Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase

Mullinax, Thomas R.; Stepp, Larry R.; Brown, James R.; Reed, Lester J.

doi:10.1016/0003-9861(85)90543-0

Cited by 29 publications

(18 citation statements)

References 17 publications

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“…However, our data do not support the suggestion that Arg 287 -␣ is important for recognition by the BCKD kinase (14). A synthetic oligopeptide in which the equivalent arginine residue in the conserved phosphorylation loop region is not included can be phosphorylated by the related pyruvate dehydrogenase kinase (38).…”

Section: Discussioncontrasting

confidence: 99%

Cross-talk between Thiamin Diphosphate Binding and Phosphorylation Loop Conformation in Human Branched-chain α-Keto Acid Decarboxylase/Dehydrogenase

Li¹,

Wynn

Machius³

et al. 2004

Journal of Biological Chemistry

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Section: Discussioncontrasting

confidence: 99%

Cross-talk between Thiamin Diphosphate Binding and Phosphorylation Loop Conformation in Human Branched-chain α-Keto Acid Decarboxylase/Dehydrogenase

Li¹,

Wynn

Machius³

et al. 2004

Journal of Biological Chemistry

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“…Intact mitochondria (28,34) were extracted from fresh muscle, and an aliquot was used to determine total PDP activity (19,26) using a synthesized polypeptide substrate (30), synthesized and purified by New England Peptide (Gardner, MA). Small pieces of frozen muscle were homogenized, and citrate synthase maximal enzyme activity (39) and PDH in the active form (PDHa; 37) were measured.…”

Section: Methodsmentioning

confidence: 99%

Skeletal muscle type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression: effects of obesity and endurance training

LeBlanc¹,

Mulligan²,

Antolić³

et al. 2008

American Journal of Physiology-Regulatory, Integrative and Comp

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SJ. Skeletal muscle type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression: effects of obesity and endurance training. Am J Physiol Regul Integr Comp Physiol 295: R1224 -R1230, 2008. First published August 20, 2008 doi:10.1152/ajpregu.90320.2008.-Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate metabolism in skeletal muscle. PDH is activated by PDH phosphatase (PDP) and deactivated by PDH kinase (PDK). Obesity has a large negative impact on skeletal muscle carbohydrate metabolism, whereas endurance training has been shown to improve regulatory control of skeletal muscle carbohydrate metabolism, more so when coupled with obesity. A majority of this literature has focused on PDK, with little information available on PDP. To determine the relative role of PDP in regulating skeletal muscle PDH activity with obesity and endurance training, obese and lean Zucker rats remained sedentary or were endurance trained (1 h/day, 5 days/wk) for a period of 8 wk. Soleus, red gastrocnemius, (RG), and white gastrocnemius (WG) muscles were sampled after the training period. The main findings were 1) obesity resulted in a 46% decrease in PDP activity expressed per milligram extracted mitochondrial protein only in RG, while PDP isoform content was unchanged; 2) 8 wk of endurance training led to a significant 1.4 -2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; 3) 8 wk of endurance training led to a trending 1.4 -2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; and 4) PDP2 protein content was not affected by obesity or training. These results suggest that decreased PDP activity in oxidative skeletal muscles may play a role in the impairment of carbohydrate metabolism in obese rats, which is reversible with endurance training. PDP1 and 2; carbohydrate oxidation; exercise PYRUVATE DEHYDROGENASE (PDH) is a key component to glucose homeostasis, as it is the first nonreversible step in mitochondrial glucose oxidation. It is a complex enzyme with many copies of the catalytic subunits and proteins (E1␣, E1␤, E2, E3, and E3 binding protein), as well as its associated regulatory kinase and phophatase enzymes. Decreased carbohydrate flux through PDH is mediated through its phosphorylation and deactivation via PDH kinase (PDK; as reviewed by Refs. 7 and 42). The activation of PDH, via dephosphorylation by PDH phosphatase (PDP), promotes carbohydrate oxidation. The complexity of PDH control by PDK and PDP is enhanced by the presence of multiple isoforms, four PDK [PDK1-4 (4)] and two PDP [PDP1 and 2 (18)]. The two PDP isoforms are unique in their skeletal muscle type distribution and allosteric regulators. PDP1 is the predominant isoform expressed in skeletal muscle, with ϳ2.5 times more found in red gastrocnemius (RG) compared with soleus and white gastrocnemius [WG; (26)] and is ...

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“…Intact mitochondria were extracted from fresh muscle (26,30), and citrate synthase (CS) activities were measured to assess mitochondrial recovery and quality (28). An aliquot of extracted mitochondria was used to determine PDP activity by utilizing the nonradioactive phosphatase assay system (Promega, Madison, WI) (19), with the exception that the assay was conducted at 37°C and the synthesized polypeptide substrate (27) was synthesized and purified by New England Peptide (Gardner, MA). PDP activity is expressed as nanomoles of phosphate released per minute per milligram of extracted mitochondrial protein.…”

Section: Methodsmentioning

confidence: 99%

Skeletal muscle fiber type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression in fed and food-deprived rats

LeBlanc¹,

Harris²,

Peters³

2007

American Journal of Physiology-Endocrinology and Metabolism

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LeBlanc PJ, Harris RA, Peters SJ. Skeletal muscle fiber type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression in fed and food-deprived rats. Am J Physiol Endocrinol Metab 292: E571-E576, 2007. First published October 3, 2006; doi:10.1152/ajpendo.00327.2006.-Fiber type specificity of pyruvate dehydrogenase (PDH) phosphatase (PDP) was determined in fed (CON) and 48-h food-deprived (FD) rats. PDP activity and isoform protein content were determined in soleus (slow-twitch oxidative), red gastrocnemius (RG; fast-twitch oxidative glycolytic), and white gastrocnemius (WG; fast-twitch glycolytic) muscles. When normalized for mitochondrial volume, there was no difference in PDP activity between muscle types or CON and FD. When expressed per gram wet tissue weight, PDP activity was higher in RG compared with soleus and WG in both CON and FD rats. PDP activities from CON muscles were 1.48 Ϯ 0.19, 2.68 Ϯ 0.65, and 1.20 Ϯ 0.33 nmol ⅐ min Ϫ1 ⅐ g wet tissue wt Ϫ1 in soleus, RG, and WG, respectively, and decreased in FD muscles (1.22 Ϯ 0.22, 2.00 Ϯ 0.57, and 0.84 Ϯ 0.18 nmol ⅐ min Ϫ1 ⅐ g wet tissue wt Ϫ1 ). This correlated with increased PDP2 protein, however, only in RG, as PDP2 was not detectable in soleus or WG. PDP1 protein was not responsive to food deprivation in all fiber types. In conclusion, PDP activity and protein content were higher in fasttwitch oxidative glycolytic muscles from CON and FD rats, identifying a unique inter-and intramuscular distribution. FD induced a small but significant decrease in PDP activity that was partially due to decreases in PDP2 protein. As a result, coordinate changes to PDP activity opposite to those of the other regulatory enzyme, PDH kinase, during food deprivation would maximize the inactivation of skeletal muscle PDH and enhance carbohydrate conservation during periods of limited carbohydrate supply.pyruvate dehydrogenase phosphatase-1; pyruvate dehydrogenase phosphatase-2; starvation; carbohydrate oxidation PYRUVATE DEHYDROGENASE (PDH) is a key component of glucose homeostasis, as it regulates mitochondrial carbohydrate oxidation. The activation of PDH (PDHa), via dephosphorylation by PDH phosphatase (PDP), promotes carbohydrate oxidation. During periods of carbohydrate conservation (e.g., food deprivation), PDH is deactivated via phosphorylation by PDH kinase (PDK; as reviewed in Refs. 10 and 38). The complexity of PDH control by PDK and PDP is enhanced by the presence of multiple isoforms, four PDK [PDK1-4 (5)] and two PDP [PDP1 and -2 (18)], with PDK2 and -4 (5) and PDP1 (18) predominantly expressed in skeletal muscle. Intercomparison of both PDK and PDP isoforms reveals differences in specific activities and kinetic properties, tissue-specific mRNA, and protein content and responsiveness to different allosteric regulators, resulting in unique responses to certain metabolic demands. Alterations in activities of either PDK or PDP can occur due either to altered total activity or to modulation through intramitochondrial effectors. Thus, the ratio o...

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Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase

Cited by 29 publications

References 17 publications

Cross-talk between Thiamin Diphosphate Binding and Phosphorylation Loop Conformation in Human Branched-chain α-Keto Acid Decarboxylase/Dehydrogenase

Cross-talk between Thiamin Diphosphate Binding and Phosphorylation Loop Conformation in Human Branched-chain α-Keto Acid Decarboxylase/Dehydrogenase

Skeletal muscle type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression: effects of obesity and endurance training

Skeletal muscle fiber type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression in fed and food-deprived rats

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