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Isolation and characterization of βA3-crystallin associated proteinase from α-crystallin fraction of human lenses
PURPOSE: The purpose was to characterize the properties of a proteinase activity associated with βA3-crystallin, which was isolated from the α-crystallin fraction of human lenses. METHODS: An inactive, Arg-bond hydrolyzing proteinase in the α-crystallin fraction, which was isolated from the water so...
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Formato: | Texto |
Lenguaje: | English |
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Molecular Vision
2008
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2571948/ https://www.ncbi.nlm.nih.gov/pubmed/18949065 |
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author | Srivastava, O. P. Srivastava, K. Chaves, J. M. |
author_facet | Srivastava, O. P. Srivastava, K. Chaves, J. M. |
author_sort | Srivastava, O. P. |
collection | PubMed |
description | PURPOSE: The purpose was to characterize the properties of a proteinase activity associated with βA3-crystallin, which was isolated from the α-crystallin fraction of human lenses. METHODS: An inactive, Arg-bond hydrolyzing proteinase in the α-crystallin fraction, which was isolated from the water soluble (WS) protein fraction of 60- to 70-year-old human lenses, was activated by sodium deoxycholate treatment. The activated enzyme was purified by a three-step procedure that included a size-exclusion Agarose A1.5 m chromatography, non-denaturing preparative gel-electrophoresis, and size-exclusion HPLC. The purified proteinase was characterized for the proteinase type, proteolysis of bovine recombinant γB-, γC-, and γD-crystallins, and its presence in three different protein fractions of human lenses (i.e., α-crystallin, β(H)-crystallin, and membrane fractions). RESULTS: An inactive, Arg-bond hydrolyzing proteinase present in the α-crystallin fraction showed activity on treatment with detergents such as sodium deoxycholate, Triton X-100, octyl β-D-glucopyranoside, and CHAPS (3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate). The sodium deoxycholate-activated enzyme was released from the α-crystallin fraction since it eluted at a lower molecular weight species than α-crystallin during size-exclusion Agarose A1.5 m chromatography. Following a three-step purification procedure, the enzyme showed three species between 22 kDa and 25 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) analysis. The three protein bands were identified as βA3-, βB1-, and βB2-crystallin by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem mass spectrometric (ES-MS/MS) methods. Inhibitor studies revealed that the enzyme was a serine-type proteinase. Among the recombinant βA3-, βB1-, or βB2-crystallins, only the βA3-crystallin exhibited the proteinase activity following detergent treatment and size-exclusion chromatography. The proteinase also exhibited proteolysis of γC- and γD- crystallins, and the cleavage of γD-crystallin at M(1)-G(2), Q(54)-Y(55), M(70)-G(71), and Q(103)-M(104) bonds. Further, the enzyme was also present in three fractions of human lenses (α-crystallin, β(H)-crystallin, and membrane fractions). CONCLUSIONS: A serine-type βA3-crystallin proteinase existed in an inactive state in the α-crystallin fraction and was activated by detergents. The enzyme proteolyzed αA-, αB-, γC-, and γD-crystallins and was present in three fractions (α-crystallin, β(H)-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses. |
format | Text |
id | pubmed-2571948 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | Molecular Vision |
record_format | MEDLINE/PubMed |
spelling | pubmed-25719482008-10-23 Isolation and characterization of βA3-crystallin associated proteinase from α-crystallin fraction of human lenses Srivastava, O. P. Srivastava, K. Chaves, J. M. Mol Vis Research Article PURPOSE: The purpose was to characterize the properties of a proteinase activity associated with βA3-crystallin, which was isolated from the α-crystallin fraction of human lenses. METHODS: An inactive, Arg-bond hydrolyzing proteinase in the α-crystallin fraction, which was isolated from the water soluble (WS) protein fraction of 60- to 70-year-old human lenses, was activated by sodium deoxycholate treatment. The activated enzyme was purified by a three-step procedure that included a size-exclusion Agarose A1.5 m chromatography, non-denaturing preparative gel-electrophoresis, and size-exclusion HPLC. The purified proteinase was characterized for the proteinase type, proteolysis of bovine recombinant γB-, γC-, and γD-crystallins, and its presence in three different protein fractions of human lenses (i.e., α-crystallin, β(H)-crystallin, and membrane fractions). RESULTS: An inactive, Arg-bond hydrolyzing proteinase present in the α-crystallin fraction showed activity on treatment with detergents such as sodium deoxycholate, Triton X-100, octyl β-D-glucopyranoside, and CHAPS (3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate). The sodium deoxycholate-activated enzyme was released from the α-crystallin fraction since it eluted at a lower molecular weight species than α-crystallin during size-exclusion Agarose A1.5 m chromatography. Following a three-step purification procedure, the enzyme showed three species between 22 kDa and 25 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) analysis. The three protein bands were identified as βA3-, βB1-, and βB2-crystallin by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem mass spectrometric (ES-MS/MS) methods. Inhibitor studies revealed that the enzyme was a serine-type proteinase. Among the recombinant βA3-, βB1-, or βB2-crystallins, only the βA3-crystallin exhibited the proteinase activity following detergent treatment and size-exclusion chromatography. The proteinase also exhibited proteolysis of γC- and γD- crystallins, and the cleavage of γD-crystallin at M(1)-G(2), Q(54)-Y(55), M(70)-G(71), and Q(103)-M(104) bonds. Further, the enzyme was also present in three fractions of human lenses (α-crystallin, β(H)-crystallin, and membrane fractions). CONCLUSIONS: A serine-type βA3-crystallin proteinase existed in an inactive state in the α-crystallin fraction and was activated by detergents. The enzyme proteolyzed αA-, αB-, γC-, and γD-crystallins and was present in three fractions (α-crystallin, β(H)-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses. Molecular Vision 2008-10-20 /pmc/articles/PMC2571948/ /pubmed/18949065 Text en http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Srivastava, O. P. Srivastava, K. Chaves, J. M. Isolation and characterization of βA3-crystallin associated proteinase from α-crystallin fraction of human lenses |
title | Isolation and characterization of βA3-crystallin associated proteinase from α-crystallin fraction of human lenses |
title_full | Isolation and characterization of βA3-crystallin associated proteinase from α-crystallin fraction of human lenses |
title_fullStr | Isolation and characterization of βA3-crystallin associated proteinase from α-crystallin fraction of human lenses |
title_full_unstemmed | Isolation and characterization of βA3-crystallin associated proteinase from α-crystallin fraction of human lenses |
title_short | Isolation and characterization of βA3-crystallin associated proteinase from α-crystallin fraction of human lenses |
title_sort | isolation and characterization of βa3-crystallin associated proteinase from α-crystallin fraction of human lenses |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2571948/ https://www.ncbi.nlm.nih.gov/pubmed/18949065 |
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