Elution of Acid Phosphatase from the Cell Surface of Saccharomyces mellis by Potassium Chloride.

Weimberg, Ralph (Northern Regional Research Laboratory, Peoria, Ill.), and William L. Orton. Elution of acid phosphatase from the cell surface of Saccharomyces mellis by potassium chloride. J. Bacteriol. 90:82-94. 1965.-Acid phosphatase of Saccharomyces mellis may be eluted from intact resting cells by 0.5 m KCl or other salts. However, the enzyme is not eluted at higher salt concentrations of about 2 m unless a thiol, such as beta-mercaptoethanol, is included in the reaction mixture. These treatments do not significantly affect viability of the cells. Neutral compounds like sorbitol or sucrose cannot substitute for ionic compounds in eluting the enzyme from resting cells. Furthermore, the neutral compounds are also inadequate for stabilizing the protoplast structure. It is suggested that the enzyme is held on the cell surface by a combination of electrostatic forces and disulfide bonds. Thiol alone dissociates protein and carbohydrate from the cell surface, but the eluate has no acid phosphatase activity. Salts also remove protein and carbohydrate from the cell surface, but the amount of protein removed is considerably less than that dissociated by thiol. A concentration of 0.5 m KCl elutes more protein than does a 2 m concentration, and enzymatic activity is present only in the 0.5 m KCl eluate. The carbohydrate eluted by either reagent has been identified as a mannan. Conditions for eluting acid phosphatase from acetonedried cells of S. mellis are essentially the same as those for resting cells. Significantly, though, thiol is required at all salt concentrations to dissociate the enzyme. Pretreatment of the cells with thiol, followed by KCl, elutes acid phosphatase, whereas the reverse procedure does not. Acid phosphatase is excreted by growing cells of S. mellis into growth media if the medium contains 0.25 m KCl. The total yield of enzymatic activity may be 8 to 10 times greater than is usually present on derepressed cells grown in a salt-free medium. The enzyme can be precipitated from the culture fluid with acetone. The acetone-precipitated fraction contains mannan and protein in a ratio of 12:1 by weight. Partial purification of the enzyme by calcium phosphate gel and elution resulted in an enzyme fraction in which the specific activity on the basis of protein increased 12-fold, and the carbohydrate-protein ratio was reduced to 1:1.