Regulation of Pea Mitochondrial Pyruvate Dehydrogenase Complex

Abstract

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

Monovalent Cation Activation of Plant Pyruvate Dehydrogenase Kinase

“…In this paper, we characterize further the mechanism involved in the monovalent cation stimulation and show that NH4+ stimulation of PDH kinase activity persists in the presence of saturating Kf. This latter result supports our hypothesis that photorespiratory NH4+ may be important in regulating PDC activity during photosynthesis and photorespiration (Schuller and Randall, 1989).…”

“…It was consistently observed that at subsaturating concentrations of ATP, kinase activity in the presence of K+ was less than in the presence of NH4+. Previous studies by Schuller and Randall (1989) demonstrated that NH4+ and K+ had no effect on PDC activity even at concentrations up to 100 mM; therefore, the monovalent cation effects described here are on the kinase activity .…”

“…In contrast to mammalian PDH kinase, acetyl-COA and NADH inhibit the plant PDH kinase . We previously reported that K+ or NH4+ stimulated the plant PDH kinase (Schuller and Randall, 1989). In this paper, we characterize further the mechanism involved in the monovalent cation stimulation and show that NH4+ stimulation of PDH kinase activity persists in the presence of saturating Kf.…”

“…PDC activity was assayed by following NADH production at 340 nm using a Gilford- Response spectrophotometer. The PDC assay mixture contained 50 mM Tes-NaOH (pH 7.5), 3.3 mM MgC12, 2.5 mM NAD, 0.2 mM TPP, 0.13 mM LiCoA, 2.6 mM Cys, 1.5 mM Na pyruvate and about 50 pg of mitochondrial protein (Schuller and Randall, 1989). One unit of PDC activity was defined as the amount required to catalyze the formation of 1 pmol of NADH min-'.…”

Light Regulation of Leaf Mitochondrial Pyruvate Dehydrogenase Complex

“…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.…”