Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle

The rabbit muscle is one of the most frequently used sources for glyceraldehyde-3-phosphate dehydrogenase. This chapter describes the assay method and properties of the enzyme, glyceraldehyde-3-phosphate dehydrogenase, isolated from the rabbit muscle. Classical isolation procedures involve the high solubility of the nicotinamide adenine dinucleotide kinase (NAD+)-containing enzyme in (NH4)2SO4 solutions; several recrystallization steps from concentrated (NH4)2SO4 solutions at pH values above 8.0 are also involved. The steps involved in the purification procedure of the enzyme are (1) the extraction of the rabbit muscle, (2) ammonium sulfate fractionation, and (3) chromatography. The preparation of holoenzyme, apoenzyme, and tetra (3-phosphoglyceroyl) enzyme are also discussed in the chapter. The specific activity of the enzyme prepared is about 185 units per milligram of protein. The enzyme migrates as a single band in polyacrylamide gel electrophoresis and has a molecular weight of 145,000. It contains 3.8–4.0 highly reactive cysteine residues, as determined with dithionitrobenzoate, and can bind 4.0 molecules of NAD+ or nicotinamide adenine dinucleotide dehydrogenase (NADH) per enzyme molecule