A rapid purification procedure for glyceraldehyde 3-phosphate dehydrogenase from Bakers' yeast.

The traditional Krebs procedure (1) for preparing yeast glyceraldehyde 3-phosphate dehydrogenasc has many advantages, but it also has some serious disadvantages. The procedure requires long periods of waiting during the first few steps of making the crude extract, and is llot reproducible in the quality of the enzyme obtained. Lazarus et al. (2) have shown that proteolytic enzymes survive the toluenc plnsmolysis step in the purification of yeast hesokinasc and affect the st.ability, specific activity, and even the number of forms of the enzyme. We find levels of proteolytic activity in t,hc classic Krebs procedure for glyceraldehyde-3-P dehgtlrogennsc comparable to those reported by Lazarus. Since new resins and methods are available today, the purification of yeast glyceraldehyde-3-l’ dehydrogenase has been re-examined and a scheme for rapid purification has been devised. This scheme requires 3 days and consistently gives enzyme of superior specific activity: 200 ~molcs per min per mg in the back reaction (NADH + 1,3-diphosphoglycerate) and 100 ~molcs per min per mg in the forward reaction (NAT) + glyccraldchyde3-l’ + arsenate). One pound of pressed yeast yields 150 to 200 mg of pure enzyme. The yeast enzyme has been shown to consist of a mix;ture of isozymes (3), and the species isolated in this procedure is the most acidic of the set.