Abstract
This chapter describes the assay method of glyceraldehyde-3-phosphate dehydrogenase isolated from the pig liver. Liver glyceraldehyde-3- phosphate dehydrogenase (GAPD) operates in the backward direction when conditions require gluconeogenesis. With the rabbit muscle enzyme, the backward reaction is strongly inhibited by nicotinamide adenine dinucleotide (NAD), whereas the liver enzyme is only weakly inhibited by NAD. The pig liver enzyme is highly soluble in metal ion and high salt concentrations; a major step is the initial precipitation of large amounts of unwanted proteins with ZnCl 2 . The reaction velocity shows a decrease with time, unless precautions are taken to avoid: (a) product inhibition by nicotinamide adenine dinucleotide dehydrogenase (NADH); (b) product inhibition by 1,3-diphosphoglycerate; and (c) the deterioration of glyceraldehyde-3-P with time. The use of arsenate in the assay, in place of phosphate, avoids the inhibition by 1,3-diphosphoglycerate. The steps involved in the purification of GAPD are: (1) homogenization in ZnCl 2 solutions, (2) ammonium sulfate fractionation, (3) chromatography on diethylaminoethyl (DEAE)-Sephadex, and (4) crystallization. Sodium dodecyl sulfate polyacrylamide gel electrophoresis experiments show a single protein band, indicating that the enzyme is pure and consists of similar subunits.
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