Chemical Properties
White crystalline material, suspended in 3.2 mol/L ammonium sulfate solution (containing 0.1 mmol/L EDTA). Stability: under the condition of 5℃, the suspension can be kept for several months with the same activity; changing the new medium regularly can keep the activity unchanged for a long time; freezing or thawing can inactivate it. Each molecule of glyceraldehyde-3-phosphate dehydrogenase contains 3.8-4.0 highly active cysteine residues, and can bind to 4 molecules of coenzyme I (NAD+) or reduced coenzyme I (NADH), the enzyme reaction: D-glyceraldehyde-3-phosphate + coenzyme I + inorganic phosphorus ═1,3-bisphosphate-D-glyceroic acid + reduced coenzyme I.
Uses
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
General Description
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.
Biochem/physiol Actions
Glyceraldehyde-3-phosphate dehydrogenase catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate as part of glycolysis. It has also been shown to have roles in initiation of apoptosis, transcription activation and the shuttling of ER to Golgi vesicles<<<,New>>>.
Purification Methods
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.]
