Purification and some properties of glyceraldehyde 3-phosphate dehydrogenase from Synechococcus sp.
Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was purified 386 fold to apparent homogeneity from the thermophilic cyanobacterium Synechococcus sp. grown at optimum light intensities in batch cultures. The molecular mass of the tetrameric form of the enzyme was 160 kDa as determined by gel filtration and sucrose gradient centrifugation in a phosphate buffer containing DTT. The pH optimum for the oxidation of NADPH was broad (6-8) and the enzyme had a pI of 4.5. The turnover number was 36,000 min-1 at 40 degrees C. The activation energy was 12.4 Kcal for t > 29 degrees C and 20.6 Kcal for t < 29 degrees C. The specific absorption coefficient, A 1% 1cm 280 mm of the pure enzyme in phosphate buffer at pH 6.8 was 15.2. By SDS gel electrophoresis molecular masses of 78 kDa and 39 kDa were found, indicating that the purified enzyme is a tetramer, probably a homotetramer. When Tris was used as buffer in the homogenization and phosphate and DTT were omitted, a high molecular form with a molecular mass above 500 kDa was found. This form was less active than the purified tetrameric form. Acetone and other organic solvents stimulated the native enzyme several fold.
Institute of Biochemistry, Odense University, Denmark.
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Petersen
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Jørgen
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Iversen
D000458:Cyanobacteria / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D011487:Protein Conformation; D013816:Thermodynamics
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases