Glycerol-3-Phosphate Dehydrogenase (G3PDH)
|(L-glycerol-3-phosphate:NAD+ oxidoreductase; EC 22.214.171.124)
Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the following reaction:
Dihydroxyacetone phosphate + NADH + H+ ------> L-glycerol-3-phosphate + NAD+
GPDH activity has been demonstrated in a wide variety of species belonging to animal as well as plant kingdoms (Lin, E.C.C., Ann. Rev. Biochem, 46, 765, 1977). A marked reduction in GPDH activity was observed in chronic plaques of multiple sclerosis (MS) (Hirsch, H.E., Blanco, C.E. and M.E. Parks, J. Neurochem., 34(3), 760, 1980). This may be of diagnostic and clinical significance. GPDH can also be used for glycerol determination in triglyceride kits (Sugiura, M., Oikawa, R., Hirano, K., Maeda, H., Yoshimura, H., Sugiyama, M. and T. Kuratsu, Clin Chem Acta, 81, 125, 1977).
The amount of enzyme which will convert one micromole of dihydroxyacetone phosphate to alpha-glycerophosphate per minute at pH 7.9 at 25°C.
The rate of decrease in the absorbancy at 340 nm, resulting from the oxidation of NADH, is a measure of GPDH activity.
- 0.05 M Triethanolamine hydrochloride buffer, pH 7.9.
- 0.006 M NADH disodium salt (3.9 mg/ml) in buffer. ,LI> 0.024 M Dihydroxyacetone phosphate, dimethylketal dicyclohexylamine salt (10 mg/ml). Dissolve 250 mg dihydroxyacetone phosphate, dimethylketal dicyclohexylamine salt in about 8 ml distilled water. Add Dowex-50 W, hydrogen form, until pH reaches approximately 2. Filter, wash and incubate filtrate at 40°C for 4-6 hours. Cool and adjust pH to 4.5 with 0.4 M sodium carbonate. Bring the volume up to 25 ml with distilled water. Store frozen in 1 ml aliquots (substrate).
- Glycerol-3-phosphate dehydrogenase (GPDH) solution (10 mg/ml). Dilute further, using 1% cold bovine serum albumin to give a final concentration of 0.5-1.0 U/ml. Must be prepared fresh immediately prior to assay.
- Set spectrophotometer (equipped with strip chart recorder and temperature
control) at 340 nm and 25°C.
- Into a cuvette pipette the following reagents:
Triethanolamine HCl buffer 2.7 ml
NADH 0.1 ml
Dihydroxyacetone phosphate 0.1 ml
- Mix and incubate at 25°C for 5 minutes in the spectrophotometer, to attain
- Record blank rate, if any, at 340 nm for 2-3 minutes.
- Initiate the reaction by adding 0.1 ml of fresh GPDH (enzyme) solution to
the cuvette. Monitor the reaction for 5-10 minutes at 340 nm.
- Calculate E340 nm/min