Sensitivity of pyruvate dehydrogenase phosphate phosphatase to magnesium ions. Similar effects of spermine and insulin

Thomas, Andrew P.; Diggle, Tricia A.; Denton, R M

doi:10.1042/bj2380083

Cited by 85 publications

(68 citation statements)

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“…With P-El complexed with E2 (i.e., P-PDC) as substrate, but not with uncomplexed P-E1, and in the presence of a saturating concentration of Mg2+, Ca2+ stimulates the activity of PDP about 10-fold. Ca2+ decreases the Km of PDP for complexed P-E1 (7) and for Mg2+ (6). As shown in Fig.…”

mentioning

confidence: 66%

“…Ca>2 apparently mediates the specific binding of PDP to E2 in juxtaposition to P-El (7). This orientation decreases the apparent Km of PDP for P-El (7) and for Mg2> (6). There appears to be two Ca2+-binding sites, one intrinsic to PDPc and a second produced by association of PDP with E2 (2).…”

Section: Introductionmentioning

confidence: 99%

“…The apparent Km of PDP for Mg2> varies with the assay buffer from about 1.5 to 5 mM. The apparent Km for Ca>2 is -1 ,uM (5,6).…”

mentioning

confidence: 99%

“…Like Ca2+, spermine decreases the apparent Km of PDP for Mg2+ but by a different but hitherto unknown mechanism (6,8,9). This paper reports that PDPr decreases the sensitivity of PDPc to Mg2+ and that this effect is reversed by spermine, which apparently interacts with PDPr.…”

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confidence: 99%

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Role of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.

Yan

Lawson

Reed

1996

Proc. Natl. Acad. Sci. U.S.A.

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Bovine pyruvate dehydrogenase phosphatase (PDP) is a Mg2+-dependent and Ca2+-stimulated heterodimer that is a member of the protein phosphatase 2C family and is localized to mitochondria. Insight into the function of the regulatory subunit of PDP (PDPr) has been gained. It Both phosphorylated El (P-E1) and PDP must be bound to the 60-mer icosahedral dihydrolipoamide acetyltransferase (E2) component of PDC to obtain a maximum rate of dephosphorylation. Ca>2 apparently mediates the specific binding of PDP to E2 in juxtaposition to P-El (7). This orientation decreases the apparent Km of PDP for P-El (7) and for Mg2> (6). There appears to be two Ca2+-binding sites, one intrinsic to PDPc and a second produced by association of PDP with E2 (2).At subsaturating concentrations of Mg2>, PDP activity is stimulated by polyamines, particularly spermine (8). Like Ca2+, spermine decreases the apparent Km of PDP for Mg2+ but by a different but hitherto unknown mechanism (6,8,9). This paper reports that PDPr decreases the sensitivity of PDPc to Mg2+ and that this effect is reversed by spermine, which apparently interacts with PDPr. These observations are potentially significant in understanding the molecular basis of the insulin-induced activation of the mitochondrial PDP.MATERIALS AND METHODS Materials. Highly purified PDC and PDP were prepared from bovine kidney mitochondria (10, 11). Recombinant PDPc was expressed in Escherichia coli and purified to near homogeneity (3). EB and E2 were obtained by resolution of the PDC (11). The E2 contained small amounts of tightly bound E3-binding protein (protein X) and PDH kinase (E2-X-K complex). The peptides RRASVA and RRATVA were prepared manually by solid-phase synthesis using Boc-Ala-Pam resin and Boc-protected amino acids (Peptides International 32P-labeled P-El was prepared by incubating a solution containing 10 mg of E1, 50 ,ug of E2-X-K complex, and 0.1 mM [,y-32P]ATP in 2 ml of buffer A for 1 h at 30°C. The solution was adjusted to pH 5.8 to precipitate P-El, which was dissolved in a small volume of buffer A, the solution was dialyzed against three changes of buffer A, and then centrifuged at 35,000 rpm and 4°C for 1-5 h in a Beckman model Optima TLX ultracentrifuge. The P-El contained 9-12 nmol of phosphate groups per mg of protein. P-El was also prepared by resolution of P-PDC (11).The peptides RRASVA and RRATVA were phosphorylated by incubating for 8-10 h at 30°C a solution containing 10 mg peptide, 25 mM Tris HCl (pH 7.3), 2.5 mM MgCl2, 0.1% 2-mercaptoethanol, 10% glycerol, 0.1 mM [,y-32P]ATP, and 40 units of protein kinase A catalytic subunit in a final volume of 2 ml (12). The reaction was terminated by adding 2 ml of 60% acetic acid, and the solution was passed through an anionexchange column (1.0 x 4 cm) of Dowex 2X8-100 equilibrated with 30% acetic acid. The flowthrough, which contained the phosphopeptide, was lyophilized. The peptide was dissolved in 1 ml of 25 mM Tris-HCl (pH 7.3), 0.1% 2-mercaptoethanol, and the pH was adjusted to 7.3 with 1 M Tris (pH 10...

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mentioning

confidence: 66%

Section: Introductionmentioning

confidence: 99%

“…The apparent Km of PDP for Mg2> varies with the assay buffer from about 1.5 to 5 mM. The apparent Km for Ca>2 is -1 ,uM (5,6).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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Role of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.

Yan

Lawson

Reed

1996

Proc. Natl. Acad. Sci. U.S.A.

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“…PDP1, the dominant isoform in Ca 2ϩ -sensitive tissues, requires Mg 2ϩ and is stimulated by Ca 2ϩ (18). Ca 2ϩ stimulation of PDP1 arises in part because PDP1 binds to E2 via an interaction that requires micromolar concentrations of Ca 2ϩ (38) and in part because Ca 2ϩ decreases the K m of PDP1 for Mg 2ϩ (61). PDP1 comprises a catalytic and a regulatory subunit (42,56).…”

Section: Regulation Of Pdpmentioning

confidence: 99%

Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs

Sugden

Holness

2003

American Journal of Physiology-Endocrinology and Metabolism

450

416

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The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic acid cycle and fatty acid (FA) synthesis. Knowledge of the mechanisms that regulate PDC activity is important, because PDC inactivation is crucial for glucose conservation when glucose is scarce, whereas adequate PDC activity is required to allow both ATP and FA production from glucose. The mechanisms that control mammalian PDC activity include its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDKs 1–4) and its dephosphorylation (activation, reactivation) by the pyruvate dehydrogenase phosphate phosphatases (PDPs 1 and 2). Isoform-specific differences in kinetic parameters, regulation, and phosphorylation site specificity of the PDKs introduce variations in the regulation of PDC activity in differing endocrine and metabolic states. In this review, we summarize recent significant advances in our knowledge of the mechanisms regulating PDC with emphasis on the PDKs, in particular PDK4, whose expression is linked with sustained changes in tissue lipid handling and which may represent an attractive target for pharmacological interventions aimed at modulating whole body glucose, lipid, and lactate homeostasis in disease states.

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Phosphorylation status of pyruvate dehydrogenase in the mousebird Colius striatus undergoing torpor

et al. 2021

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Torpor is a heterothermic response that occurs in some animals to reduce metabolic expenditure. The speckled mousebird (Colius striatus) belongs to one of the few avian taxa possessing the capacity for pronounced torpor, entering a hypometabolic state with concomitant decreases in body temperature in response to reduced food access or elevated thermoregulatory energy requirements. The pyruvate dehydrogenase complex (PDC) is a crucial site regulating metabolism by bridging glycolysis and the Krebs cycle. Three highly conserved phosphorylation sites are found within the E1 enzyme of the complex that inhibit PDC activity and reduce the flow of carbohydrate substrates into the mitochondria. The current study demonstrates a marked increase in S232 phosphorylation during torpor in liver, heart, and skeletal muscle of C. striatus. The increase in S232 phosphorylation during torpor was particularly notable in skeletal muscle where levels were ~49-fold higher in torpid birds compared to controls. This was in contrast to the other two phosphorylation sites (S293 and S300) which remained consistently phosphorylated regardless of tissue. The relevant PDH kinase (PDHK1) known to phosphorylate S232 was found to be substantially upregulated (~5-fold change) in the muscle during torpor as well as increasing moderately in the liver (~2.2-fold increase). Additionally, in the heart, a slight (~23%) decrease in total PDH levels was noted. Taken together the phosphorylation changes in PDH suggest that inhibition of the complex is a common feature across several tissues in the mousebird during torpor and that this regulation is mediated at a specific residue.

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Sensitivity of pyruvate dehydrogenase phosphate phosphatase to magnesium ions. Similar effects of spermine and insulin

Cited by 85 publications

References 50 publications

Role of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.

Role of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.

Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs

Phosphorylation status of pyruvate dehydrogenase in the mousebird Colius striatus undergoing torpor

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