scholarly journals Long-term regulation of pyruvate dehydrogenase complex. Evidence that kinase-activator protein (KAP) is free pyruvate dehydrogenase kinase

1991 ◽  
Vol 275 (3) ◽  
pp. 781-784 ◽  
Author(s):  
B S Jones ◽  
S J Yeaman
Keyword(s):  
Pyruvate Dehydrogenase ◽  
High Speed ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Phosphorylation Site ◽  
Pyruvate Dehydrogenase Kinase ◽  
Activator Protein ◽  
Major Phosphorylation Site ◽  
Dehydrogenase Complex

The kinase-activator protein (KAP) of pyruvate dehydrogenase complex (PDC) has been purified approx. 2250-fold from high-speed supernatants of mitochondrial extracts from the liver of 48 h-starved rats. Purified KAP demonstrates kinase activity towards both the E1 component of PDC and towards a synthetic peptide corresponding to the major phosphorylation site on E1. Furthermore, the activities of KAP and PDC kinase co-fractionate through several stages of purification and have the same apparent mass. We conclude that KAP is not a distinct protein, but is kinase which has dissociated from the complex.

scholarly journals Interaction between the individual isoenzymes of pyruvate dehydrogenase kinase and the inner lipoyl-bearing domain of transacetylase component of pyruvate dehydrogenase complex

2002 ◽  
Vol 366 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Alina TUGANOVA ◽  
Igor BOULATNIKOV ◽  
Kirill M. POPOV
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Tight Binding ◽  
Enzymic Activity ◽  
Pyruvate Dehydrogenase Kinase ◽  
Size Exclusion ◽  
Apparent Dissociation Constant ◽  
The Individual ◽  
Dehydrogenase Complex

Protein—protein interactions play an important role in the regulation of enzymic activity of pyruvate dehydrogenase kinase (PDK). It is generally believed that the binding of PDK to the inner lipoyl-bearing domain L2 of the transacetylase component E2 of pyruvate dehydrogenase complex largely determines the level of kinase activity. In the present study, we characterized the interaction between the individual isoenzymes of PDK (PDK1—PDK4) and monomeric L2 domain of human E2, as well as the effect of this interaction on kinase activity. It was found that PDK isoenzymes are markedly different with respect to their affinities for L2. PDK3 demonstrated a very tight binding, which persisted during isolation of PDK3—L2 complexes using size-exclusion chromatography. Binding of PDK1 and PDK2 was readily reversible with the apparent dissociation constant of approx. 10μM for both isoenzymes. PDK4 had a greatly reduced capacity for L2 binding (relative order PDK3>PDK1 = PDK2>PDK4). Monomeric L2 domain alone had very little effect on the activities of either PDK1 or PDK2. In contrast, L2 caused a 3-fold increase in PDK3 activity and approx. 37% increase in PDK4 activity. These results strongly suggest that the interactions between the individual isoenzymes of PDK and L2 domain are isoenzyme-specific and might be among the major factors that determine the level of kinase activity of particular isoenzyme towards the pyruvate dehydrogenase complex.

scholarly journals Pyruvate dehydrogenase kinase/activator in rat heart mitochondria, Assay, effect of starvation, and effect of protein-synthesis inhibitors of starvation

1982 ◽  
Vol 206 (1) ◽  
pp. 103-111 ◽  
Author(s):  
A L Kerbey ◽  
P J Randle
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
High Speed ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Active Form ◽  
Pyruvate Dehydrogenase Kinase ◽  
Heart Mitochondria ◽  
Supernatant Fraction ◽  
Kidney Mitochondria

Purified pig heart pyruvate dehydrogenase complex is denuded of its intrinsic pyruvate dehydrogenase kinase activity by sedimentation from dilute solution (60 munits/ml). Kinase activity is restored by a supernatant fraction prepared by high-speed centrifugation of rat heart mitochondrial extracts; the factor responsible is referred to as kinase/activator. Kinase/activator was also assayed by its ability to accelerate NgATP-induced inactivation in dilute solutions of unprocessed complex (50 munits/ml). With this assay it has been shown that the activity of kinase/activator in heart mitochondria is increased 3-6 fold by starvation of rats for 48 h. This increase was prevented completely by cycloheximide treatment and prevented partially by puromycin treatment of rats during starvation. The concentration of kinase/activator in heart mitochondria fell during 20 h of re-feeding of 48 h-starved rats; this fall was correlated with an increase in the proportion of complex in the active form. Kinase/activator was also extracted from ox kidney mitochondria, and on gel filtration (Sephadex G-100, superfine grade) was eluted close to the void volume. Kinase/activator (ox kidney or rat heart) was thermolabile, non-diffusable on dialysis, and inactivated by trypsin. The results of this study appear to show increased cytoplasmic synthesis in starvation of pyruvate dehydrogenase kinase and/or of an activator of the kinase.

scholarly journals Starvation and diabetes increase the amount of pyruvate dehydrogenase kinase isoenzyme 4 in rat heart

1998 ◽  
Vol 329 (1) ◽  
pp. 197-201 ◽  
Author(s):  
Pengfei WU ◽  
Juichi SATO ◽  
Yu ZHAO ◽  
Jerzy JASKIEWICZ ◽  
M. Kirill POPOV ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Insulin Treatment ◽  
Pyruvate Dehydrogenase Complex ◽  
Immunoblot Analysis ◽  
Diabetic Rats ◽  
Pyruvate Dehydrogenase Kinase ◽  
Specific Effects ◽  
Dehydrogenase Complex

This study investigated whether conditions known to alter the activity and phosphorylation state of the pyruvate dehydrogenase complex have specific effects on the levels of isoenzymes of pyruvate dehydrogenase kinase (PDK) in rat heart. Immunoblot analysis revealed a remarkable increase in the amount of PDK4 in the hearts of rats that had been starved or rendered diabetic with streptozotocin. Re-feeding of starved rats and insulin treatment of diabetic rats very effectively reversed the increase in PDK4 protein and restored PDK enzyme activity to levels of chow-fed control rats. Starvation and diabetes also markedly increased the abundance of PDK4 mRNA, and re-feeding and insulin treatment reduced levels of the message to that of controls. In contrast with the findings for PDK4, little or no changes in the amounts of PDK1 and PDK2 protein and the abundance of their messages occurred in response to starvation and diabetes. The observed shift in the relative abundance of PDK isoenzymes probably explains previous studies of the effects of starvation and diabetes on heart PDK activity. The results indicate that control of the amount of PDK4 is important in long-term regulation of the activity of the pyruvate dehydrogenase complex in rat heart.

scholarly journals Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex

1998 ◽  
Vol 329 (1) ◽  
pp. 191-196 ◽  
Author(s):  
Melissa M. BOWKER-KINLEY ◽  
I. Wilhelmina DAVIS ◽  
Pengfei WU ◽  
A. Robert HARRIS ◽  
M. Kirill POPOV
Keyword(s):  
Tissue Distribution ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
Synthetic Analogue ◽  
Complex Activity ◽  
Acetyl Coa ◽  
Tissue Specific ◽  
Specific Regulation ◽  
Dehydrogenase Complex

Tissue distribution and kinetic parameters for the four isoenzymes of pyruvate dehydrogenase kinase (PDK1, PDK2, PDK3 and PDK4) identified thus far in mammals were analysed. It appeared that expression of these isoenzymes occurs in a tissue-specific manner. The mRNA for isoenzyme PDK1 was found almost exclusively in rat heart. The mRNA for PDK3 was most abundantly expressed in rat testis. The message for PDK2 was present in all tissues tested but the level was low in spleen and lung. The mRNA for PDK4 was predominantly expressed in skeletal muscle and heart. The specific activities of the isoenzymes varied 25-fold, from 50 nmol/min per mg for PDK2 to 1250 nmol/min per mg for PDK3. Apparent Ki values of the isoenzymes for the synthetic analogue of pyruvate, dichloroacetate, varied 40-fold, from 0.2 mM for PDK2 to 8 mM for PDK3. The isoenzymes were also different with respect to their ability to respond to NADH and NADH plus acetyl-CoA. NADH alone stimulated the activities of PDK1 and PDK2 by 20 and 30% respectively. NADH plus acetyl-CoA activated these isoenzymes nearly 200 and 300%. Under comparable conditions, isoenzyme PDK3 was almost completely unresponsive to NADH, and NADH plus acetyl-CoA caused inhibition rather than activation. Isoenzyme PDK4 was activated almost 2-fold by NADH, but NADH plus acetyl-CoA did not activate above the level seen with NADH alone. These results provide the first evidence that the unique tissue distribution and kinetic characteristics of the isoenzymes of PDK are among the major factors responsible for tissue-specific regulation of the pyruvate dehydrogenase complex activity.

Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites

2001 ◽  
Vol 358 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Elena KOLOBOVA ◽  
Alina TUGANOVA ◽  
Igor BOULATNIKOV ◽  
Kirill M. POPOV
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Phosphorylation Site ◽  
Enzymic Activity ◽  
Mammalian Tissues ◽  
Activity State ◽  
The Individual ◽  
Pyruvate Dehydrogenase Phosphatase ◽  
Thiamin Pyrophosphate ◽  
Dehydrogenase Complex

The enzymic activity of the mammalian pyruvate dehydrogenase complex is regulated by the phosphorylation of three serine residues (sites 1, 2 and 3) located on the E1 component of the complex. Here we report that the four isoenzymes of protein kinase responsible for the phosphorylation and inactivation of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their abilities to phosphorylate the enzyme. PDK1 can phosphorylate all three sites, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. Although PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporated by each isoenzyme decreases in the order PDK1>PDK3PDK4>PDK2. Significantly, binding of the coenzyme thiamin pyrophosphate to pyruvate dehydrogenase alters the rates and stoichiometries of phosphorylation of the individual sites. First, the rate of phosphorylation of site 1 by all isoenzymes of kinase is decreased. Secondly, thiamin pyrophosphate markedly decreases the amount of phosphate that PDK1 incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenzyme does not significantly affect the total amount of phosphate incorporated in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphorylation of this site. Furthermore, pyruvate dehydrogenase complex phosphorylated by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyruvate dehydrogenase is phosphorylated by other isoenzymes, the rates of reactivation decrease in the order PDK4PDK3> PDK1. Taken together, results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.

Increased pyruvate dehydrogenase kinase activity in response to sepsis

1991 ◽  
Vol 260 (5) ◽  
pp. E669-E674 ◽  
Author(s):  
T. C. Vary
Keyword(s):  
Skeletal Muscle ◽  
Pyruvate Dehydrogenase ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
Sterile Inflammation ◽  
Stable Factor ◽  
Skeletal Muscle Mitochondria ◽  
Dependent Inactivation ◽  
Significant Difference

The effect of sterile inflammation and sepsis on the proportion of active pyruvate dehydrogenase complex (PDH) in mitochondria isolated from skeletal muscle has been investigated. The proportion of active PDH in mitochondria isolated from septic animals was significantly reduced compared with control under all incubation conditions examined, even in the presence of inhibitors of the PDH kinase. There was no significant difference between control and sterile inflammation in any of the incubations examined. The rate constant for ATP-dependent inactivation of the PDH complex in mitochondrial extracts from control animals was -0.42 min-1 (r = 0.993; P less than 0.001) and was not altered in mitochondrial extracts from sterile inflammatory animals (-0.43 min-1; r = 0.999; P less than 0.001). However, rate constants for inactivation in septic animals was significantly increased over twofold to -1.08 min-1 (r = 0.987; P less than 0.001) (P less than 0.001 vs. control or sterile inflammation). In the presence of inhibitors of the PDH kinase reaction (2.5 mM pyruvate or 1 mM dichloroacetate), inactivation of PDH after addition of ATP was significantly greater in mitochondrial extracts from septic than either control or sterile inflammatory animals. These results suggest that sepsis, but not sterile inflammation, induces a stable factor in skeletal muscle mitochondria that increased PDH kinase activity.

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