Purification and characterization of zinc-dependent acid phosphatase from bovine brain.
Zn(2+)-Dependent acid phosphatase (Zn(2+)-APase) was purified to homogeneity from bovine brain. The apparent molecular weight of the enzyme was estimated to be about 62000 by gel filtration and 31000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme exhibited an isoelectric point of approximately 4.8. The enzyme required Zn2+ ions for catalytic activity, but other cations had little or no effect. The maximum enzyme activity was obtained in the presence of about 5 mM of Zn2+ at pH 5.5 in 50 mM 2-(N-morpholino)ethanesulfonic acid-NaOH buffer. The enzyme significantly catalyzed the hydrolysis of p-nitrophenyl phosphate, phenyl phosphate, and phosphotyrosine. The enzyme was also active for myo-inositol-2-monophosphate and adenosine 2'-monophosphate of the other common phosphate esters tested, though significantly less active than for p-nitrophenyl phosphate. The optimum activity pH of the enzyme was around 5.5 with p-nitrophenyl phosphate and myo-inositol-2-monophosphate. The enzyme was resistant to fluoride ions. Two types of Zn(2+)-APases, a high molecular weight (molecular weight, M(r)., about 110,000) and a low molecular weight (M(r), about 62,000) type, were found to exist in various tissues of rat. Brain, lung, spleen, stomach, heart, skeletal muscle, and erythrocytes contained only the lower molecular weight type. On the other hand, liver and kidney contained mainly the higher molecular weight type, and the small intestine contained significant quantities of the both types.
Fujimoto
S
S
Kyoto Pharmaceutical University, Japan.
Gotoh
H
H
Ohbayashi
T
T
Yazaki
H
H
Ohara
A
A
D000135:Acid Phosphatase / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology; D008297:Male; D008970:Molecular Weight; D009439:Neuraminidase; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*
D000135:Acid Phosphatase; D009439:Neuraminidase; D015032:Zinc