PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “LIPOAMIDE DEHYDROGENASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 415 records · Page 23Linked to original sources
PubMed1978

Lactate-dehydrogenase activity in the rat testis: a comparison between fluorometric assay of freeze-dried sections and histochemical localization with phenazine methosulphate.

The activity of lactate dehydrogenase (LDH) in freeze-dried sections of rat testes was determined by using a fluorometric assay method and found to be 4.47 +/- 0.23 moles/Kg dry weight/hr (MKDH +/- S.E.M.) in whole sections, 3.31 +/- 0.16 in tubules and 12.0 +/- 1.9 in interstitial tissue. The activities and regional variation are similar to those measured in nervous tissue and are well correlated with the histochemical localization of LDH activity when phenazine methosulphate (PMS) is not used as an electron carrier. LDH and lipoamide dehydrogenase activity have the same histochemical distribution and there is no nonspecific staining with either method. The use of PMS results in reduced dependence on substrate and coenzyme and does not indicate higher interstitial activity but may provide an indication of developing lactate metabolism in maturing sperm. It is recomended that methods with and without PMS be used in studies of LDH activity in the testis.

Animals↗
PubMed1995

Structural analysis of the L-methionine gamma-lyase gene from Pseudomonas putida.

The gene encoding L-methionine gamma-lyase from Pseudomonas putida was cloned and the primary structure of the enzyme was deduced from its nucleotide sequence. The L-methionine gamma-lyase gene was expressed in Escherichia coli. The amino acid sequences of BrCN-digested peptides agreed with the corresponding parts of the L-methionine gamma-lyase sequence determined from the gene structure. The polypeptide is composed of 398 amino acid residues with a calculated molecular weight of 42,626, corresponding to the subunit of the homotetrameric enzyme. The deduced amino acid sequence of L-methionine gamma-lyase only showed extensive homology with other well known alpha,gamma-elimination and/or gamma-replacement pyridoxal 5'-phosphate-dependent enzymes, such as cystathionine gamma-lyase, cystathionine gamma-synthase, and O-acetylhomoserine O-acetylserine sulfhydrylase, that participate in the biosynthesis of sulfur amino acids. However, the deduced essential cysteine residue of L-methionine gamma-lyase was not conserved in these enzymes. We confirmed the presence of a part of an open reading frame in the 3'-flanking region of the L-methionine gamma-lyase gene, which showed high homology with the N-terminal region of pyruvate dehydrogenase (lipoamide) from E. coli, suggesting that it participates in the degradative pathway for L-methionine together with L-methionine gamma-lyase.

Amino Acid Sequence↗
PubMed2000

Symbiotic induction of pyruvate dehydrogenase genes from Sinorhizobium meliloti.

Genes coding for components of the pyruvate dehydrogenase (PDH) multienzyme complex (PDHc) from Sinorhizobium meliloti, the alfalfa symbiont, have been isolated on the basis of their high expression in symbiotic bacteria. The Elp component, PDH, is encoded by two genes, pdhAalpha (1,047 bp) and pdhAbeta (1,383 bp), a situation encountered in the alpha-proteobacteria Rickettsia prowazekii and Zymomonas mobilis as well as in some gram-positive bacteria and in mitochondria. pdhAalpha and pdhAbeta precede pdhB (1,344 bp), which encodes the E2p component, dihydrolipoamide acetyltransferase, of the PDHc. No gene encoding the E3 component, lipoamide dehydrogenase, was found in the immediate vicinity of pdhA and pdhB genes. pdhAalpha, pdhAbeta and pdhB likely constitute an operon. Here, we provide evidence that pdhA expression is induced in the symbiotic stage, compared with free-living conditions. We demonstrate that symbiotic expression of pdhA genes does not depend on the fix LJ regulatory cascade that regulates nitrogen fixation and respiration gene expression in symbiotic S. meliloti cells. Induction of pdhA expression could be obtained under free-living conditions upon the addition of pyruvate to the culture medium. Induction by pyruvate and symbiotic activation of pdh gene expression take place at the same promoter.

Amino Acid Sequence↗
PubMed1991

Cloning, sequencing, and expression of the Pseudomonas testosteroni gene encoding 3-oxosteroid delta 1-dehydrogenase.

Pseudomonas testosteroni ATCC 17410 is able to grow on testosterone. This strain was mutagenized by Tn5, and 41 mutants defective in the utilization of testosterone were isolated. One of them, called mutant 06, expressed 3-oxosteroid delta 1- and 3-oxosteroid delta 4-5 alpha-dehydrogenases only at low levels. The DNA region around the Tn5 insertion in mutant 06 was cloned into pUC19, and the 1-kbp EcoRI-BamHI segment neighbor to the Tn5 insertion was used to probe DNA from the wild-type strain. The probe hybridized to a 7.8-kbp SalI fragment. Plasmid pTES5, which is a pUC19 derivative containing this 7.8-kbp SalI fragment, was isolated after the screening by the 1-kbp EcoRI-BamHI probe. This plasmid expressed delta 1-dehydrogenase in Escherichia coli cells. The 2.2-kbp KpnI-KpnI segment of pTES5 was subcloned into pUC18, and pTEK21 was constructed. In E. coli containing the lacIq plasmid pRG1 and pTEK21, the expression of delta 1-dehydrogenase was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The induced level was about 40 times higher than the induced level in P. testosteroni. Delta 1-Dehydrogenase synthesized in E. coli was localized in the inner membrane fraction. The minicell experiments showed that a 59-kDa polypeptide was synthesized from pTEK21, and this polypeptide was located in the inner membrane fraction. The complete nucleotide sequence of the 2.2-kbp KpnI-KpnI segment of pTEK21 was determined. An open reading frame which encodes a 62.4-kDa polypeptide and which is preceded by a Shine-Dalgarno-like sequence was identified. The first 44 amino acids of the putative product exhibited significant sequence similarity to the N-terminal sequences of lipoamide dehydrogenases.

Amino Acid Sequence↗
PubMed1995

Modelling cortical cataractogenesis 17: in vitro effect of a-lipoic acid on glucose-induced lens membrane damage, a model of diabetic cataractogenesis.

The effect of R, S, and racemic forms of a-lipoic acid was tested on the formation of opacity in normal rat lenses incubated with 55.6 mM glucose, as a model for in vivo diabetic cataractogenesis. Control lenses, incubated 8 days with 5.56 mM glucose, did not develop opacities. Formation of lens opacities in vitro was correlated with lactate dehydrogenase (LDH) leakage into the incubation medium. Opacity formation and LDH leakage, resulting from incubation in medium containing 55.6 mM glucose to model diabetes, were both suppressed by the addition of 1 mM R-lipoic acid. Addition of 1 mM racemic lipoic acid reduces these damaging effects to the lens by one-half, while S-lipoic acid potentiated LDH leakage, consistent with the hypothesis that R-lipoic acid is the active form. Although HPLC analysis demonstrated that both stereoisomers of lipoic acid were reduced to dihydrolipoate at comparable rates by the intact lens, the mitochondrial lipoamide dehydrogenase system is highly specific for reduction of exogenous R-lipoic to dihydrolipoic acid. Therefore, stereospecific protection against this opacity is consistent with specific reduction of R-lipoic acid in mitochondria of the vulnerable cells at the lens equator where the first globular degeneration is seen in glucose cataract.

Animals↗
PubMed1979

Inactivation and disassembly of the pyruvate dehydrogenase multienzyme complex from bovine kidney by limited proteolysis with an enzyme from rat liver.

Mammalian pyruvate dehydrogenase multienzyme complex is inactivated when treated with a leupeptin-sensitive enzyme (termed 'inactivase') obtained from rat liver lysosomes. However, the inactivation of the overall reaction does not affect any of the component activities of the enzyme complex. By several methods it is demonstrated that treatment with the inactivase provokes the disassembly of the complex into its constituent enzyme components which, though being enzymatically active when assayed separately, are unable to catalyze the coordinated reaction sequence of pyruvate oxidation. The dissociation occurs as a consequence of limited proteolysis of the lipoate acetyltransferase core of the multienzyme complex. Isolated nicked acetyltransferase retains its complete enzymatic activity and behaves as a high-molecular-weight aggregate. The lipoamide dehydrogenase and pyruvate dehydrogenase components, however, are not cleaved by the inactivase.

Animals↗
PubMed2001

Involvement of matrix NADP turnover in the oxidation of NAD-linked substrates by pea leaf mitochondria.

The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane [NDin(NADPH)] in the oxidation of strictly NAD+-linked substrates by pea (Pisum sativum L.) leaf mitochondria was measured. As estimated by the inhibition caused by 5 µM diphenyleneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of NDin(NADPH) during glycine oxidation (measured both as O2 uptake and as CO2 release) was 40-50 nmol mg-1 protein min-1. No significant activity of NDin(NADPH) could be detected during the oxidation of 2-oxoglutarate, another strictly NAD+-linked substrate; this was possibly due to its relatively low oxidation rate. Control experiments showed that, even at 125 µM, DPI had no effect on the activity of glycine decarboxylase complex (GDC) and lipoamide dehydrogenase. The relative activity of complex I, NDin(NADPH), and NDin(NADH) during glycine oxidation, estimated using rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria with NAD+ and NADP+, both of which were taken up by the organelle. We conclude that the involvement of NADP turnover during glycine oxidation is not due to the direct production of NADPH by GDC but is an indirect result of this process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant mitochondria.

Journal Article↗
PubMed2006

Lipoic acid as a novel treatment for Alzheimer's disease and related dementias.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that destroys patient memory and cognition, communication ability with the social environment and the ability to carry out daily activities. Despite extensive research into the pathogenesis of AD, a neuroprotective treatment - particularly for the early stages of disease - remains unavailable for clinical use. In this review, we advance the suggestion that lipoic acid (LA) may fulfil this therapeutic need. A naturally occurring precursor of an essential cofactor for mitochondrial enzymes, including pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH), LA has been shown to have a variety of properties which can interfere with pathogenic principles of AD. For example, LA increases acetylcholine (ACh) production by activation of choline acetyltransferase and increases glucose uptake, thus supplying more acetyl-CoA for the production of ACh. LA chelates redox-active transition metals, thus inhibiting the formation of hydroxyl radicals and also scavenges reactive oxygen species (ROS), thereby increasing the levels of reduced glutathione. Via the same mechanisms, downregulation redox-sensitive inflammatory processes is also achieved. Furthermore, LA can scavenge lipid peroxidation products such as hydroxynonenal and acrolein. The reduced form of LA, dihydrolipoic acid (DHLA), is the active compound responsible for most of these beneficial effects. R-alpha-LA can be applied instead of DHLA, as it is reduced by mitochondrial lipoamide dehydrogenase, a part of the PDH complex. In this review, the properties of LA are explored with particular emphasis on how this agent, particularly the R-alpha-enantiomer, may be effective to treat AD and related dementias.

Aged↗
PubMed2001

Developmental expression of the mitochondrial pyruvate dehydrogenase complex in pea (Pisum sativum) seedlings.

In order to better understand control of the mitochondrial pyruvate dehydrogenase complex (PDC), total catalytic activity was determined during development of the primary leaves of pea (Pisum sativum L.) seedlings, as well as in each leaf pair of 21-day-old plants. Activity of the PDC in clarified homogenates was highest in the youngest organs and then dropped dramatically as the leaves matured and became photosynthetically competent. As leaves began to senesce, total PDC activity dropped to zero. Steady-state mRNA levels were determined using E1 and E3 cDNA probes. The overall pattern of transcript abundance matched the pattern observed for total PDC activity; transcript levels for E1alpha and E1beta approached zero during senescence. Levels of the E1alpha, E1beta, E2 and E3 subunits of the PDC were analyzed in the same samples, using specific antibodies. Quantitation of the immunoblotting results throughout this developmental series showed a pattern in parallel with that of catalytic activity and mRNA levels, although the relative changes in subunit protein levels were not as extreme as the changes in activity. The exception to the global pattern was that of the E3 subunit: lipoamide dehydrogenase. Expression of this enzyme was highest in mature, fully expanded leaves, which were active in photosynthesis and photorespiration, reflecting the additional role of E3 as a component of glycine decarboxylase.

Journal Article↗
PubMed1983

Temperature-dependence of intramolecular coupling of active sites in pyruvate dehydrogenase multienzyme complexes.

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complexes of Escherichia coli, ox heart and Bacillus stearothermophilus was measured at various temperatures. As the temperature was raised, the extent of active-site coupling was found to increase, approaching a maximum near the physiological growth temperature of the organism. Under these conditions, a single pyruvate dehydrogenase (lipoamide) dimer appeared able to cause a rapid (20s) reductive acetylation of probably all 24 polypeptide chains in the dihydrolipoamide acetyltransferase core of the enzyme complex from E. coli at 37 degrees C, and of most if not all of the 60 polypeptide chains in the dihydrolipoamide acetyltransferase cores of the enzymes from ox heart and B. stearothermophilus at 37 degrees C and 60 degrees C respectively. Experiments designed to measure the inter-core and intra-core migration of enzyme subunits suggested that, in the bacterial enzymes at least, this was not a major contributor to active-site coupling.

Acetylation↗
PubMed1999

[Vitamin B1].

Vitamin B1 (thiamin), taken-up into cells, is converted to thiamin diphosphate (TDP), and TDP acts as a cofactor for several enzymes involving in carbohydrate metabolism. CoA-dependent oxidative decarboxylation of pyruvate is catalyzed by pyruvate dehydrogenase multienzyme complex (PDC) with NAD+ as an electron acceptor in most organisms involving mammals and higher plants. PDC consists of three component enzymes, one of which is pyruvate dehydrogenase (lipoamide) which contains TDP as a prosthetic group. Similar multienzyme complex for 2-oxoglutarate or branched chain 2-oxoacids is also found in mammals. In anaerobic bacteria, archaebacteria and anaerobic protozoa, pyruvate:ferredoxin oxidoreductase (PFOR) functions for the oxidative decarboxylation of pyruvate with ferredoxin in place of NAD+. PFOR contains TDP as a cofactor; however its structure is quite different from PDC and 1-3[4Fe-4S] clusters are involved as redox centers. Pyruvate:NADP+ oxidoreductase (PNOR), which catalyzes the oxidative decarboxylation of pyruvate with NADP+ as an electron acceptor, occurs in mitochondria of Euglena gracilis, a protist containing chloroplasts. PNOR consists of two functional domains, one of which contains TDP and 3[4Fe-4S] clusters and resembles PFOR. Another domain involves FMN and FAD as redox centers and its structure is similar to NADPH-cytochrom P450 reductase.

Carboxy-Lyases↗
PubMed1988

Biosynthesis, import and processing of precursor polypeptides of mammalian mitochondrial pyruvate dehydrogenase complex.

An immunological analysis has been conducted of early events in the biosynthesis, import and assembly of the mammalian pyruvate dehydrogenase complex (PDC). For this purpose, monospecific polyclonal antisera were produced against the intact assembly from ox heart, Mr 8.5 x 10(6), and each of its component polypeptides, E1 alpha, E1 beta, E2, E3 and protein X. Optimal detergent-based incubation mixtures were developed for obtaining clean immunoprecipitation of PDC polypeptides and their precursors from [35S]methionine-labelled extracts of PK-15 (pig kidney), NBL-1 (bovine kidney) and BRL (Buffalo Rat liver) cells. In PK-15 cells, independent higher Mr species, corresponding to precursors of the E2, E1 alpha and E1 beta subunits of PDC, could be detected by immune precipitation and fluorography after incubation of intact cells for 4 h with [35S]methionine and 1-2 mM-2,4-dinitrophenol or 10-15 microM-carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similar precursor states could be observed in uncoupler-treated BRL or NBL-1 cells. Pre-E1 alpha, pre-E1 beta and also pre-E3, have signal sequences in the Mr range 1500-3000 while pre-E2 contains a long additional segment of Mr 7000-9000. All of these forms exhibit similar kinetics of processing to the mature subunits with a transit time of 10-12 min. In NBL-1 cells, E3 is present in the immune complexes formed with anti-PDC serum whereas this is not the case in PK-15 cells. Thus, there are significant variations in the affinity of lipoamide dehydrogenase (E3) for the E2 core structure in different species. Pre-E1 alpha accumulates only poorly in PK-15 cells and is aberrantly processed on removal of uncoupler. This precursor is markedly more stable in NBL-1 and BRL cells. The lack of detection of a precursor form of component X is also discussed.

2,4-Dinitrophenol↗
PubMed2000

The role of taurine in the pathogenesis of the cardiomyopathy of insulin-dependent diabetes mellitus.

The cellular and molecular physiology and pathology of insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) are mostly studied and understood through the use of animal models. Fundamental differences between the IDDM and NIDDM animal models may help to explain the etiology behind diabetic cardiomyopathy, one of the most severe complications of IDDM. Experimental rat models of IDDM exhibit a characteristic increase in tissue levels of taurine in the heart, a change that is not seen in NIDDM rats. This article deals with the causes and possible consequences of this observation which may contribute to the development of diabetic cardiomyopathy. Modulation of pyruvate dehydrogenase (lipoamide) (PDH; EC 1.2.4.1) activity was found to be a possible mode for taurine involvement. PDH is a mitochondrial protein and is the rate-limiting step in the generation of acetyl CoA from glycolysis. In IDDM, PDH activity is decreased through a mechanism that includes the stimulation of the de novo synthesis of a kinase activator protein (KAP) which phosphorylates PDH and inactivates the enzyme. This lesion does not occur in NIDDM rat hearts. Taurine is known to inhibit the phosphorylation of PDH in vitro, and in taurine-depleted rats PDH phosphorylation is known to increase. Thus, the increased levels of taurine in the diabetic heart may be inhibiting this phosphorylation which in turn may be stimulating the synthesis of KAP through a negative feedback process. The main argument for this theory would be the lack of change in both the taurine levels and the activity of PDH in the NIDDM rat model.

Animals↗
PubMed1999

Chondrocyte death is linked to development of a mitochondrial membrane permeability transition in the growth plate.

In the companion article, we reported that the local phosphate (Pi) concentration triggers apoptosis in epiphyseal chondrocytes. The goal of the current investigation was to evaluate the apoptotic process in relationship to the energy status of cells in the growth plate. For these studies, we used sections of the adolescent growth plate, as well as cells isolated from the tissue. We found that there was a maturation-dependent loss of mitochondrial function in growth plate chondrocytes and these cells generated energy by glycolysis. Since treatment with the uncoupler 2,4-dinitrophenol as well as the site-specific inhibitors antimycin A and rotenone failed to elicit a further increase in the activity of the glycolytic pathway, we concluded that oxidative metabolism was minimum in these cells. Flow cytometric studies of growth plate cells and confocal microscopy of growth plate sections using the mitochondrial probes Rh123 and DiOC6(3) provided unequivocal evidence that there was loss of mitochondrial membrane potential in hypertrophic cells. Furthermore, the intrinsic fluorescence of the flavoprotein lipoamide dehydrogenase complex of the electron transport chain revealed that the mitochondria were in an oxidized state. Finally, we assessed Bcl-2 expression in these cells. Although immunohistochemical and Western blot analysis showed that the chick cells contained a low level of the anti-apoptotic protein Bcl-2, reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that transcripts were present in chondrocytes. Based on these observations, we suggest that terminally differentiated chondrocytes undergo a maturation-dependent loss of mitochondrial function. In concert with the low expression of Bcl-2, they become sensitive to signals for programmed cell death. We hypothesize that Pi triggers apoptosis in these energy-compromised cells by promoting a mitochondrial membrane transition, thereby inducing the death process.

Adenosine Triphosphate↗