Alpha Subunit Of Pyruvate Dehydrogenase Research Articles

Calcium-stimulated protein phosphorylation in synaptic membranes.

Synaptic membranes from rat brain contain several calcium-requiring protein kinase (PK) activities with different substrate specificities: (a) an activity (CaH-PK) effective at high concentrations of Ca2+ ion in the absence of Mg2+ (active on class F substrates); (b) a (Ca + Mg)-PK activity that is mediated by Ca2+ ion in the presence of Mg2+ (active on class B substrates); (c) (Ca-CaM)-PK activities that exhibit simultaneous requirements for both Ca2+ ion and CaM (for class C and D substrates). Also described are three activities (d-f) that do not require Ca2+ ion: (d) a Mg-PK activity in which the presence of Ca2+ causes the inhibition of phosphorylation (active on class A substrates); (e) an activity affecting a diverse group of substrates (class E substrates), the phosphorylation of which occurs in the presence of Mg2+ ion alone (Mg-PK activity) and is unaffected by the addition of Ca2+ ion and CaM, the substrates of which show different responses to several types of inhibitors; and, finally, (f) the previously well characterized cAMP-dependent PK activities. Several of the substrates of these kinases have been identified in a fairly unambiguous manner: among them are P43 (class A), as the alpha subunit of pyruvate dehydrogenase; P54 (class B), as the presynaptic protein B50; and the doublet P75-P80, as proteins IA and IB of Ueda and Greengard. The most interesting activity is that requiring both Ca2+ and CaM. The half-maximal stimulation (K0.5) for Ca2+ in the presence of CaM was found to be 1.0 microM Ca2+F in untreated membranes. There is little change in this value on prior EGTA extraction of the membranes, which removes the bulk of its Ca2+ and reduces its residual CaM by greater than or equal to 50%. The apparent K0.5 for CaM in the presence of excess Ca2+ ion was found to equal 0.4 microgram per reaction mixture (8 micrograms/ml) or 1.35 micrograms per reaction mixture (27 micrograms/ml), for the untreated and EGTA-treated membranes, respectively.

The effect of dichloroacetate on the phosphorylation of mitochondria proteins

Succinyl-CoA synthetase and the alpha-subunit of pyruvate dehydrogenase are phosphorylated after incubation of mitochondria from brain, heart, and liver with [gamma- 32P]ATP. Dichloroacetate, a known specific inhibitor for pyruvate dehydrogenase kinase, inhibits not only the phosphate incorporation into the alpha-subunit of pyruvate dehydrogenase but also the autophosphorylation of succinyl-CoA synthetase. AMP also inhibits the phosphorylation of both proteins. Phosphorylation of the alpha-subunit of pyruvate dehydrogenase in liver mitochondria is significantly lower than in mitochondria from other tissues.

The chemical mediators of insulin action: Possible targets for postreceptor defects

An insulin-sensitive subcellular system was developed from rat adipocytes consisting of plasma membranes and mitochondria. Direct addition of insulin, concanavalin A or anti-insulin receptor antibody to this system resulted in the production of a mediator substance from the plasma membrane that caused dephosphorylation of the alpha subunit of pyruvate dehydrogenase in the mitochondria with concomitant activation of the enzyme. The mediator activated pyruvate dehydrogenase by activating the pyruvate dehydrogenase phosphatase and not by inhibiting the pyruvate dehydrogenase kinase. This was similar to the mechanism by which insulin causes activation of the enzyme in the intact cell. The insulin-sensitive mediator material from the adipocyte plasma membrane was acid-stable with a molecular weight of 1,000 to 1,500. Our laboratory has shown that the mediator that activates pyruvate dehydrogenase was present in intact adipocytes, hepatoma cells, and IM-9 lymphocytes. Insulin altered the amount or activity of the mediator consistent with the effect of the hormone on the cell. Other laboratories have shown similar effects on skeletal muscle and liver. We have shown the mediator to mimic insulin action on the low Km cyclic adenosine monophosphate (AMP) phosphodiesterase and the (calcium ++-magnesium ++)-adenosine triphosphatase (Ca ++-Mg ++)-ATPase of adipocyte plasma membranes in addition to pyruvate dehydrogenase. Other laboratories have shown the mediator to activate glycogen synthase. A body of direct and indirect evidence exists that demonstrates that more than one mediator exists. The chemical nature of the mediator is unknown but probably represents a new family of intracellular mediators of hormone action. These mediators may have clinical relevance in postreceptor defects of obesity and type II diabetes (noninsulin-dependent diabetes mellitus).

Brain pyruvate dehydrogenase activity: regulation by phosphorylation-dephosphorylation

The alpha-subunit of pyruvate dehydrogenase (PDHa) decarboxylates pyruvate and is the rate-limiting step in the PDH-complex reaction sequence. Using different phosphorylation conditions which inhibit PDHa-kinase of PDHa-phosphatase, we demonstrate that PDHa in brain is regulated by a phosphorylation (inactivation) - dephosphorylation (activation) cycle.

Phosphoproteins of the adrenal chromaffin granule membrane.

A fraction of chromaffin granule membranes contained a number of substrates for endogenous protein kinase activity as well as endogenous phosphatase activity. The major 32P-labelled polypeptide of molecular weight 43,000 appeared to be the alpha-subunit of pyruvate dehydrogenase of residual mitochondria. Several polypeptides showed cyclic AMP stimulation of phosphorylation of which the major polypeptide of molecular weight 59,000 shows half-maximal phosphorylation with 0.49 microM cyclic AMP. The phosphorylation of several other polypeptides is inhibited at high cyclic AMP concentrations. From studies with immunoprecipitation and two-dimensional electrophoresis it was found that alpha- and beta-tubulin and actin were absent from the granule membranes. However 32P labelling of a proportion of the copies of dopamine-beta-hydroxylase was demonstrated. The majority of the substrates for endogenous protein kinase activity are probably on the cytoplasmic side of the granule membrane.

Brain pyruvate dehydrogenase: phosphorylation and enzyme activity altered by a training experience.

The active portion of the alpha subunit of pyruvate dehydrogenase in rat frontal cortex was elevated after a training experience. No change in total pyruvate dehydrogenase activity was observed. The phosphorylation in vitro of pyruvate dehydrogenase (band F-2) was also elevated after training. Since activation of pyruvate dehydrogenase requires its dephosphorylation, the following sequence is proposed. Training alters frontal cortex and reduces the phosphate content of pyruvate dehydrogenase in vivo; this leads to enzyme activation; and an increase in back-titration of sites available for phosphorylation in vitro.

Evidence that the 40,000 Mr phosphoprotein influenced by high frequency synaptic stimulation is the alpha subunit of pyruvate dehydrogenase

We have previously shown that brief periods of high frequency synaptic stimulation of the rat hippocampus influence the endogenous phosphorylation of a 40,000 Mr brain protein (Browning et al. 6). The results of the present study demonstrate that this brain phosphoprotein is enriched in a purified mitochondrial fraction and co-migrates with the α-subunit of pyruvate dehydrogenase in sodium dodecyl sulfate polyacrylamide gels. Comparisons of total and partial proteolytic fingerprints indicate that the two proteins are essentially identical. In addition, the phosphorylation of the 40,000 Mr brain protein is sensitive to both dichloroacetate and magnesium as has been reported for pyruvate dehydrogenase 35. Taken together these data provide persuasive evidence that the brain protein is the a-subunit of pyruvate dehydrogenase and thereby raise the possibility that even very short periods of synaptic activity influence an enzyme of particular importance to mitochondrial metabolism in brain.

Studies on the incorporation of [32P]phosphate into pyruvate dehydrogenase in intact rat fat-cells. Effects of insulin.

1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35--45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells.