Affinity separation of an acid phosphatase from rat tissues and Gaucher spleen with immobilized Cibacron Blue.
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 KCl. This enzyme bound to Cibacron Blue-immobilized Sepharose (Blue-Sepharose) and was desorbed by 2 M KCl 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 triphosphates. 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.
D000135:Acid Phosphatase / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D005776:Gaucher Disease / Q000201:enzymology*; D006801:Humans; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D051381:Rats; D012016:Reference Values; D012685:Sepharose / Q000031:analogs & derivatives; D013154:Spleen / Q000201:enzymology*
C017842:cibacron blue-sepharose; D012685:Sepharose; D007501:Iron; D000135:Acid Phosphatase