Purification and characterization of purple acid phosphatase from developing rat bone.
Tartrate-resistant acid phosphatase active on nucleoside di- and triphosphate substrates was isolated from developing rat bone and purified 2500-fold. The enzyme concentration had a purple coloration and activity that was sensitive to reducing agents. Mild reducing agents such as ferrous ion and ascorbic acid caused loss of purple color and increased activity toward substrates severalfold; however, a strong reductant such as dithionite caused loss of both color and activity which were partially restored by addition of ferrous ion and ascorbic acid. Enzyme activity was homogeneous with protein during the final gel permeation steps of chromatography and gave an apparent molecular size of about 40,000 Da. Determination of iron in the most pure preparation revealed the presence of 1.3 atoms of iron per molecule of the tartrate-resistant enzyme E2. Other properties of the purified enzyme include a pI of approximately 9.5 and sensitivity to inhibition by ions of copper, zinc, fluoride, and molybdate. Antibody prepared to the pre-concanavalin A (Con A)-Sepharose purified enzyme reacted with all protein from the Con A step, but it did not react with tartrate-sensitive acid phosphatase from rat bone or with potato acid phosphatase. Purple acid phosphatase from rat bone has many properties that parallel the iron-containing purple acid phosphatases from rat spleen, bovine spleen, and pig uterine secretions.
D000135:Acid Phosphatase / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001846:Bone Development; D001842:Bone and Bones / Q000201:enzymology*; D002846:Chromatography, Affinity; D004589:Electrophoresis, Disc; D004789:Enzyme Activation; D005779:Immunodiffusion; D007525:Isoelectric Focusing; D011489:Protein Denaturation; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013644:Tartrates / Q000494:pharmacology
D013644:Tartrates; D000135:Acid Phosphatase; C029768:tartaric acid