The isolation and specific activity of rabbit-muscle glyceraldehyde phosphate dehydrogenase

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Abstract
Some factors that affect the specific activity of glyceraldehyde phosphate dehydrogenase were investigated. Traces of heavy-metal salts, even in the presence of EDTA, decrease the activity. The concentration of phosphate or arsenate has a critical effect on the activity, which is maximal in 20 m[image]-phosphate or 40 m[image]-arsenate. Glassware that has been treated witn dichlorodimethylsilane gives higher and more reproducible values for specific activity than untreated glassware. Abnormally high activities can be detected in dilute solutions of the enzyme (approx. 10 [mu]g./ml.) during the first few minutes after dilution at 0-3[degree]. The activity falls rapidly to the "normal" value, and the phenomenon does not appear to be due to adsorption on to the surfaces of the containing vessels nor to oxidation during dilution. It may be due to a molecular rearrangement accompanying dilution. Only traces of zinc could be detected in active samples of the enzyme, and the enzymic activity was rapidly abolished by 16m[image]-zinc sulphate, although not by 16 m[image]-magnesium sulphate. The isolation of highly active glyceraldehyde phosphate dehydrogenase from rabbit-muscle extracts was made difficult by the tendency of the enzyme to form mixed crystals. A method is described which avoids this difficulty and gives good yields (1.2g./kg. of muscle) of material of specific activity 164 [plus or minus] 10 units/mg. at 25[degree]. The enzyme isolated in this way contains less bound NAD (less than 1 mole/mole) than that of previous workers (2.4-3.6 moles/mole), but precipitation with ammonium sulphatr in the presence of an excess of NAD yields a product containing 3.9-4.0 moles of bound NAD/mole.