Subunit Association and Catalytic Activity of Uridine

It was recently observed' that uridine diphosphate galactose4-epimerase from Saccharomyces fragilis exhibits greatly enhanced activity in the presence of various cations. This activation appears to be primarily due to an increase in the affinity of the enzyme for its substrate, UDP-galactose.1' 2 Many different types of cation are effective in this regard, including univalent and divalent metal ions, and the protonated forms of a number of amines. Studies of the sedimentation behavior of epimerase in a sucrose gradient at 40 have revealed that under conditions of low ionic strength where epimerase shows suboptimal catalytic activity, the sedimentation coefficient is approximately 6S. In the presence of 10-2 M spermine hydrochloride, which is a highly effective activator per unit ionic strength, the sedimentation coefficient is 11S. It appears clear, however, that formation of the 11S particle is not a prerequisite for activation of the enzyme. For example, the 11S particle does not appear at very low spermine concentrations (10-4 M) which nevertheless produce marked activation. Furthermore, when the sedimentation is carried out at 250 instead of at 40, no 11S particle is seen, even in the presence of 10-2 M spermine which gives maximal activation. It has been known for some time that treatment with pCMNB completely inactivates yeast epimerase, and that when the inactivated epimerase is in turn treated with a sulfhydryl compound such as cysteine, and oxidized DPN, 10-20 per cent of the activity is restored.3 In this paper it is shown that inactivation of yeast epimerase by pCMB is accompanied by a decrease in its sedimentation coefficient from 6S to 4S. Upon reactivation with mercaptoethanol and DPN, the enzyme again sediments at 6S. Such reconstituted epimerase, however, has lost its ability to form the 11S particle in the presence of spermine. We interpret these sedimentation coefficients of 4S, 6S, and 11S as representing successive stages of subunit association corresponding to n, 2n, and 4n, where n is the molecular weight of the 4S particle. Methods.-Enzyme preparation: Epimerase was prepared from Saccharomyces fragilis (Candida pseudotropicalis) 10022 (A.T.C.C.), by a method similar to that published by Maxwell and de Robichon-Szulmajster.4 Two main departures from this method were found useful: (1) Disruption of the yeast cells with glass beads was replaced by plasmolysis with toluene, using the procedure employed by Cori and his collaborators5 for the purification of hexokinase. (2) Fractionation on a DEAE-cellulose column was replaced by a step in which the enzyme was precipitated in 75% saturated ammonium sulfate (4°C). The precipitate was then washed successively with 35, 30, and 25% saturated ammonium sulfate equilibrated with 0.02 M succinate buffer, pH 5.6, and finally with buffer alone. This final wash contained most of the activity, which was reprecipitated by adding solid ammonium sulfate to 50% saturation. The precipitate was then dissolved in a minimal volume of 0.1 M Tris HCl pH 7.5, containing 0.0001 M EDTA and 1 part in 1,000 of 2-mercaptoethanol, and stored at -90°C. The new preparation had 3 times as much firmly bound DPN per milligram of protein, and 10 times the specific catalytic activity reported earlier,4 although the comparison of activity is inexact since the earlier assays were done at an undefined cation concentration. The fluorescence proper-