibition depending on the type of enzyme involved.     

Stimulation of respiration by Pb2+ in detached leaves and mitochondria of C3 and C4 plants

The exposure of detached leaves of C₃ plants (pea, barley) and C₄ plant (maize) to 5 m M Pb (NO₃)₂ for 24 h caused a reduction of their photosynthetic activity by 40–60%, whereas the respiratory rate was stimulated by 20–50%. Mitochondria isolated from Pb²⁺-treated pea leaves oxidized substrates (glycine, succinate, malate) at higher rates than mitochondria from control leaves. The respiratory control (RCR) and the ADP/O ratio were not affected. Pb²⁺ caused an increase in ATP content and the ATP/ADP ratio in pea and maize leaves. Rapid fractionation of barley protoplasts incubated at low and high CO₂ conditions, indicated that the increased ATP/ADP ratio in Pb²⁺-treated leaves resulted mainly from the production of mitochondrial ATP. The measurements of membrane potential of mitochondria with a TPP⁺-sensitive electrode further showed that mitochondria isolated from Pb²⁺-treated leaves had at least as high membrane potential as mitochondria from control leaves. The activity of NAD-malate dehydrogenase in the protoplasts from barley leaves treated with Pb²⁺ was 3-fold higher than in protoplasts from control leaves. The activities of photorespiratory enzymes NADH-hydroxypyruvate reductase and glycolate oxidase as well as of NAD-malic enzyme were not affected. The presented data indicate that stimulation of respiration in leaves treated by lead is in a close relationship with activation of malate dehydrogenase and stimulation of the mitochondrial ATP production. Thus, respiration might fulfil a protective role during heavy metal exposure.     

Isolation, characterization, and sequence analysis of a cDNA clone encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria.

L-protein is the dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a lambda gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an Mr of about 60000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected. Second, Northern-blot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.     

Purification and partial characterization of the glycine decarboxylase multienzyme complex from Eubacterium acidaminophilum.

The proteins P1, P2, and P4 of the glycine cleavage system have been purified from the anaerobic, glycine-utilizing bacterium Eubacterium acidaminophilum. By gel filtration, these proteins were determined to have Mrs of 225,000, 15,500, and 49,000, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein P1 was determined to have two subunits with Mrs of 59,500 and 54,100, indicating an alpha 2 beta 2 tetramer, whereas the proteins P2 and P4 showed only single bands with estimated Mrs of 15,500 and 42,000, respectively. In reconstitution assays, proteins P1, P2, P4 and the previously reported lipoamide dehydrogenase (P3) had to be present to achieve glycine decarboxylase or synthase activity. All four glycine decarboxylase proteins exhibited highest activities when NADP+ was used as the electron acceptor or when NADPH was used as the electron donor in the glycine synthase reaction. The oxidation of glycine depended on the presence of tetrahydrofolate, dithioerythreitol, NAD(P)+, and pyridoxal phosphate. The latter was loosely bound to the purified protein P1, which was able to catalyze the glycine-bicarbonate exchange reaction only in combination with protein P2. Protein P2 could not be replaced by lipoic acid or lipoamide, although lipoic acid was determined to be a constituent (0.66 mol/mol of protein) of protein P2. Glycine synthase activity of the four isolated proteins and in crude extracts was low and reached only 12% of glycine decarboxylase activity. Antibodies raised against P1 and P2 showed cross-reactivity with crude extracts of Clostridium cylindrosporum.