Regulation of Steady State Pyruvate Dehydrogenase Complex Activity in Plant 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: 98%

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

Huber

Hite²,

Outlaw³

1991

“…Pyruvate, TPP and Na+ are inhibitory while K' and NH4+ are stimulatory. Other studies have shown that Mg2e is an inhibitor (2). The relative importance of these effectors and the interactions between them are still unclear.…”

Section: Mm)mentioning

confidence: 99%

“…Plant mitochondrial PDH kinase activity, assayed in intact or detergent-permeabilized mitochondria, purified from green leaf tissue of pea, is inhibited by pyruvate, dichloroacetate and Mg2e but is not affected by ADP, TPP or Ca2" (1)(2)(3) …”

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

“…Initial studies of the regulatory properties of plant mPDC have been conducted in vitro using preparations that had been purified 20-to 300-fold (18)(19)(20)(21). Recently, we (2)(3)(4)16) have developed a method for assaying mPDC activity in situ using purified intact pea seedling mitochondria. Using this method, several observations on the regulation by reversible phosphorylation have been clarified and extended (2)(3)(4).…”

mentioning

confidence: 99%

“…Recently, we (2)(3)(4)16) have developed a method for assaying mPDC activity in situ using purified intact pea seedling mitochondria. Using this method, several observations on the regulation by reversible phosphorylation have been clarified and extended (2)(3)(4). This report continues our investigation of the role of product inhibition in regulation of PDC activity within intact mitochondria.…”

mentioning

confidence: 99%

Acetyl-Coenzyme A Can Regulate Activity of the Mitochondrial Pyruvate Dehydrogenase Complex in Situ

Budde

Fang²,

Randall³

et al. 1991

Self Cite

In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon Km and K1 relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [19871 Plant Physiol 83: 306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or Lcamitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition.The mPDC' occupies a crucial branch point in intermediary metabolism, and activity is regulated by several mechanisms including compartmentation, product and metabolite inhibition, and reversible phosphorylation (reviewed in refs. 13,17). Initial studies of the regulatory properties of plant mPDC have been conducted in vitro using preparations that had been purified 20-to 300-fold (18-21). Recently, we (2-4, 16) have developed a method for assaying mPDC activity in situ using purified intact pea seedling mitochondria. Using this method, several observations on the regulation by reversible phosphorylation have been clarified and extended (2-4). This report continues our investigation of the role of product inhibition in regulation of PDC activity within intact mitochondria.Much like mammalian and microbial PDCs, plant mPDCs