Purification and Characterization of the Pea Chloroplast Pyruvate Dehydrogenase Complex

Camp, Pamela J.; Randall, Douglas D.

doi:10.1104/pp.77.3.571

Cited by 141 publications

(73 citation statements)

References 25 publications

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“…attributable to the chloroplast isoform of PDC that does not undergo reversible phosphorylation (10,11) and is not inactivated in the light (data not shown). Details of this calculation were described by Budde and Randall (7).…”

Section: Mitochondria Isolationmentioning

confidence: 99%

“…Plants are unique in having an additional isoform of PDC in their plastids (10,11) that is also quite sensitive to product feedback regulation but does not undergo regulation by reversible phosphorylation (10,27). In vitro studies of the PDC kinase have also shown that the phosphorylation-inactivation reaction is stimulated by micromolar NH4+ (29) and is inhibited by pyruvate, the substrate for the PDC (26,30).…”

mentioning

confidence: 98%

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Light Regulation of Leaf Mitochondrial Pyruvate Dehydrogenase Complex

Gemel¹,

Randall²

1992

Plant Physiol.

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Light-dependent inactivation of mitochondrial pyruvate dehydrogenase complex (mtPDC) in pea (Pisum sativum L.) leaves was further characterized, and this phenomenon was extended to several monocot and dicot species. The light-dependent inactivation of mtPDC in vivo was rapidly reversed in the dark, even after prolonged illumination. The mtPDC can be efficiently cycled through the inactivated-reactivated status by rapid light-dark cycling. Light-dependent inactivation of mtPDC was shown to be suppressed by inhibitors of photorespiratory carbon metabolism, including 2-pyridylhydroxymethane sulfonate, isonicotinic acid hydrazide, and aminoacetonitrile, and by an inhibitor of photosynthesis, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Glycine fed to pea leaf strips in the dark yielded partially inactivated leaf mtPDC, and this inactivation was blocked by inhibitors of glycine oxidation. It is concluded that the photorespiratory glycine to serine conversion that occurs in C3 leaf mitochondria can provide the NADH to drive oxidative phosphorylation and subsequent inactivation of mtPDC. Glycine oxidation also produces ammonium ion, which has been shown to enhance the inactivation of mtPDC in vitro by stimulating the pyruvate dehydrogenase kinase that catalyzes the phosphorylation (inactivation) of the mtPDC. Thus, light-dependent, photorespiration-stimulated inactivation of the mtPDC can regulate carbon entry into the Krebs cycle during C3 photosynthesis.The mtPDC2 links glycolytic carbon metabolism with the Krebs cycle by catalyzing the oxidative decarboxylation of pyruvate to acetyl-CoA. Pyruvate provides the primary substrate for the Krebs cycle, which, in turn, provides the reducing equivalents for ATP production by oxidative phosphorylation. for regulation of the mitochondrial complex through product feedback by NADH and acetyl-CoA (23) and inactivationreactivation by reversible phosphorylation (26,27). Plants are unique in having an additional isoform of PDC in their plastids (10, 11) that is also quite sensitive to product feedback regulation but does not undergo regulation by reversible phosphorylation (10,27). In vitro studies of the PDC kinase have also shown that the phosphorylation-inactivation reaction is stimulated by micromolar NH4+ (29) and is inhibited by pyruvate, the substrate for the PDC (26,30). In situ studies with purified pea leaf mitochondria have shown that the PDC phosphorylation status is increased when the mitochondria are oxidizing substrates other than pyruvate (6), in particular, glycine, an intermediate of the photorespiratory carbon oxidation pathway.The controversy of whether or not mitochondrial respiration is occurring during photosynthesis has been long standing (16), and recent reports by Kromer and colleagues (20, 21) working with barley (Hordeum vulgare) leaf protoplasts provide convincing evidence that mitochondrial ATP production is required for optimal photosynthesis. However, Budde and Randall (7,8) recently reported that the pea leaf mtPDC is primarily in an inactivate...

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Section: Mitochondria Isolationmentioning

confidence: 99%

mentioning

confidence: 98%

Light Regulation of Leaf Mitochondrial Pyruvate Dehydrogenase Complex

Gemel¹,

Randall²

1992

Plant Physiol.

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“…56 The quaternary structure and organization of the plastid PDC is largely unknown because the plastid PDC, unlike the mitochondrial form, dissociates rapidly in vitro. 57 The presence of all four subunits in the membrane-depleted 30k × g fraction characterized here (bands [17][18][19][20][21][25][26][27] indicates this complex can be isolated under rapid extraction conditions for further structural analyses (Table 2). Since this complex sedimented at relatively low centrifugal forces association with other enzymes is possible and may signal the presence of a larger, metabolic enzyme assembly in plastids including the heteromeric acetyl-CoA carboxylase ( Table 2).…”

Section: Phinney and Thelenmentioning

confidence: 99%

Proteomic Characterization of A Triton-Insoluble Fraction from Chloroplasts Defines A Novel Group of Proteins Associated with Macromolecular Structures

Phinney

Thelen

2005

J. Proteome Res.

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Proteomic analysis of a Triton X-100 insoluble, 30 000 × g pellet from purified pea chloroplasts resulted in the identification of 179 nonredundant proteins. This chloroplast fraction was mostly depleted of chloroplast membranes since only 23% and 9% of the identified proteins were also observed in envelope and thylakoid membranes, respectively. One of the most abundant proteins in this fraction was sulfite reductase, a dual function protein previously shown to act as a plastid DNA condensing protein.Approximately 35 other proteins known (or predicted) to be associated with high-density protein-nucleic acid particles (nucleoids) were also identified including a family of DNA gyrases, as well as proteins involved in plastid transcription and translation. Although nucleoids appeared to be the predominant component of 30k × g Triton-insoluble chloroplast preparations, multi-enzyme protein complexes were also present including each subunit to the pyruvate dehydrogenase and acetyl-CoA carboxylase multienzyme complexes, as well as a proposed assembly of the first three enzymes of the Calvin cycle. Approximately 18% of the proteins identified were annonated as unknown or hypothetical proteins and another 20% contained "putative" or "like" in the identifier tag. This is the first proteomic characterization of a membrane-depleted, high-density fraction from plastids and demonstrates the utility of this simple procedure to isolate intact macromolecular structures from purified organelles for analysis of protein-protein and protein-nucleic acid interactions.

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“…Why chloroplasts should contain a GR preferring NADH is at present unclear, although it is already known that chloroplasts contain at least one enzyme that can generate NADH, i.e. pyruvate dehydrogenase complex (3).…”

Section: Discussionmentioning

confidence: 99%

Subcellular Location of NADPH-Dependent Hydroxypyruvate Reductase Activity in Leaf Protoplasts of Pisum sativum L. and Its Role in Photorespiratory Metabolism

Kleczkowski¹,

Givan²,

Hodgson³

et al. 1988

Plant Physiol.

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Protoplasts purified from pea (Pisum sativum L.) leaves were lysed and fractionated to assess the subcellular distribution of NADPH-dependent hydroxypyruvate reductase (NADPH-HPR) activity. Rate-zonal centrifugation and sucrose-gradient experiments demonstrated that most (about 70%) of the NADPH-HPR activity was located in the supernatant or cytosol fraction. Detectable, but relatively minor activities were associated with the chloroplast fraction (up to 10% on a chlorophyll basis when compared to the lysate) and with peroxisomes. The minor NADPH-HPR activity in the peroxisomes could be fully accounted for by the secondary NADPH-dependent activity of NADH-dependent HPR. The subcellular distribution of NADPH-HPR followed closely that previously determined for NADPH-dependent glyoxylate reductase (NADPH-GR), an enzyme localized predominantly in the cytosol of pea leaf protoplasts (CV Givan et al. 1988 J Plant Physiol 132: 593-599). Low activities of both NADPH-HPR and NADPH-GR were also found in purified chloroplasts prepared by mechanical homogenization of Pisum and Spinacia leaves. In pea and spinach chloroplasts, rates of both NADPH-HPR and NADPH-GR were lower than the activity of the NADH-dependent GR. The results are discussed in relation to a possible role for NADPH-HPR in the oxidative carbon pathway of photorespiration. Both NADPH-HPR and the GRs could function as auxiliary reactions to photorespiration, utilizing hydroxypyruvate and/or glyoxylate 'leaked' or otherwise exported from peroxisomes. NADPH-HPR function might be especially significant under conditions of limiting NADH supply to peroxisomes, with extraperoxisomal reduced pyridine nucleotide acting as the reductant.Hydroxypyruvate is a primary metabolite in the photorespiratory carbon pathway. Hydroxypyruvate formation in leaf cells occurs primarily in peroxisomes as a result ofthe transamination of serine by serine-glyoxylate aminotransferase (8). Serine-glyoxylate aminotransferase is accompanied by the peroxisomal

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Purification and Characterization of the Pea Chloroplast Pyruvate Dehydrogenase Complex

Cited by 141 publications

References 25 publications

Light Regulation of Leaf Mitochondrial Pyruvate Dehydrogenase Complex

Light Regulation of Leaf Mitochondrial Pyruvate Dehydrogenase Complex

Proteomic Characterization of A Triton-Insoluble Fraction from Chloroplasts Defines A Novel Group of Proteins Associated with Macromolecular Structures

Subcellular Location of NADPH-Dependent Hydroxypyruvate Reductase Activity in Leaf Protoplasts of Pisum sativum L. and Its Role in Photorespiratory Metabolism

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