Recombinant Human Dihydrolipoamide Dehydrogenase/DLD, CF

R&D Systems | Catalog # 8646-DH

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human Dihydrolipoamide Dehydrogenase/DLD (8646-DH)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P09622

Applications

Enzyme Activity
View all Dihydrolipoamide Dehydrogenase/DLD Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human Dihydrolipoamide Dehydrogenase/DLD protein
GGS HHHHHH GMASLENLYFQ Human DLD
(Ala36-Phe509)
Accession # P09622
N-terminus C-terminus

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Gly

Predicted Molecular Mass

52 kDa

SDS-PAGE

52-62 kDa, reducing conditions

Activity

Measured by its ability to produce NADH during the oxidation of lipoic acid.
The specific activity is >5,000 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

8646-DH
Formulation Supplied as a 0.2 μm filtered solution in Sodium Phosphate and Sucrose.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: Dihydrolipoamide Dehydrogenase/DLD

Dihydrolipoamide dehydrogenase (DLD), also known as LADH, is an NAD-dependent oxidoreductase in the mitochondrial matrix (1). DLD serves as the E3 subunit of four mitochondrial enzyme complexes: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, branched chain alpha-ketotacid dehydrogenase, and the glycine cleavage system (2, 3). It is active as a 120 kDa dimer that catalyzes oxidation within these enzyme complexes. Several mutations of human DLD have been described, some of which contribute to the loss of respiratory function during oxidative stress (4, 5). DLD mutations located at the interface between dimer subunits can impair dimer formation (5, 6). The involvement of DLD in the regulation of lipid peroxidation and lactate metabolism is important for mouse hippocampal neuroblast proliferation and hamster sperm capacitation, respectively (7, 8). DLD polymorphisms in insects can increase their resistance to the pesticide phosphine gas, while they can increase the sensitivity to arsenite in the nematode C. elegans (9). Mature human DLD shares 95-96% amino acid (aa) sequence identity with hamster, mouse, and rat DLD. Alternative splicing generates additional human DLD isoforms that lack the N-terminal 99 aa or carry a deletion of aa 147-194.

References

  1. Brand, M.D. (2010) Exp. Gerontol. 45:466.
  2. Johnson, M.T. et al. (1997) Proc. Natl. Acad. Sci. USA 94:14512.
  3. Sundquist, A.R. and R.C. Fahey (1988) J. Bacteriol. 170:3459.
  4. Ambrus, A. et al. (2011) Hum. Mol. Genet. 20:2984.
  5. Vaubel, R.A. et al. (2011) J. Biol. Chem. 286:40232.
  6. Babady, N.E. et al. (2007) Proc. Natl. Acad. Sci. USA 104:6158.
  7. Calingasan, N.Y. et al. (2008) Neuroscience 153:986.
  8. Panneerdoss, S. et al. (2012) J. Androl. 33:699.
  9. Schlipalius, D.I. et al. (2012) Science 338:807.

Alternate Names

Diaphorase, DLD, DLDD, DLDH, GCSL, LAD, Lipoamide Dehydrogenase, PHE3

Entrez Gene IDs

1738 (Human); 13382 (Mouse); 298942 (Rat)

Gene Symbol

DLD

UniProt

P09622

Additional Dihydrolipoamide Dehydrogenase/DLD Products

Product Documents for Recombinant Human Dihydrolipoamide Dehydrogenase/DLD, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human Dihydrolipoamide Dehydrogenase/DLD, CF

For research use only

Related Research Areas

Redox Enzymes

Citations for Recombinant Human Dihydrolipoamide Dehydrogenase/DLD, CF

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Protocols

View specific protocols for Recombinant Human Dihydrolipoamide Dehydrogenase/DLD, CF (8646-DH):

Materials
  • Assay Buffer: 50 mM Sodium Phosphate, 1 mM EDTA, 1 mg/mL BSA, pH 5.5
  • Recombinant Human Dihydrolipoamide Dehydrogenase/DLD (rhDLD) (Catalog # 8646-DH)
  • NADH (Sigma, Catalog # N8129), 20 mM stock in 0.1 M Sodium Borate, pH 9.0
  • NAD (Sigma, Catalog # N6522), 100 mM stock in deionized water
  • (±)-alpha -Lipoic acid (Sigma, Catalog # T1395), 20 mM stock in 95% Ethanol
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Create a Substrate Mixture containing 0.4 mM NADH, 0.2 mM NAD and 2 mM Lipoic Acid in Assay Buffer.
  2. Incubate Substrate Mixture at room temperature for 5 minutes in the dark.
  3. Dilute rhDLD to 2 µg/mL in Assay Buffer.
  4. Load 50 µL of 2 µg/mL rhDLD in a plate, and start the reaction by adding 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
  5. Read plate in kinetic mode for 5 minutes at an absorbance of 340 nm.
  6. Calculate specific activity:

Specific Activity (pmol/min/µg) =

Adjusted Vmax* (OD/min) x -1 x well volume (L) x 1012 pmol/mol
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

*Adjusted for Substrate Blank
** Using the extinction coefficient 6220 M-1cm-1
*** Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD

Per Well:

  • rhDLD: 0.1 µg
  • NADH: 0.2 mM
  • NAD: 0.1 mM
  • Lipoic Acid: 1 mM

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