GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

Glyceraldehyde-3-phosphate Dehydrogenase from rabbit muscle has been used:

• for measurements of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity assay
• to generate a linear standard curve to analyse GAPDH activity in the experimental samples collected from mice
• in anti-aggregation assays
• in purified GAPDH studies

GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) catalyzes the conversion of glyceraldehyde-3-phosphate into D-glycerate-1,3-bisphosphate as part of the glycolysis pathway. GAPDH has also been found to function in additional cellular process, such as transcription, apoptosis, oxidative stress and ER to Golgi transport.

Purify the dehydrogenase from rabbit muscle by extraction with 0.03N KOH and precipitate it with (NH4)2SO4 (0.52 of saturation). The clear supernatant is adjusted to pH 7.5, and NH3 is added dropwise to pH 8.2-8.4. Crystals appear sometimes even without seeding. The crystals are dissolved in H2O, filtered to remove suspended material and 2 volumes of saturated (NH4)2SO4 at pH 8.2-8.4 is added. After 1hour the crystals appear. Recrystallise it in the same way. [Cori et al. J Biol Chem 173 605 1948, Furfine & Velick J Biol Chem 240 844 1965, The Enzymes 7 243 1963, Lui & Huskey Biochemistry 31 6998 1992.] The Km values are: NADH (3.3WM) and 1,3-diphosphoglycerate (8x10-7M) in pH 7.4 imidazole buffer at 26o, NAD (13WM), glyceraldehyde-3-P (90WM), Pi (2.9x10-4M), and arsenate (69WM) in 8.6 M NaHCO3 buffer at 26oC. [Orsi & Cleland Biochemistry 11 102 1972.]