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PubMed2006

Activity of pyruvate dehydrogenase A (PDHA) in hamster spermatozoa correlates positively with hyperactivation and is associated with sperm capacitation.

Unravelling the molecular basis of capacitation is crucial to our understanding the basis of acquisition of fertilization competence by spermatozoa. In two recent studies, we have demonstrated that dihydrolipoamide dehydrogenase, which is a post-pyruvate metabolic enzyme and one of the components of pyruvate dehydrogenase complex, undergoes capacitation-dependent tyrosine phosphorylation, and that the activity of the enzyme correlates with capacitation events in the hamster spermatozoa. However, it is not clear as to whether other components of the pyruvate dehydrogenase complex are also crucial for sperm capacitation. In this report, we have identified pyruvate dehydrogenase A2 (PDHA2), a constituent of pyruvate dehydrogenase A (PDHA), which is a component of pyruvate dehydrogenase complex that exhibits tyrosine phosphorylation during hamster spermatozoal capacitation. This is the first report showing that hamster sperm PDHA2 is a testis-specific phosphotyrosine that is associated with the fibrous sheath of hamster spermatozoa. The localization of PDHA2 in spermatozoa was investigated using antibodies to PDHA, which is the active tetrameric protein that consists of a homodimer of PDHA2 and PDHB. Both immunofluorescence and confocal studies indicated a unique non-canonical, extramitochondrial localization for PDHA in the principal piece of hamster spermatozoa. It was also observed that PDHA colocalized with AKAP4 in the fibrous sheath of the spermatozoon. The enzymatic activity of PDHA was positively correlated with hyperactivation but not with the acrosome reaction. Given the localization of PDHA and the evidence that its activity correlates positively with hyperactivation and that its PDHA2 subunit exhibits capacitation-associated protein tyrosine phosphorylation, it appears that PDHA2 is associated with the process of capacitation.

Animals↗
PubMed1979

Regulation of kinase reactions in pig heart pyruvate dehydrogenase complex.

1. Pig heart pyruvate dehydrogenase complex is inactivated by phosphorylation (MgATP2-) of an alpha-chain of the decarboxylase component. Three serine residues may be phosphorylated, one of which (site 1) is the major inactivating site. 2. The relative rates of phosphorylation are site 1 greater than 2 greater than site 3. 3. The kinetics of the inactivating phosphorylation were investigated by measuring inactivation of the complex with MgATP2-. The apparent Km for the Mg complex of ATP was 25.5 microM; ADP was a competitive inhibitor (Ki 69.8 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 2.8 microM). Inactivation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA. 4. The kinetics of additional phosphorylations (predominantly site 2 under these conditions) were investigated by measurement of 32P incorporation into non-radioactive pyruvate dehydrogenase phosphate containing 3-6% of active complex, and assumed from parrallel experiments with 32P labelling to contain 91% of protein-bound phosphate in site 1 and 9% in site 2. 5. The apparent Km for the Mg complex of ATP was 10.1 microM; ADP was a competitive inhibitor (Ki 31.5 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 1.1 mM). 6. Incorporation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA, although it was less marked at the highest ratios.

Adenosine Triphosphate↗
PubMedDate not supplied

[Activity of the pyruvate dehydrogenase complex in Walker-256 carcinosarcoma].

Pyruvate dehydrogenase complex activity in the coarsely purified enzyme fractions (supernatant and mitochondrial extract) isolated from Walker-256 carcinosarcoma is shown to be much lower than the complex activity in the rat brain. Acetoin was not found in the incubation medium containing pyruvate dehydrogenase tumour complex. The rate of the reaction of nonoxidative formation of acetaldehyde by pyruvate dehydrogenase complex of the tumour was approximately five times lower than the rate of reaction catalyzed by the complex from the rat brain. It is shown that in the presence of adenine nucleotides AMP, ADP or ATP (0.5 mM) in the medium pyruvate: NAD+ oxidoreductase activity of the tumour complex is inhibited by 40-56% as compared to the control and reaction of nonoxidative decarboxylation of pyruvate is approximately 2 times activated. It is supposed that the accumulation of nonoxidative reaction products may be an essential factor for development and functioning of the tumour.

Adenosine Diphosphate↗
PubMed1975

Regulatory function of pyruvate dehydrogenase and the mitochondrion in lipogenesis.

The activity of pyruvate dehydrogenase from freshly isolated mitochondria was shown to be dependent upon the nutritional and metabolic state of the animal prior to sacrifice, such that mitochondria from the livers of 48 hr starved, diabetic, or high fat fed rats had lower enzyme activity than normal, chow fed rats. The activity of pyruvate dehydrogenase and the rate of lipogenesis were shown to correlate to a certain extent when a reconstituted, cell free system consisting of 105,000 x g supernatant of rat liver and isolated mitochondria was used. This system was employed so that the role of the mitochondrion and pyruvate dehydrogenase in lipogenesis could be investigated. Dichloroacetate increased the activity of pyruvate dehydrogenase and increased the rate of lipogenesis, suggesting that the activity of pyruvate dehydrogenase is an important factor in determining the rate of lipogenesis in the reconstituted system. It was observed, however, that dichloroacetate was more effective in stimulating the activity of pyruvate dehydrogenase than the rate of lipogenesis when mitochondria from starved animals were used to reconstitute lipogenesis. Furthermore, the cytoplasmic adenosine triphosphate/adenosine diphosphate ratios and phosphorylation potentials (ATP/ADP x Pi) maintained in the reconstituted system by mitochondria isolated from starved animals were found to be significantly lower than those maintained by mitochondria isolated from chow fed animals. It is proposed that the lower "energy pressure" maintained in the reconstituted system by mitochondria isolated from starved animals severely limits lipogenesis at the ATP requiring steps of the process.

Acetates↗
PubMed1984

The pyruvate dehydrogenase complex during aging.

Acetylcholine synthesis and pyruvate oxidation decline with age. To determine the role of the pyruvate dehydrogenase complex in these age-related deficits, its activity and activation state were measured in vivo and in vitro in the brains of 3-, 10- and 30-month-old mice. Aging did not alter the active form of pyruvate dehydrogenase complex in vivo, although the total complex was 17% lower at 30 than at 3 months of age. In vitro, total or active forms of pyruvate dehydrogenase complex did not change with age. The results suggest that neither changes in total activity nor in the activation state of the pyruvate dehydrogenase complex account for the age-related deficits in oxidative or acetylcholine metabolism.

Acetylcholine↗
PubMed1984

Partial purification from human mononuclear cells and placental plasma membranes of an insulin mediator which stimulates pyruvate dehydrogenase and suppresses glucose-6-phosphatase.

A substance capable of stimulating pyruvate dehydrogenase (PDH) and suppressing glucose-6-phosphatase (G-6-Pase) in a cell-free system was prepared from insulin-treated human placental plasma membranes and peripheral blood mononuclear cells by formic acid extraction. This material was partially purified by molecular-exclusion chromatography, ion-exchange chromatography, and hydroxylapatite chromatography. This was found to stimulate pyruvate dehydrogenase and inhibit glucose-6-phosphatase in a dose-dependent manner. The amount or ability of this substance to stimulate pyruvate dehydrogenase was increased in the proportion to the concentration of insulin. The stimulation of pyruvate dehydrogenase by the factor was eliminated when sodium fluoride was presented in the assay of the activation. This result implied that the activation of pyruvate dehydrogenase was mediated by the stimulation of the phosphatase of pyruvate dehydrogenase complex. Each material isolated from insulin-treated human placental plasma membranes and mononuclear cells shared a number of important characteristics of putative second messengers of insulin action as follows: (i) heat and acid stability; (ii) a similar molecular weight; (iii) increased activity of pyruvate dehydrogenase in a insulin-dependent manner; and (iv) stimulated pyruvate dehydrogenase by the sodium fluoride-sensitive mechanism. This human putative second messenger of insulin action was eluted from the anion-exchange resin AG1-X8 at an ionic strength of 0.3-0.4 M, as well as from the hydroxylapatite column at a phosphate concentration of 0.2-0.3 M.

Adenosine Triphosphate↗
PubMed1992

Atomic structure of the cubic core of the pyruvate dehydrogenase multienzyme complex.

The highly symmetric pyruvate dehydrogenase multienzyme complexes have molecular masses ranging from 5 to 10 million daltons. They consist of numerous copies of three different enzymes: pyruvate dehydrogenase, dihydrolipoyl transacetylase, and lipoamide dehydrogenase. The three-dimensional crystal structure of the catalytic domain of Azotobacter vinelandii dihydrolipoyl transacetylase has been determined at 2.6 angstrom (A) resolution. Eight trimers assemble as a hollow truncated cube with an edge of 125 A, forming the core of the multienzyme complex. Coenzyme A must enter the 29 A long active site channel from the inside of the cube, and lipoamide must enter from the outside. The trimer of the catalytic domain of dihydrolipoyl transacetylase has a topology identical to chloramphenicol acetyl transferase. The atomic structure of the 24-subunit cube core provides a framework for understanding all pyruvate dehydrogenase and related multienzyme complexes.

Amino Acid Sequence↗
PubMed1969

Alpha-keto acid dehydrogenase complexes. XI. Comparative studies of regulatory properties of the pyruvate dehydrogenase complexes from kidney, heart, and liver mitochondria.

The activity of the multienzyme pyruvate dehydrogenase complexes, isolated from mitochondria of beef kidney, beef heart, and pork liver, is regulated by phosphorylation and dephosphorylation. Phosphorylation and concomitant inactivation of each of the three complexes are catalyzed by an ATP-specific kinase, and dephosphorylation and concomitant reactivation are catalyzed by a phosphatase. The phosphatase has been separated from the other component enzymes of each pyruvate dehydrogenase complex, and the three phosphatases are functionally interchangeable. The kinase has been isolated from the beef kidney complex, and it is functional with the beef heart and pork liver complexes. ADP is competitive with ATP, and the ADP effect is more pronounced with the kidney kinase than with the liver and heart kinases. Pyruvate protects strongly the heart and liver pruvate dehydrogenase complexes and, to a lesser extent, the kidney complex against inactivation by ATP. Pyruvate apparently exerts its effect on the pyruvate dehydrogenase component of the complex, rather than on the kinase.

Adenine Nucleotides↗
PubMed1990

Insulin-like effects of ATP on adipocyte pyruvate dehydrogenase and phosphorylase.

Extracellular ATP stimulated adipocyte pyruvate dehydrogenase in a time- and dose-dependent manner with an EC50 of 0.1 mM. The maximal effect was observed at 0.5 mM ATP after a 15-min incubation with a lag period of about 5 min. Depletion of intracellular Ca2+ with ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid reduced the effect of ATP by 50% and completely abolished the stimulatory effect of vasopressin on adipocyte pyruvate dehydrogenase but had no effect on the stimulation induced by insulin or adenosine. The effects of insulin and ATP on pyruvate dehydrogenase were glucose-dependent whereas the effect of adenosine was glucose-independent. Furthermore, ATP, like insulin, partially blocked the stimulatory effect of isoproterenol on phosphorylase. Adenosine, at a concentration of 1 mM, did not affect either basal or isoproterenol-stimulated phosphorylase activities. It is concluded that ATP activates adipocyte pyruvate dehydrogenase by at least two separate mechanisms: one is Ca2(+)-dependent and the other is Ca2(+)-independent. However, neither is the result of the formation of adenosine from ATP through hydrolysis.

Adenosine↗
PubMed1981

A third unlinked gene controlling the pyruvate dehydrogenase complex in Aspergillus nidulans.

Pyruvate dehydrogenase complex mutants of Aspergillus nidulans were obtained by ultraviolet treatment and enrichment procedures. Among 160 glycolytic mutants, 86 pyruvate dehydrogenase complex mutants (including some temperature-sensitive mutants) were found. In addition to genes pdhA and pdhB, which are described in previous studies, a third gene, pdhC, controlling the function of the enzyme complex, was identified. The three genes were not linked and were mapped in the following linkage groups: pdhA in group I, pdhB in group V, and pdhC in group VIII, where it was the first marker on the left arm.

Aspergillus nidulans↗
PubMed1997

Effects of aging on the activities of pyruvate dehydrogenase complex and its kinase in rat heart.

Effects of aging on the activities of heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase were examined using 7, 35 and 60 wk old rats. Aging did not affect the total activity of pyruvate dehydrogenase complex but decreased the activity state (percentage of active form) of the complex in rats under the fed condition (52%, 36% and 26% for 7, 35 and 60 wk old rats, respectively). This decrease in the complex activity with aging was suggested to be associated with an age-related decrease in the blood glucose disposal. Starvation for 24 h decreased the activity state to less than 3% in all of the age groups. The activity of pyruvate dehydrogenase kinase associated with the complex was not related to the alteration in the activity state of the complex; the kinase activity was slightly lower in 60 wk old rats than in the younger rats under the fed condition and activation of the kinase by starvation was greater in the younger rats. The mechanism for the decrease in activity of pyruvate dehydrogenase complex was discussed on the basis of glucose and fatty acid utilization of heart muscle cells.

Aging↗
PubMed1979

Amino acid sequences around the sites of phosphorylation in the pig heart pyruvate dehydrogenase complex.

1. When pig heart pyruvate dehydrogenase complex was phosphorylated to completion with [gamma-32P]ATP by its intrinsic kinase, three phosphorylation sites were observed. The amino acid sequences around these sites were: sequence 1, Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg; and sequence 2, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser(P)-Tyr-Arg. 2. When phosphorylated to inactivation by repetitive additions of limiting quantities of [gamma-32P]ATP, phosphate was incorporated mainly (more than 90%) into Ser-5 of sequence 2. Phosphorylation of this site thus results in activation of pyruvate dehydrogenase. 3. If Ser-5 is phosphorylated with ATP and the enzyme then incubated with [gamma-32P]ATP, phosphorylation of the remaining sites occurred. Ser-12 of sequence 2 is phosphorylated about twice as rapidly as Ser-6 of sequence 1. 4. Incubation of pyruvate dehydrogenase with excess [gamma-32P]ATP with termination of phosphorylation at about 50% complete inactivation showed that Ser-5 of sequence 2 was phosphorylated most rapidly, but also that Ser-12 of sequence 2 was significantly (15% of total) phosphorylated. Ser-6 sequence 1 contained about 1% total P. 5. These results suggest that addition of limiting amounts of ATP produces primarily phosphorylation of Ser-5 of sequence 2 (inactivating site). This also occurs during incubation with excess ATP before complete inactivation occurs, but a greater occupancy of other sites also occurs during this treatment.

Amino Acid Sequence↗
PubMed1978

The effect of Mycoplasma contamination on the in vitro assay of pyruvate dehydrogenase activity in cultured fibroblasts.

Cultured fibroblasts contaminated with mycoplasma were shown to have increased in levels of apparent pyruvate dehydrogenase and pyruvate dehydrogenase complex enzyme activity. The apparent pyruvate dehydrogenase specific activity was about 1000-fold greater in cultured Mycoplasma pneumoniae than in cultured fibroblasts. Several acid hydrolytic enzyme activities were also shown to be present in M. pneumoniae though a lower apparent specific activity than in cultured fibroblasts.

Cell Line↗
PubMed1984

Domain structure and 1H-n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

The pyruvate dehydrogenase complex of Bacillus stearothermophilus was treated with Staphylococcus aureus V8 proteinase, causing cleavage of the dihydrolipoamide acetyltransferase polypeptide chain (apparent Mr 57 000), inhibition of the enzymic activity and disassembly of the complex. Fragments of the dihydrolipoamide acetyltransferase chains with apparent Mr 28 000, which contained the acetyltransferase activity, remained assembled as a particle ascribed the role of an inner core of the complex. The lipoic acid residue of each dihydrolipoamide acetyltransferase chain was found as part of a small but stable domain that, unlike free lipoamide, was able still to function as a substrate for reductive acetylation by pyruvate in the presence of intact enzyme complex or isolated pyruvate dehydrogenase (lipoamide) component. The lipoyl domain was acidic and had an apparent Mr of 6500 (by sedimentation equilibrium), 7800 (by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) and 10 000 and 20 400 (by gel filtration in the presence and in the absence respectively of 6M-guanidinium chloride). 1H-n.m.r. spectroscopy of the dihydrolipoamide acetyltransferase inner core demonstrated that it did not contain the segments of highly mobile polypeptide chain found in the pyruvate dehydrogenase complex. 1H-n.m.r. spectroscopy of the lipoyl domain demonstrated that it had a stable and defined tertiary structure. From these and other experiments, a model of the dihydrolipoamide acetyltransferase chain is proposed in which the small, folded, lipoyl domain comprises the N-terminal region, and the large, folded, core-forming domain that contains the acetyltransferase active site comprises the C-terminal region. These two regions are separated by a third segment of the chain, which includes a substantial region of polypeptide chain that enjoys high conformational mobility and facilitates movement of the lipoyl domain between the various active sites in the enzyme complex.

Chemical Phenomena↗
PubMed1980

[Familial partial pyruvic dehydrogenase deficiency (author's transl)].

Pyruvic dehydrogenase deficiency was found in two living children of a Turkish family, whereas lactate acidosis was found in a dead child. Two further children died with a pattern of a therapy-refractory metabolic acidosis without any recognisable cause. The prerequisites for the creation of lactate acidosis are demonstrated by the example of pyruvic dehydrogenase deficiency. The extent of remaining activity in case of peruvic dehydrogenase deficiency determines the pattern of clinical signs and the course of the disease which can be influenced to a varying degree by additional complications and different attempts at treatment.

Acidosis↗
PubMed1985

Immunochemical characterization of pyruvate dehydrogenase complex in rat brain.

Pyruvate dehydrogenase complex (PDHC) in rat brain was studied immunochemically, using antibodies against the bovine kidney PDHC, by immunoblotting, immunoprecipitation, inhibition of enzyme activity, and enzyme-linked immunoabsorbent assay (ELISA). The immunoblots showed that the antibodies bound strongly to the alpha peptide of the pyruvate dehydrogenase (E1) component, and to the dihydrolipoyl transacetylase (E2) and the dihydrolipoyl dehydrogenase (E3) components of PDHC. A similar immunoblotting pattern was observed in all eight brain regions examined. On immunoblotting of the subcellular fractions, these PDHC peptides were observed in mitochondria and synaptosomes but not in the postmitochondrial supernatants. This agrees with other evidence that brain PDHC is localized in the mitochondria. These results, together with those from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitin, also showed that the alpha E1, beta E1, and E3 peptides of rat brain PDHC are very similar in sizes to those of the bovine kidney PDHC, being 42, 36, and 58 kD, respectively. The size of the E2 peptide, 66 kD, is different from that of bovine kidney E2, 73 kD. The relative abundance of PDHC protein in nonsynaptic mitochondria was compared by enzyme activity titration and ELISA. Both methods demonstrated that the amount of PDHC antigen in the mitochondria from cerebral cortex is greater than that in the olfactory bulb mitochondria. This is consistent with the results of the activity measurement. The ELISA also showed that the PDHCs in both mitochondrial populations are antigenically similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

A possible mechanism for selective cerebellar damage in partial pyruvate dehydrogenase deficiency.

In patients with partial deficiencies of pyruvate dehydrogenase, cerebellar ataxia has been the most prominent and sometimes the only neurologic abnormality. It is not clear how this generalized enzyme deficiency (with activity 15 to 30 percent of normal in several tissues) might lead to clinical signs referable to a limited part of the nervous system. We therefore compared the normal activity of pyruvate dehydrogenase with the normal rate of pyruvate oxidation in different parts of animal brains and then calculated the effect on pyruvate oxidation of partial deficiencies of the enzyme. The data indicate that pyruvate oxidation could be impaired in an area of anterior cerebellar vermis by deficiencies of pyruvate dehydrogenase too mild to affect pyruvate oxidation in the other areas of the brain we examined.

Animals↗