Acid phosphatases of rabbit spermatozoa: II. Partial purification and biochemical characterization of the multiple forms of rabbit spermatozoan acid phosphatase

doi:10.1016/0304-4165(73)90177-3

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 320, Issue 1, 17 August 1973, Pages 180-194

https://doi.org/10.1016/0304-4165(73)90177-3 Get rights and content

Abstract

Five acid phosphatases, S4, S3, S2, Szn and S1 (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) of ejaculated rabbit spermatozoa were either partially purified by DEAE-Sephadex column chromatography or prepared by specific extraction methods.

The pH optimum of S4 was 6.0–6.5 in acetate buffer and 7.0 in Tris-HCl buffer; the pH optima of S3, S2, Szn, and S1 were 4.5, 5.5., 6.0 and 5.2, respectively, in acetate buffer. The apparent molecular weights of S3, S, Szn and S1, determined by disc gel electrophoresis, were 123 000, 86 000, 64 000 and 45 000–49 000, respectively. Incubation with neuraminidase did not alter the electrophoretic mobilities of any of the enzymes.

Ten natural phosphoric esters were tested as substrates. S4 preferentially hydrolyzed ATP, ADP, PP_i and 3′-AMP. S3 hydrolyzed only β-glycerophosphate and glucose 6-phosphate to a significant extent. S2 hydrolyzed β-glycerophosphate, glucose 1-phosphate, the phosphoproteins, casein and phosvitin. S1 hydrolyzed ADP and β-glycerophosphate most readily. Szn may be an ATPase since it exhibits very high Zn²⁺-stimulated against ATP.

These characteristics combined with the effects of NaF, ZnCl₂, l-(+)-tartaric acid, and formaldehyde on the activity of each partially purified enzyme with α-naphthyl phosphate as substrate indicate that these phosphatases are structurally and functionally different.

References (49)

L.W. Gonzales et al.
Biochim. Biophys. Acta
(1973)
W.A. Anderson
J. Ultrastruct. Res.
(1968)
J.L. Hedrick et al.
Arch. Biochem. Biophys.
(1968)
A. Chrambach et al.
Anal. Biochem.
(1967)
D.M. Campbell et al.
Clin. Chim. Acta
(1961)
O.H. Lowry et al.
J. Biol. Chem.
(1946)
H.R. Revel et al.
Biochim. Biophys. Acta
(1960)
M.H. Meisler et al.
J. Biol. Chem.
(1969)
S. Bramhall et al.
Anal. Biochem.
(1969)
J.K. Smith et al.
Biochim. Biophys. Acta
(1968)

K. Paigen et al.

J. Biol. Chem.

(1959)

L. Nelson

Biochim. Biophys. Acta

(1954)

K. Ogawa et al.

Biochim. Biophys. Acta

(1972)

L.A. Heppel et al.

J. Biol. Chem.

(1953)

P.E. Lindahl

Exp. Cell Res.

(1968)

P.E. Lindahl et al.

Exp. Cell Res.

(1972)

M. Gordon et al.

Exp. Cell Res.

(1967)

M.R. Stetten et al.

Biochim. Biophys. Acta

(1969)

R.J. Teichman et al.

J. Reprod. Fert.

(1971)

R.J. Teichman et al.

J. Cell Biol.

(1969)

J.M. Allen et al.

Ann. N.Y. Acad. Sci.

(1964)

E. Baginski et al.

Clin. Chem. Acta

(1960)

J.L. Peel et al.

Biochem. J.

(1955)

E. Dziembor et al.

Acta Biochim. Pol.

(1971)

Cited by (23)

The spermatozoon
2006, Knobil and Neill's Physiology of Reproduction
The Spermatozoon
2005, Knobil and Neill's Physiology of Reproduction
Purification and some properties of a Mg<sup>2+</sup>-activated acid phosphatase from rat testis
1994, International Journal of Biochemistry
- 1.
  1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity.
- 2.
  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.
  3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein.
- 4.
  4. The rat testis AcPase IV is a metal activated enzyme in which Mg²⁺ is the metal activating agent with a K_a, = 0.88 × 10⁻³ M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg²⁺ ions, is 0.23 × 10⁻³ M.
- 5.
  5. The enzyme preferentially hydrolizes p-nitrophenylphosphate, phenylphosphate and ATP.
Presence of ATPase and alkaline phosphatase activities in the starfish sperm acrosome
1988, Cell Biology International Reports
ATPase activity was cytochemically detected in the peripheral acrosomal component of ionophore-reacted sperm, while alkaline phosphatase activity was demonstrated in the upper and central components of the acrosome and, at fertilization, at the site of sperm-oocyte binding. Supernatants of ionophore treated sperm suspensions were assayed for ATPase, alkaline and acid phosphatase activities. Results suggest that alkaline phosphatase may be involved both in the acrosomal reaction and oocyte jelly lysis but the function of the acrosomal ATPase remains unknown.
Starfish acrosomal acid phosphatase: a cytochemical and biochemical study
1988, Biology of the Cell
The acrosome reaction of spermatozoa from the starfish Marthasterias glacialis was induced with the ionophore A23187. Reacted cells were then processed for acid phosphatase ultrastructural cytochemistry, but significant enzyme activity was not detected. However, when the supernates from suspensions of ionophore-treated sperm were assayed for acid phosphatase, a net enzyme activity was observed. Supernatant proteins were run in starch gel electrophoresis and fluorescent zymograms revealed a single band of acid phosphatase. SDS-PAGE of proteins eluted from the active spots of starch gels showed one major band of about 63 kDa. The results obtained support the hypothesis that the acid phosphatase whose activity has been detected only at the time of binding of sperm and egg originates from the sperm acrosome.
Zn<sup>2+</sup>-dependent acid p-nitrophenylphosphatase from chicken liver. purification, characterization and subcellular localization
1988, International Journal of Biochemistry
- 1.
  1. Zn²⁺-dependent acid p-nitrophenylphosphatase from chicken liver was purified to homogeneity.
- 2.
  2. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity.
- 3.
  3. Gel filtration on Sephadex G-200 gave an apparent molecular weight of 110,000 with two apparent identical subunits of 54,000–56,000 as determined by sodium dodecyl sulphate gel electrophoresis.
- 4.
  4. The maximum of enzyme activity was obtained in the presence of 3–5 mM ZnCl₂ at pH 6–6.2, however, higher concentrations of metal are inhibitory. The enzyme hydrolyses p-nitrophenylphosphate, o-carboxyphenylphosphate and phenylphosphate, was insensitive to NaF and was inhibited by phosphate and ATP. The K_m for p-nitrophenylphosphate was 0.28 × 10⁻³ M at pH 6 in 50 mM sodium acetate/100 mM NaCl.
- 5.
  5. Phosphate is a competitive inhibitor (K_i = 0.5 x 10⁻³ M) whereas ATP seems to be a non-competitive inhibitor (K_i = 0.35 × 10⁻³ M). The isoelectric point determined by isolectric focusing on polyacrylamide gel is 7.5.
- 6.
  6. Cell fractionation studies indicate that the Zn²⁺-dependent acid p-nitrophenylphosphatase of chicken liver is a soluble enzyme form.

View all citing articles on Scopus

^★: Present address: Department of Neurology, School of Medicine, University of California, Davis, Calif. 95616 (U.S.A.).

View full text

Acid phosphatases of rabbit spermatozoa: II. Partial purification and biochemical characterization of the multiple forms of rabbit spermatozoan acid phosphatase

Abstract

Biochim. Biophys. Acta

J. Ultrastruct. Res.

Arch. Biochem. Biophys.

Anal. Biochem.

Clin. Chim. Acta

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Anal. Biochem.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

Exp. Cell Res.

Exp. Cell Res.

Exp. Cell Res.

Biochim. Biophys. Acta

J. Reprod. Fert.

J. Cell Biol.

Ann. N.Y. Acad. Sci.

Clin. Chem. Acta

Biochem. J.

Acta Biochim. Pol.