Purification and some properties of a Mg(2+)-activated acid phosphatase from rat testis.
1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity. 2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5-20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein. 3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein. 4. The rat testis AcPase IV is a metal activated enzyme in which Mg2+ is the metal activating agent with a Ka = 0.88 x 10(-3) M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg2+ ions, is 0.23 x 10(-3) M. 5. The enzyme preferentially hydrolyzes p-nitrophenylphosphate, phenylphosphate and ATP.
Panara
F
F
Istituto di Anatomia Comparata, Università degli Studi di Perugia, Italy.
Angiolillo
A
A
Di Rosa
I
I
Fagotti
A
A
Contenti
S
S
Simoncelli
F
F
Lorvik
S
S
Pascolini
R
R
D000135:Acid Phosphatase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008274:Magnesium / Q000494:pharmacology*; D008297:Male; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D051381:Rats; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D013737:Testis / Q000201:enzymology*
D002413:Cations, Divalent; D009596:Nitrophenols; D010755:Organophosphates; D009943:Organophosphorus Compounds; C028326:concanavalin A-sepharose; C008644:nitrophenylphosphate; C074782:phenylphosphate; D000255:Adenosine Triphosphate; D012685:Sepharose; D004492:Edetic Acid; D000135:Acid Phosphatase; D008274:Magnesium