Dihydrolipoamide dehydrogenase (DLD), also known as LADH, is an NAD-dependent oxidoreductase in the mitochondrial matrix. 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. 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. DLD mutations located at the interface between dimer subunits can impair dimer formation. 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. 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. 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.
Post-translational: Tyrosine phosphorylated.
Store at -20°C for 1 year. Avoid freeze/thaw cycle.
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Recombinant Human Dihydrolipoamide Dehydrogenase/DLD Protein (rp188307) - SDS-PAGE 3 μg/lane of Recombinant Human Dihydrolipoamide Dehydrogenase/DLD Protein was resolved with SDS-PAGE under reducing (R) and non-reducing (N) conditions and visualized by Coomassie® Blue staining, showing a band at 52.0 kDa.
Recombinant Human Dihydrolipoamide Dehydrogenase/DLD Protein - Enzyme 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. Materials: Assay Buffer: 50 mM Sodium Phosphate, 1 mM EDTA, 1 mg/mL BSA, pH 5.5 Recombinant Human Dihydrolipoamide Dehydrogenase/DLD Protein (rp188307) NADH (N196977), 20 mM stock in 0.1 M Sodium Borate, pH 9.0 NAD (N111610), 100 mM stock in deionized water DL-alpha-Lipoic acid(T106640), 20 mM stock in 95% Ethanol Procedures： 1. Create a Substrate Mixture containing 1.6 mM NADH, 0.8 mM NAD and 8 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:
*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: 0.8 mM, NAD: 0.4 mM, Lipoic Acid: 4 mM
ZJ24F1216118
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