Some Kinetic and Regulatory Properties of the Pea Mitochondrial Pyruvate Dehydrogenase Complex

1992

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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...

Section: Plant Materialsmentioning

confidence: 99%

Light Regulation of Leaf Mitochondrial Pyruvate Dehydrogenase Complex

Gemel¹,

1992

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“…Pea seedlings were grown, mitochondria isolated, the PDC partially purified, and assayed according to established procedures (1,12,16). Incubation of the PDC with ATP resulted in inactivation through phosphorylation catalyzed by the intrinsic PDH-kinase.…”

Section: Methodsmentioning

confidence: 99%

Some Properties of Pea Mitochondrial Phospho-Pyruvate Dehydrogenase-Phosphatase

Miernyk

1987

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“…Activity of the complex is regulated in part by reversible phosphorylation and in part by product inhibition (4,12,17,19). Phosphorylation, catalyzed by pyruvate dehydrogenase (PDH) kinase, inactivates the complex and dephosphorylation, catalyzed by P-PDH phosphatase, results in reactivation.…”

Section: Biochemicalsmentioning

confidence: 99%

“…Activity of the complex is regulated in part by reversible phosphorylation and in part by product inhibition (4,12,17,19 …”

Section: Biochemicalsmentioning

confidence: 99%

Regulation of Pea Mitochondrial Pyruvate Dehydrogenase Complex

Schuller¹,

1989

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Inactivation of the pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase kinase was studied using intact mitochondria purified from green leaf tissue of pea (Pisum sativum L.) and dialyzed mitochondrial extracts. Thiamine pyrophosphate was inhibitory in dialyzed extracts but not in intact mitochondria, except in the presence of high concentraffons of Na+. NH4+, at concentrations as low as 20 micromolar, markedly stimulated inactivation in dialyzed extracts. K+ in the range I to 10 millimolar also enhanced inactivation. In contrast, Na+ was without affect at lower concentrations but was inhibitory at 10 to 100 millimolar levels. The effect of NHW+ is discussed in relation to a possible regulatory interaction between photorespiratory NH.+ production and the entry of carbon into the tricarboxylic acid cycle by way of the pyruvate dehydrogenase complex.inhibited by TPP in dialyzed extracts but not in intact mitochondria. Furthermore, K+ and NH4+ are shown to markedly stimulate PDH kinase activity with NH4+ being more effective than K+. The latter result is discussed in relation to a potential role for photorespiratory NHE+ in regulating PDC activity and consequently the carbon supply for dark respiration during photosynthesis. This is important because it is not known to what extent dark respiration proceeds simultaneously with photosynthesis, let alone what regulates the balance between these two processes in meeting energy demands (9). MATERIALS AND METHODS BiochemicalsBiochemicals were purchased from either Sigma (St. Louis, MO) or Pharmacia (Piscataway, NJ). ATP and pyruvate were sodium salts and TPP was the free acid.The mitochondrial pyruvate dehydrogenase complex (PDC2) catalyzes a key reaction regulating entry of carbon into the tricarboxylic acid cycle (4) and thereby indirectly affects the rate of dark respiration. Activity of the complex is regulated in part by reversible phosphorylation and in part by product inhibition (4,12,17,19