Phosphorylation-dephosphorylation of pyruvate dehydrogenase complex from pea leaf mitochondria

Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with ['-32P]ATP Arch Biochem Biophys 270: 681-690). Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 250C before assay. The "spontaneous" inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg2+ and was relatively insensitive to Ca2+ and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with [32PJPi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 250C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate. We postulate that highly activated SPS contains phosphorylated residue(s) that increase activation state, and that spontaneous inactivation occurs by removal of these phosphate group(s). Inactivation of SPS in vivo caused by feeding uncouplers to darkened leaf tissue that had previously been fed mannose in the dark, may occur by this mechanism. However, there is no evidence that this mechanism is involved in light-dark regulation of SPS in vivo.Regulation of SPS2 activity is thought to be one of the components that controls the flux of carbon into sucrose, and hence translocation, in situ (for review see ref. 19 (9). Thus, phosphorylation of SPS occurred both in vivo and in vitro, and in both systems, the more highly phosphorylated enzyme had a lower activation state. Attempts to study the biochemical mechanism involved have been successful because the spinach leaf enzyme is often relatively stable (for short periods of time) at room temperature. The work we report here focused on the highly activated enzyme extracted from illuminated leaves that were fed mannose. We compared this SPS form with the relatively inactive (extracted from darkened leaves) and intermediate enzymeforms (extracted from leaves illuminated without mannose). We observed that highly activated enzyme was not stable when desalted crude extracts were preincubated at 25°C. Our study of this loss in activation state indicates that the "spontaneous" inactivation is mediated by an endogenous phosphatase(s), which constitutes evidence for a novel regulatory covalent modification of SPS.

Section: Discussionmentioning

confidence: 99%

Inactivation of Highly Activated Spinach Leaf Sucrose-Phosphate Synthase by Dephosphorylation

Huber

Hite²,

Outlaw³

1991

“…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%

“…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%

Regulation of Pea Mitochondrial Pyruvate Dehydrogenase Complex

Schuller¹,

Randall²

1989

Self Cite

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

“…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).…”

Section: Introductionmentioning

confidence: 99%

“…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).…”

Section: Introductionmentioning

confidence: 99%

Light Regulation of Leaf Mitochondrial Pyruvate Dehydrogenase Complex

Gemel¹,

Randall²

1992

Self Cite

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...