Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Properties of the branched-chain 2-hydroxy acid/2-oxo acid shuttle in mouse spermatozoa.

ABSTRACT: Operation of the branched-chain 2-hydroxy acid/2-oxo acid shuttle for the transfer of reducing equivalents in mitochondria of mouse spermatozoa was studied in vitro in reconstituted systems. Results show that the branched-chain 2-oxo acids within the mitochondria are offered several metabolic pathways. (a) Decarboxylation: mouse sperm mitochondria possess high branched-chain 2-oxo acid decarboxylase activity. (b) Recycling to the cytosol by using a transport system which can be inhibited by alpha-cyano-3-hydroxycinnamate and pH 6.8. (c) Transamination to the corresponding amino acids: experiments presented indicate that leucine formed from 4-methyl-2-oxopentanoate may pass to the external phase, re-initiating the cycle. These two last possibilities would allow autocatalytic operation of the shuttle. The branched-chain 2-hydroxy acids apparently do not utilize the monocarboxylate carrier to penetrate the mitochondria.

SUBMITTER: Coronel CE

PROVIDER: S-EPMC1146765 | biostudies-other | 1986 May

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Partial purification and properties of branched-chain 2-oxo acid dehydrogenase of ox liver.

Project description:1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.

| S-EPMC1184023 | biostudies-other

Subcellular localization of branched-chain amino acid aminotransferase and lactate dehydrogenase C4 in rat and mouse spermatozoa.

Project description:Spermatozoa isolated from rat and mouse epididymes show a relatively high branched-chain amino acid aminotransferase (leucine aminotransferase, EC 2.6.1.6) activity. There is a significant reduction of leucine aminotransferase and of the isoenzyme C4 of lactate dehydrogenase (EC 1.1.1.27) in the gametes during their epididymal transit. Studies of patterns of liberation of the leucine aminotransferase and of the lactate dehydrogenase C4 from intact spermatozoa, treated with increasing concentrations of digitonin, indicate that both enzymes have the same dual subcellular location, i.e. in the cytosol and in the mitochondria.

| S-EPMC1135348 | biostudies-other

Purification and properties of a protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex.

Project description:The protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex was purified greater than 1000-fold from extracts of rat liver mitochondria; the specific activity was greater than 1000 units/mg of protein (1 unit gives half-maximum re-activation of 10 munits of phosphorylated complex). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave two bands (Mr 47700 and 35300) indistinguishable from the alpha- and beta-subunits of the branched-chain dehydrogenase component of the complex. On gel filtration (Sephacryl S-300), apparent Mr was 190000. This and other evidence suggests that activator protein is free branched-chain dehydrogenase; this conclusion is provisional until identical amino acid composition of the subunits has been demonstrated. Activator protein (i.e. free branched-chain dehydrogenase) was inhibited (up to 30%) by NaF, whereas branched-chain complex was not inhibited. There was no convincing evidence for interconvertible active and inactive forms of activator protein in rat liver mitochondria. Activator protein was detected in mitochondria from liver (ox, rabbit and rat) and kidney (ox and rat), but not in rat heart or skeletal-muscle mitochondria. In rat liver mitochondrial extracts, branched-chain complex sedimented with the mitochondrial membranes, whereas activator protein remained in the supernatant. Activator protein re-activated phosphorylated (inactive) particulate complex from rat liver mitochondria, but it did not activate dephosphorylated complex. Liver and kidney, but not muscle, mitochondria apparently contain surplus free branched-chain dehydrogenase, which is bound by the complex with lower affinity than is the branched-chain dehydrogenase intrinsic to the complex. It is suggested that this functions as a buffering mechanism to maintain branched-chain complex activity in liver and kidney mitochondria.

| S-EPMC1144617 | biostudies-other

Reversible ATP-induced inactivation of branched-chain 2-oxo acid dehydrogenase.

Project description:The branched chain 2-oxo acid dehydrogenase from rat skeletal muscle, heart, kidney and liver mitochondria can undergo a reversible activation-inactivation cycle in vitro. Similar results were obtained with the enzyme from kidney mitochondria of pig and cow. The dehydrogenase is markedly inhibited by ATP and the inhibition is not reversed by removing the nucleotide. The non-metabolizable ATP analogue adenosine 5'-[beta gamma-imido] triphosphate can block the effect of ATP when added with the nucleotide, but has no effect by itself, nor can it reverse the inhibition in mitochondria preincubated with ATP. These findings suggest that the branched chain 2-oxo acid dehydrogenase undergoes a stable modification that requires the splitting of the ATP gamma-phosphate group. In skeletal muscle mitochondria the rate of inhibition by ATP is decreased by oxo acid substrates and enhanced by NADH. The dehydrogenase can be reactivated 10-20 fold by incubation at pH 7.8 in a buffer containing Mg2+ and cofactors. Reactivation is blocked by NaF (25 mM). The initial activity of dehydrogenase extracted from various tissues of fed rats varies considerably. Activity is near maximal in kidney and liver whereas the dehydrogenase in heart and skeletal muscle is almost completely inactivated. These studies emphasize that comparisons of branched chain 2-oxo acid dehydrogenase activity under various physiological conditions or in different tissues must take into account its state of activation. Thus the possibility exists that the branched chain 2-oxo acid dehydrogenase may be physiologically regulated via a covalent mechanism.

| S-EPMC1162318 | biostudies-other

Developmental changes in rat liver branched-chain 2-oxo acid dehydrogenase.

Project description:Branched-chain 2-oxo acid dehydrogenase catalyses the first irreversible step in the degradation of the branched-chain amino acids leucine, isoleucine and valine. With specifically labelled 4-methyl-2-oxo[1-14C]pentanoate as substrate, the enzyme's activity was measured in rat liver homogenates. Activity (per g wet wL of liver or per mg of protein) increased most rapidly during the perinatal period (2 days before to 1 day after birth), reaching approximately adult values by the time of weaning. The apparent Vmax, of the enzyme increased with age, but its Km appeared unchanged. The data suggest that hepatic branched-chain 2-oxo acid dehydrogenase is induced or activated during the perinatal period. The enzyme's activity at birth was unaffected by maternal diabetes, or by treating the mother with pharmacological doses of corticosterone or 3,3',5-tri-iodothyronine, during the last 5 days of pregnancy.

| S-EPMC1158376 | biostudies-other

Studies in vitro on shuttle systems of mouse spermatozoa.

Project description:Observations on systems reconstituted in vitro with different starting substrates (2-hydroxy-acids, 2-oxo-acids or leucine) indicate that a branched-chain 2-hydroxy-acid/2-oxo-acid shuttle for the transfer of reducing equivalents from cytosol to mitochondria may be operational in mouse sperm. Evidence is presented suggesting that the 2-oxo-acids produced by intramitochondrial oxidation of 2-hydroxy-acids ingressed from the cytosol can recycle back into the external phase. Observations in vitro demonstrate that, in addition to the branched-chain 2-hydroxy-acid/2-oxo-acid shuttle, the malate/aspartate system is also active in mouse sperm. On the contrary, the lactate/pyruvate redox couple does not appear to function as part of a shuttle system in mouse sperm mitochondria. The glycerol 3-phosphate shuttle probably is not functionally significant in mouse spermatozoa, since the activity of the 'soluble' glycerol 3-phosphate dehydrogenase is very low.

| S-EPMC1153978 | biostudies-other

Activities of branched-chain amino acid aminotransferase and branched-chain 2-oxo acid dehydrogenase complex in tissues of maternal and fetal sheep.

Project description:Branched-chain amino acid aminotransferase (BCAAT) and branched-chain 2-oxo acid dehydrogenase (BCODH) activities were determined in sheep maternal and fetal liver, kidney, skeletal muscle, adipose and placenta in the fed and 5-day-starved states at 125 days gestation (term is 147 days). BCAAT activities were quite low in maternal skeletal muscle, low in fetal and maternal liver, high in fetal muscle, and very high in placenta. No significant changes occurred with maternal starvation. The high BCAAT activity of the placenta may provide the essential branched-chain 2-oxo acids for the fetus. BCODH activity was highest in liver and kidney of both the mother and fetus; it was largely in the active (dephosphorylated) state in maternal liver and kidney, and in fetal kidney, and about 50% active in fetal liver. The enzyme was largely inactive in fetal muscle, and about 50% active in maternal muscle. Although starvation had little effect on the activity of BCODH in fetal tissues, a significant decrease in activity was observed in maternal tissues, thereby potentially sparing branched-chain amino acids or the corresponding 2-oxo acids for maintenance of the fetus during compromised maternal nutrition.

| S-EPMC1147700 | biostudies-other

Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO.

Project description:Branched-chain oxo acid dehydrogenase was purified from Pseudomonas aeruginosa strain PAO with the objective of resolving the complex into its subunits. The purified complex consisted of four proteins, of Mr 36,000, 42,000, 49,000 and 50,000. The complex was resolved by heat treatment into the 49,000 and 50,000-Mr proteins, which were separated by chromatography on DEAE-Sepharose. The 49,000-Mr protein was identified as the E2 subunit by its ability to catalyse transacylation with a variety of substrates, with dihydrolipoamide as the acceptor. P. aeruginosa, like P. putida, produces two lipoamide dehydrogenases. One, the 50,000-Mr protein, was identified as the specific E3 subunit of branched-chain oxo acid dehydrogenase and had many properties in common with the lipoamide dehydrogenase LPD-val of P. putida. The second lipoamide dehydrogenase had Mr 54,000 and corresponded to the lipoamide dehydrogenase LPD-glc of P. putida. Fragments of C-terminal CNBr peptides of LPD-val from P. putida and P. aeruginosa corresponded closely, with only two amino acid differences over 31 amino acids. A corresponding fragment at the C-terminal end of lipoamide dehydrogenase from Escherichia coli also showed extensive homology. All three peptides had a common segment of eight amino acids, with the sequence TIHAHPTL. This homology was not evident in any other flavoproteins in the Dayhoff data base which suggests that this sequence might be characteristic of lipoamide dehydrogenase.

| S-EPMC1153093 | biostudies-other

Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex.

Project description:Branched-chain 2-oxo acid dehydrogenase complex was resolved into component E1 and E2-kinase subcomplex by gel filtration in the presence of 1 M-NaC1. Essentially all the original activity of the complex can be regained after reconstitution of the component enzymes, reassociation being a rapid process. The specific activities of E1 and E2 were 25.1 and 19.0 units/mg respectively. Non-phosphorylated active E1 has an approx. 6-fold higher affinity for E2 than does phosphorylated E1. The components of the branched-chain 2-oxo acid dehydrogenase complex do not crossreact with the respective components from the pyruvate dehydrogenase complex. The significance of these results and of the tight association of the kinase with E2 are discussed.

| S-EPMC1144650 | biostudies-other

Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated adipocytes. Effects of 2-oxo acids.

Project description:Isolated adipocytes from rat epididymal fat-pads were incubated with [32P]Pi, and intracellular phosphoproteins were then analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. A phosphorylated polypeptide of apparent Mr 46,000 was identified as the alpha-subunit of branched-chain 2-oxo acid dehydrogenase complex by immunoprecipitation using antiserum raised against the homogeneous E1 component of branched-chain 2-oxo acid dehydrogenase complex. Immunoprecipitation of this phosphoprotein is blocked in a competitive manner by purified branched-chain 2-oxo acid dehydrogenase complex. Peptide mapping of the isolated phosphoprotein indicates that two sites on the polypeptide are phosphorylated in the intact cells. Addition of branched-chain 2-oxo acids to the incubation medium causes diminution in the extent of labelling of both phosphorylation sites on the alpha-subunit, an effect presumably mediated via their known inhibitory action on branched-chain 2-oxo acid dehydrogenase kinase. These observations provide direct evidence for phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in intact cells.

| S-EPMC1146807 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data