Affinity Separation of an Acid Phosphatase from Rat Tissues and Gaucher Spleen with Immobilized Cibacron Blue1

Abstract

An acid phosphatase species which was activated by Fe²⁺ was determined to be partially soluble but mainly particulate in rat spleen. The particulate enzyme could be extracted into 1 M KCI. This enzyme bound to Cibacron Blue-immobilized Sepharose (Blue-Sepharose) and was desorbed by 2 M KCI with a good yield, while the other acid phosphatases in rat spleen did not adsorb on Blue-Sepharose. The enzymes eluted on Blue-Sepharose chromatography of both the soluble and particulate fractions were electrophoretically identical. The enzyme hydrolyzed aryl monophosphates, phosphoproteins, and nucleoside di- and tri-phosphates. The activity for the three kinds of substrate was similarly activated by Fe²⁺, ascorbic acid and cysteine, and inhibited by molybdate, Cu²⁺ and F⁻. Cibacron Blue inhibited the enzyme competitively with respect to a substrate, p-nitrophenyl phosphate, but kinetic analysis suggested that more than one dye molecule binds to the enzyme. The Blue-Sepharose technique could be applied not only to quantitative separation of acid phosphatases similar to the spleen enzyme from bone and epidermis of rat, but also to that of a tartrate-resistant acid phosphatase from human spleen with Gaucher's disease.