Recombinant Human Dihydrolipoamide Dehydrogenase/DLD, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Dihydrolipoamide Dehydrogenase/DLD, CF Summary

• Details of Functionality: 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.
• Source: E. coli-derived human Dihydrolipoamide Dehydrogenase/DLD protein
  

  GGS	HHHHHH	GMASLENLYFQ	Human DLD (Ala36-Phe509) Accession # P09622
  N-terminus			C-terminus

  Ala36 - Phe509, with N-terminal GGS-6-His tag and GMASLENLYFQ insertion
• Accession #: P09622
• N-terminal Sequence: Gly
• Protein/Peptide Type: Recombinant Enzymes
• Gene: DLD
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 52 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 52-62 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Sodium Phosphate and Sucrose.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • 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 10^12 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Dihydrolipoamide Dehydrogenase/DLD, CF

• 2-oxo-glutarate complex, branched chain keto acid dehydrogenase complex)
• branched chain keto acid dehydrogenase complex
• Diaphorase
• dihydrolipoamide dehydrogenase (E3 component of pyruvate dehydrogenase complex
• Dihydrolipoamide Dehydrogenase
• dihydrolipoamide dehydrogenasemitochondrial
• DLD
• DLDD
• DLDH
• E3 component of pyruvate dehydrogenase complex, 2-oxo-glutarate complex
• E3
• EC 1.8.1
• EC 1.8.1.4
• GCSL
• Glycine cleavage system L protein
• glycine cleavage system protein L
• LAD
• Lipoamide Dehydrogenase
• lipoamide reductase
• lipoyl dehydrogenase
• PHE3

Background

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.

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.

Bioinformatics

• Gene Symbol: DLD
• Uniprot:
  • P09622()