STUDIES ON ACID PHOSPHATASES. II. CHROMATOGRAPHIC SEPARATION OF ACID PHOSPHATASES OF RAT LIVER

The separation of soluble acid phosphatases of rat liver by anion exchange chromatography and some characteristics of the fractions obtained were described. 1) Chromatogram on N 1 ,N-diethyl-aminoethyl- (DEAE) cellulose gave 3 acid phosphatase peaks if the liver homogenate was prepared with distilled water. 2) Using a sample prepared with Triton X-100 four peaks were separated. 3) The effect of Triton X-100 is to solubilize an acid phosphatase fraction. The detergent did not alter the chromatogram if it was added to the distilled water sample. 4) All the four enzymes have a pH optimum around pH 4.5. Fraction #4 retains about 75% of its maximal activity at pH 3.0. 5) The four enzyme fractions hydrolyzed adenylic acid and α-naphthyl phosphate at about the same rate and approximately 1.5 times faster than β-glycerophosphate. All the 4 fractions hydrolyzed p-nitrophenylphosphate at a considerably higher rate than β-glycerophosphate. Fraction #1 was especially active towards p-nitrophenylphosphate. The dissociation constants with the four substrates were determined. 6) Fraction #1, which represented about 65% of the total activity, was further purified on carboxymethyl cellulose. 7) The four acid phosphatase fractions differed electrophoretically (using polyacrylamide gel electrophoresis) and according to the effect of inhibitors. The observations are reconcilable with the assumption that the four chromatographic fractions represent molecular varieties of the acid phosphatase.