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Enzyme
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Query: EC:1.8.1.4 (
diaphorase
)
2,754
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
A 2288-bp cDNA sequence encoding
dihydrolipoamide dehydrogenase
(
DLDH
; dihydrolipoamide: NAD+ oxido-reductase;
EC 1.8.1.4
) was obtained by isolating a 1762-bp cDNA clone from a canine skeletal muscle library in the vector, lambda UNIZAP, combined with PCR amplification of the 5' end of the mRNA. The
DLDH
cDNA sequence contains a 49-bp G+C-rich 5'-untranslated region (UTR), followed by 1527 bp of coding region, and 695 bp of 3'-UTR preceding a 17-bp poly(A) tail. The single open reading frame encodes a precursor
DLDH
of 509 amino acids (aa) that begins with a 35-aa leader sequence. The 3'-UTR includes six possible polyadenylation signals (three AATAAA, one TATAAA and two AATGAA) and one potential stem-loop region extending from bp 1969-1991. Alignment studies of the canine and human
DLDH
demonstrate homology within the coding region of 98% at the aa level and 94% at the nt level. Northern blot analysis using the cDNA clone as probe showed wide tissue distribution of the mRNA, with differences in the level of expression among tissues and possible utilization of different polyadenylation sites.
...
PMID:The cDNA encoding canine dihydrolipoamide dehydrogenase contains multiple termination signals. 766 89
Ferric leghemoglobin reductase (FLbR), an enzyme reducing ferric leghemoglobin (Lb) to ferrous Lb, was purified from cowpea (Vigna unguiculata) root nodules by sequential chromatography on hydroxylapatite followed by Mono-Q HR5/5 FPLC and Sephacryl S-200 gel filtration. The purified cowpea FLbR had a specific activity of 216 nmol Lb(2+)O(2) formed min(-1) mg(-1) of enzyme for cowpea Lb(3+) and a specific activity of 184 nmol Lb(2+)O(2) formed min(-1) mg(-1) of enzyme for soybean Lb(3+). A cDNA clone of cowpea FLbR was obtained by screening a cowpea root nodule cDNA library. The nucleotide sequence of cowpea FLbR cDNA exhibited about 88% similarity with soybean (Glycine max) FLbR and 85% with pea (Pisum sativum)
dihydrolipoamide dehydrogenase
(
DLDH
,
EC 1.8.1.4
) cDNAs. Conserved regions for the FAD-binding site, NAD(P)H-binding site, and disulfide active site were identified among the deduced amino acid sequences of cowpea FLbR, soybean FLbR, pea
DLDH
and other enzymes in the family of the pyridine nucleotide-disulfide oxido-reductases.
...
PMID:Analysis of a ferric leghemoglobin reductase from cowpea (Vigna unguiculata) root nodules. 1072 15
Dihydrolipoamide dehydrogenase (
DLDH
;
EC 1.8.1.4
) from porcine heart is capable of using nitric oxide (NO) as an electron acceptor, with NADH as the electron donor, forming nitrate in the reaction. NADPH was not effective as an electron donor. The reaction had a pH optimum near 6 and was not inhibited by cyanide or diphenyleneiodonium ions. The Km for NADH was 10 microM, while that for NO was 0.5 microM. The rate of NO conversion was comparable to the rate of lipoamide conversion (200 micromol min(-1) mg(-1) protein at pH 6). Cytochrome c or myoglobin were poor electron acceptors by themselves but, in the presence of methylene blue,
DLDH
had an activity of 5-7 micromol min(-1) mg(-1) protein with these substrates, indicating that
DLDH
can act also as a methemoglobin reductase. While the Km of
DLDH
for NO is relatively low, it is in the physiological range of NO levels encountered in the tissue. The enzyme may, therefore, have a significant role in modifying NO levels under specific cell conditions.
...
PMID:Dihydrolipoamide dehydrogenase from porcine heart catalyzes NADH-dependent scavenging of nitric oxide. 1519 36
Mammalian mitochondrial
dihydrolipoamide dehydrogenase
(
DLDH
,
EC 1.8.1.4
) catalyzes NAD(+)-dependent oxidation of dihydrolipoamide in vivo and can also act as a
diaphorase
catalyzing in vitro nicotinamide adenine dinucleotide (reduced form) (NADH)-dependent reduction of electron-accepting molecules such as ubiquinone and nitroblue tetrazolium (NBT). In this paper, we report a gel-based method for histochemical staining and quantification of
DLDH
diaphorase
activity using blue native PAGE (BN-PAGE). Rat brain mitochondrial extracts, used as the source of
DLDH
, were resolved by nongradient BN-PAGE (9%), which was followed by
diaphorase
activity staining using NADH as the electron donor and NBT as the electron acceptor. It was shown that activity staining of
DLDH
diaphorase
was both protein amount- and time-dependent. Moreover, this in-gel activity-staining method was demonstrated to be in good agreement with the conventional spectrophotometric method that measures
DLDH
dehydrogenase activity using dihydrolipoamide as the substrate. The method was applied to determine levels of
DLDH
diaphorase
activity in several rat tissues other than the brain, and the results indicated a similar level of
DLDH
diaphorase
activity for all the tissues examined. Finally, the effects of thiol-reactive reagents such as N-ethylmaleimide (NEM) and nitric oxide donors on
DLDH
diaphorase
activity were evaluated, demonstrating that, with this method,
DLDH
diaphorase
activity can be determined without having to remove these thiol-reactive reagents that may otherwise interfere with spectrophotometric measurement of
DLDH
dehydrogenase activity. The gel-based method can also be used as a means to isolate mitochondrial
DLDH
that is to be analyzed by mass spectral techniques in studying
DLDH
post-translational modifications.
...
PMID:Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE. 1731 58
In this report we have identified for the first time a transacetylase (TAase) in a mesophilic fungi Starkeyomyces koorchalomoides catalyzing the transfer of acetyl group from polyphenolic acetate (PA) to a receptor protein glutathione S-transferase (GST). An elegant assay procedure was established for TAase based on its ability to mediate inhibition of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model PA. Utilizing this assay procedure, S. koorchalomoides TAase was purified to homogeneity. TAase was found to have MW of 50 kDa. The purified enzyme exhibited maximum activity at 45 degrees C at pH 6.8. The N-terminal sequence of purified fungal TAase (ANDASTVED) showed identity with corresponding N-terminal sequence of
dihydrolipoamide dehydrogenase
(LADH), a mitochondrial matrix enzyme and an
E3 component of pyruvate dehydrogenase
complex (PDHC). TAase was found to have all the properties of LADH and avidly interacted with the anti-LADH antibody. TAase catalyzed acetylation of GST by DAMC was identified by LC-MS/MS and a single lysine residue (Lys-113) was found to be acetylated. Further, recombinant LADH from Streptococcus pneumoniae lacking lipoyl domain was found to exhibit little TAase activity, suggesting the role of lipoyl domain in the TAase activity of LADH. These observations bear evidence for the protein acetyltransferase activity of LADH. Such an activity of LADH can be attributed as a moonlighting function of the enzyme.
...
PMID:Moonlighting protein in Starkeyomyces koorchalomoides: characterization of dihydrolipoamide dehydrogenase as a protein acetyltransferase utilizing acetoxycoumarin as the acetyl group donor. 1938 27
Ischemia postconditioning (IPo) is a promising strategy in reducing myocardial ischemia reperfusion (I/R) injury (MIRI), but its specific molecular mechanism is incompletely understood. Langendorff-perfused isolated rat hearts were subjected to global I/R and received IPo in the absence or presence of the mitochondrial ATP-sensitive potassium channel (mitoKATP) blocker 5-hydroxydecanoate (5-HD). Myocardial mitochondria were extracted and mitochondrial comparative proteomics was analyzed. IPo significantly reduces post-ischemic myocardial infarction and improved cardiac function in I/R rat hearts, while 5-HD basically cancelled IPo's myocardial protective effect. Joint application of two-dimensional polyacrylamide gel electrophoresis (2DE) and MALDI-TOF MS identified eight differentially expressed proteins between groups. Expression of cardiac succinate dehydrogenase (ubiquinone) flavoprotein subunit (SDHA) increased more than two-fold after I/R, while IPo led to overexpression of
dihydrolipoyl dehydrogenase
(
DLD
), NADH dehydrogenase (ubiquinone) flavoprotein 1 and isoform CRA_b (NDUFV1). When the mitoKATP was blocked, MICOS complex subunit Mic60 (IMMT) and Stress-70 protein (Grp75) were over expressed, while
DLDH
, ATPase subunit A (ATPA) and rCG44606 were decreased. Seven of the differential proteins belong to electron transport chain (ETC) or metabolism regulating proteins, and five of them were induced by closing mitoKATP in I/R hearts. We thus conclude that IPo's myocardial protective effect relies on energy homeostasis regulation.
DLD
, SDHA, NDUFV1, Grp75, ATPA and rCG44606 may contribute to IPo's cardial protective effect.
...
PMID:Ischemic postconditioning influences electron transport chain protein turnover in Langendorff-perfused rat hearts. 2692 30
Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high-affinity titanium dioxide (TiO
2
)-binding protein (TiBP), purified from Rhodococcus ruber, has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal
dihydrolipoamide dehydrogenase
(rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO
2
-binding capabilities with TiBP. Intrigued by the unique TiO
2
-binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti-based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein-TiO
2
surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of
DLDH
with various metal oxides is independent of their isoelectric values strengthens this notion.
DLDH
does not lose its enzymatic activity upon binding to TiO
2
, indicating that neither the enzyme undergoes major conformational changes nor the TiO
2
binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO
2
through similar regions located far from the active site and the dimerization sites. The putative TiO
2
-binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal-binding sites in proteins.
...
PMID:The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide-Localization of a putative binding site. 2824 84
