Evaluation of the proacrosin/acrosin system and its mechanism of activation in human sperm extracts
Acrosin is an acrosomal protease synthesized as a proenzyme and activated into β-acrosin during the acrosome reaction. In the present study, a set of sensitive assays was developed to identify the proacrosin/acrosin system and to evaluate its activation pattern in human sperm extracts. Immunocytoche...
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todo:paper_01650378_v54_n1-2_p43_Zahn2023-10-03T15:02:39Z Evaluation of the proacrosin/acrosin system and its mechanism of activation in human sperm extracts Zahn, A. Furlong, L.I. Biancotti, J.C. Ghiringhelli, P.D. Marín-Briggiler, C.I. Vazquez-Levin, M.H. Acrosin Activation Human spermatozoa Proteases acrosin benzamidine calcium chloride cell extract monoclonal antibody proacrosin acrosome amino acid sequence article controlled study enzyme activation enzyme activity enzyme assay human human cell immunocytochemistry male priority journal protein analysis sperm spermatozoon staining Western blotting zymography Acrosin Amino Acid Sequence Animals Enzyme Activation Enzyme Precursors Humans Hydrogen-Ion Concentration Immunohistochemistry Male Molecular Sequence Data Molecular Weight Spermatozoa Acrosin is an acrosomal protease synthesized as a proenzyme and activated into β-acrosin during the acrosome reaction. In the present study, a set of sensitive assays was developed to identify the proacrosin/acrosin system and to evaluate its activation pattern in human sperm extracts. Immunocytochemical analysis with monoclonal antibody (Mab) AcrC5F10 showed specific staining on the acrosome of permeabilized ejaculated and capacitated spermatozoa. Acrosome reaction was associated with a decrease in staining. AcrC5F10 specifically recognized a 55-kDa band (proacrosin) in Western immunoblots. Activation studies showed enzymatically active intermediates of 39 and 35 kDa after zymography. Immunoreactive bands of 52, 43, 34, 21-26 and 16 kDa were identified in the activation patterns developed with AcrC5F10. Activation was completely inhibited in the presence of 9 mM CaCl2 or 100 mM benzamidine. A multiple sequence alignment revealed partial conservation of putative cleavage sites in the proacrosin sequence. The tests described allow the detection of human proacrosin in spermatozoa and sperm protein extracts, as well as the evaluation of the proenzyme activation pattern. They can be used to study the effect of inhibitors upon proenzyme activation. In addition, alterations in proacrosin activation in semen samples with abnormal acrosin enzymatic activity can be analyzed using these assays. © 2002 Elsevier Science Ireland Ltd. All rights reserved. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_01650378_v54_n1-2_p43_Zahn |
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Universidad de Buenos Aires |
| institution_str |
I-28 |
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R-134 |
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Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Acrosin Activation Human spermatozoa Proteases acrosin benzamidine calcium chloride cell extract monoclonal antibody proacrosin acrosome amino acid sequence article controlled study enzyme activation enzyme activity enzyme assay human human cell immunocytochemistry male priority journal protein analysis sperm spermatozoon staining Western blotting zymography Acrosin Amino Acid Sequence Animals Enzyme Activation Enzyme Precursors Humans Hydrogen-Ion Concentration Immunohistochemistry Male Molecular Sequence Data Molecular Weight Spermatozoa |
| spellingShingle |
Acrosin Activation Human spermatozoa Proteases acrosin benzamidine calcium chloride cell extract monoclonal antibody proacrosin acrosome amino acid sequence article controlled study enzyme activation enzyme activity enzyme assay human human cell immunocytochemistry male priority journal protein analysis sperm spermatozoon staining Western blotting zymography Acrosin Amino Acid Sequence Animals Enzyme Activation Enzyme Precursors Humans Hydrogen-Ion Concentration Immunohistochemistry Male Molecular Sequence Data Molecular Weight Spermatozoa Zahn, A. Furlong, L.I. Biancotti, J.C. Ghiringhelli, P.D. Marín-Briggiler, C.I. Vazquez-Levin, M.H. Evaluation of the proacrosin/acrosin system and its mechanism of activation in human sperm extracts |
| topic_facet |
Acrosin Activation Human spermatozoa Proteases acrosin benzamidine calcium chloride cell extract monoclonal antibody proacrosin acrosome amino acid sequence article controlled study enzyme activation enzyme activity enzyme assay human human cell immunocytochemistry male priority journal protein analysis sperm spermatozoon staining Western blotting zymography Acrosin Amino Acid Sequence Animals Enzyme Activation Enzyme Precursors Humans Hydrogen-Ion Concentration Immunohistochemistry Male Molecular Sequence Data Molecular Weight Spermatozoa |
| description |
Acrosin is an acrosomal protease synthesized as a proenzyme and activated into β-acrosin during the acrosome reaction. In the present study, a set of sensitive assays was developed to identify the proacrosin/acrosin system and to evaluate its activation pattern in human sperm extracts. Immunocytochemical analysis with monoclonal antibody (Mab) AcrC5F10 showed specific staining on the acrosome of permeabilized ejaculated and capacitated spermatozoa. Acrosome reaction was associated with a decrease in staining. AcrC5F10 specifically recognized a 55-kDa band (proacrosin) in Western immunoblots. Activation studies showed enzymatically active intermediates of 39 and 35 kDa after zymography. Immunoreactive bands of 52, 43, 34, 21-26 and 16 kDa were identified in the activation patterns developed with AcrC5F10. Activation was completely inhibited in the presence of 9 mM CaCl2 or 100 mM benzamidine. A multiple sequence alignment revealed partial conservation of putative cleavage sites in the proacrosin sequence. The tests described allow the detection of human proacrosin in spermatozoa and sperm protein extracts, as well as the evaluation of the proenzyme activation pattern. They can be used to study the effect of inhibitors upon proenzyme activation. In addition, alterations in proacrosin activation in semen samples with abnormal acrosin enzymatic activity can be analyzed using these assays. © 2002 Elsevier Science Ireland Ltd. All rights reserved. |
| format |
JOUR |
| author |
Zahn, A. Furlong, L.I. Biancotti, J.C. Ghiringhelli, P.D. Marín-Briggiler, C.I. Vazquez-Levin, M.H. |
| author_facet |
Zahn, A. Furlong, L.I. Biancotti, J.C. Ghiringhelli, P.D. Marín-Briggiler, C.I. Vazquez-Levin, M.H. |
| author_sort |
Zahn, A. |
| title |
Evaluation of the proacrosin/acrosin system and its mechanism of activation in human sperm extracts |
| title_short |
Evaluation of the proacrosin/acrosin system and its mechanism of activation in human sperm extracts |
| title_full |
Evaluation of the proacrosin/acrosin system and its mechanism of activation in human sperm extracts |
| title_fullStr |
Evaluation of the proacrosin/acrosin system and its mechanism of activation in human sperm extracts |
| title_full_unstemmed |
Evaluation of the proacrosin/acrosin system and its mechanism of activation in human sperm extracts |
| title_sort |
evaluation of the proacrosin/acrosin system and its mechanism of activation in human sperm extracts |
| url |
http://hdl.handle.net/20.500.12110/paper_01650378_v54_n1-2_p43_Zahn |
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