Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma.
The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5-7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K(m)=2.1x10(-3)M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase. The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.
Wysocki
P
P
University of Warmia and Mazury in Olsztyn, Faculty of Animal Bioengineering, Department of Animal Biochemistry and Biotechnology, 10-718 Olsztyn, Poland. pawel.wysocki@uwm.edu.pl
Strzezek
J
J
D000135:Acid Phosphatase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods / Q000662:veterinary; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004822:Epididymis / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D012661:Semen / Q000201:enzymology; D013379:Substrate Specificity; D013552:Swine / Q000502:physiology
D004791:Enzyme Inhibitors; D000135:Acid Phosphatase