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At least 577 records · Page 32Linked to original sources
PubMed1982

Role of individual phosphorylation sites in inactivation of pyruvate dehydrogenase complex in rat heart mitochondria.

1. A method is described using trypsin/formic acid cleavage for unambiguously measuring occupancies of phosphorylation sites in rat heart pyruvate dehydrogenase [(32)P]phosphate complexes. 2. In mitochondria oxidizing 2-oxoglutarate+l-malate relative initial rates of phosphorylation were site 1>site 2>site 3. 3. Dephosphorylation and reactivation of fully phosphorylated complex was initiated in mitochondria by inhibiting the kinase reaction. Using dichloroacetate relative rates of dephosphorylation were site 2>(1=3). Using sodium dithionite or sodium pyruvate or uncouplers+sodium arsenite or steady state turnover ((31)P replacing (32)P in inactive complex) relative rates were site 2>site 1>site 3. With dithionite reactivation was faster than site 3 dephosphorylation, i.e. site 3 is apparently not inactivating. 4. The steady state proportion of inactive complex was varied (92-48%) in mitochondria oxidizing 2-oxoglutarate/l-malate by increasing extramitochondrial Ca(2+) (0-2.6mum). This action of Ca(2+) induced dephosphorylation (site 3>site 2>site 1). These experiments enable prediction of site occupancies in vivo for given steady state proportions of inactive complexes. 5. The proportion of inactive complex was related linearly to occupancy of site 1. 6. Sodium dithionite (10mm) and Ca(2+) (0.5mum) together resulted in faster dephosphorylations of each site than either agent alone; relative rates were site 2>(1=3). 7. Dephosphorylation and possibly phosphorylation of sites 1 and 2 was not purely sequential as shown by detection of complexes phosphorylated in site 2 but not in site 1. Estimates of the contribution of site 2 phosphorylation to inactivation ranged from 0.7 to 6.4%. 8. It is concluded that the primary function of site 1 phosphorylation is inactivation, phosphorylation of site 2 is not primarily concerned with inactivation and that phosphorylation of site 3 is non-inactivating.

Animals↗
PubMed1996

Stoichiometry of binding of mature and truncated forms of the dihydrolipoamide dehydrogenase-binding protein to the dihydrolipoamide acetyltransferase core of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae.

The dihydrolipoamide dehydrogenase-binding protein (E3BP), a component of the Saccharomyces cerevisiae and mammalian pyruvate dehydrogenase (PDH) complexes, anchors an E3 homodimer inside each of the 12 pentagonal faces of the 60-mer dihydrolipoamide acetyltransferase (E2). To gain further insight into the number and localization of binding sites for E3BP on the 60-mer E2, truncated forms of the E3BP lacking the lipoyl and E3-binding domains were engineered by deletion mutagenesis. The recombinant proteins contained a polyhistidine extension on the amino terminus to facilitate purification to near-homogeneity. The stoichiometry of binding of the truncation mutants to a truncated form (inner core) of E2 (tE2, residues 181-454), lacking the lipoyl domain and the E1-binding domain, was determined. Mixtures containing tE2 and excess intact or truncated forms of E3BP were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or tE3BP, and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer. The results showed that tE2 binds about 20 copies of intact E3BP-H, about 24 copies of tE3BP-H144 (residues 144-380), lacking the lipoyl domain, and about 31 copies of tE3BP-H218 (residues 218-380), lacking both the lipoyl and E3-binding domains. The results indicate that there apparently is a binding site for E3BP on each E2 subunit and that steric hindrance by segments of E3BP prevents full stoichiometric binding of E3BP to the pentagonal dodecahedron-like E2.

Acetyltransferases↗
PubMed1994

Evidence for a role of protein kinase C in the activation of the pyruvate dehydrogenase complex by insulin in Zajdela hepatoma cells.

The signal transduction pathway involved in the activation of pyruvate dehydrogenase (PDH) by insulin is still unknown. In this study, we have examined the possible involvement of protein kinase C (PKC) in the process. In addressing this question, we examined (1) the insulin-like effects of the PKC activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) on the PDH complex, (2) the effects of various PKC inhibitors on the PDH activation by insulin, and (3) the response of PKC-depleted cells to insulin. We used as an experimental model Zajdela hepatoma cultured (ZHC) cells, which have been demonstrated to be responsive to physiological doses of insulin. Half-maximal and maximal stimulations of the PDH complex by insulin were observed at 0.05 and 5 nmol/L, respectively. Stimulation of PDH activity by insulin (5 nmol/L) occurred within 5 minutes of incubation and was maximal (+70%) at 7.5 minutes. In the presence of PMA (162 nmol/L), enzyme activity increased within 30 seconds, was maximal (+90%) at 5 minutes, and was no longer detectable after 10 minutes. Total PDH activity was unchanged by insulin or PMA treatment. The effects of PMA and insulin on basal PDH activity were not additive. Moreover, various inhibitors of PKC--staurosporine, sphingosine, acridine orange--completely blocked the stimulation of PDH activity induced by insulin or PMA. A 17-hour treatment of ZHC cells with 500 nmol/L PMA efficiently downregulated PKC, as attested by the marked decrease in the enzyme activity and the loss of phorbol 12,13-dibutyrate binding to intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1994

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus: preparation and characterization of its binding to the dihydrolipoyl dehydrogenase component.

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli. The domain was characterized by N-terminal sequence analysis, electrospray m.s. and c.d. spectroscopy. It was found to be identical in all respects to a chemically synthesized peptide of the same sequence. The association of the di-domain and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated. As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.

Amino Acid Sequence↗
PubMed1994

Production of an enzymatically active E1 component of human pyruvate dehydrogenase complex in Escherichia coli: supporting role of E1 beta subunit in E1 activity.

A co-expression plasmid containing the coding sequence of both the human liver pyruvate dehydrogenase (PDH) E1 alpha and E1 beta subunits was constructed. Functionally active PDH E1 protein was produced when this co-expression plasmid was introduced into the host Escherichia coli cell, BL21 (DE3)/plysS. In contrast, the production of E1 alpha alone resulted in a catalytically inactive protein, suggesting an important role of the E1 beta subunit in constituting enzyme activity. The PDH E1 protein produced in E. coli was capable of being phosphorylated by PDH-specific kinase. This co-expression system will provide a useful tool for studying the biochemical properties of human PDH E1.

Base Sequence↗
PubMed1997

The pyruvate dehydrogenase complex of the chemolithoautotrophic bacterium Thiobacillus ferrooxidans has an unusual E2-E3 subunit fusion.

The genes encoding pyruvate dehydrogenase (PDH) of Thiobacillus ferrooxidans were previously located by cloning and sequence analysis of the region upstream of the genes encoding the citrate synthase and gamma glutamylcysteine synthetase genes. The pdh genes of T. ferrooxidans were able to complement an Escherichia coli aroP-lpd mutant for growth on minimal medium lacking acetate, indicating that the T. ferrooxidans PDH complex was functional in E. coli. The predicted amino acid sequence of the T. ferrooxidans PDH complex contained three ORFs. The first ORF encoded a 36.7 kDa homologue of the PDH complex E1 alpha subunit, the second ORF a 37.4 kDa E1 beta subunit and the third ORF an unusual 102 kDa fusion of the E2 and E3 subunits. In spite of T. ferrooxidans being a Gram-negative bacterium, its PDH complex had more features in common with Gram-positive bacteria and eukaryotes.

Amino Acid Sequence↗