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Isolation and characterization of betaA3-crystallin associated proteinase from alpha-crystallin fraction of human lenses.

Srivastava OP, Srivastava K, Chaves JM - Mol. Vis. (2008)

Bottom Line: The purpose was to characterize the properties of a proteinase activity associated with betaA3-crystallin, which was isolated from the alpha-crystallin fraction of human lenses.A serine-type betaA3-crystallin proteinase existed in an inactive state in the alpha-crystallin fraction and was activated by detergents.The enzyme proteolyzed alphaA-, alphaB-, gammaC-, and gammaD-crystallins and was present in three fractions (alpha-crystallin, beta(H)-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses.

View Article: PubMed Central - PubMed

Affiliation: Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA. srivasta@uab.edu

ABSTRACT

Purpose: The purpose was to characterize the properties of a proteinase activity associated with betaA3-crystallin, which was isolated from the alpha-crystallin fraction of human lenses.

Methods: An inactive, Arg-bond hydrolyzing proteinase in the alpha-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 gammaB-, gammaC-, and gammaD-crystallins, and its presence in three different protein fractions of human lenses (i.e., alpha-crystallin, beta(H)-crystallin, and membrane fractions).

Results: An inactive, Arg-bond hydrolyzing proteinase present in the alpha-crystallin fraction showed activity on treatment with detergents such as sodium deoxycholate, Triton X-100, octyl beta-D-glucopyranoside, and CHAPS (3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate). The sodium deoxycholate-activated enzyme was released from the alpha-crystallin fraction since it eluted at a lower molecular weight species than alpha-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 betaA3-, betaB1-, and betaB2-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 betaA3-, betaB1-, or betaB2-crystallins, only the betaA3-crystallin exhibited the proteinase activity following detergent treatment and size-exclusion chromatography. The proteinase also exhibited proteolysis of gammaC- and gammaD- crystallins, and the cleavage of gammaD-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 (alpha-crystallin, beta(H)-crystallin, and membrane fractions).

Conclusions: A serine-type betaA3-crystallin proteinase existed in an inactive state in the alpha-crystallin fraction and was activated by detergents. The enzyme proteolyzed alphaA-, alphaB-, gammaC-, and gammaD-crystallins and was present in three fractions (alpha-crystallin, beta(H)-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses.

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Comparison of elution profiles at 280 nm during size-exclusion HPLC  of purified proteinase from the α-crystallin fraction, recombinant βA3/A1-crystallin, and recombinant βB1-crystallin. The recombinant proteins were treated with 1% sodium deoxycholate (w/v) before their chromatography (TSK G3000 PWXL). The proteinase activity was determined by the incubation of column fractions with BAPNA at 37 °C for 10 h.
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f5: Comparison of elution profiles at 280 nm during size-exclusion HPLC of purified proteinase from the α-crystallin fraction, recombinant βA3/A1-crystallin, and recombinant βB1-crystallin. The recombinant proteins were treated with 1% sodium deoxycholate (w/v) before their chromatography (TSK G3000 PWXL). The proteinase activity was determined by the incubation of column fractions with BAPNA at 37 °C for 10 h.

Mentions: To determine whether the activity was associated with βA3-crystallin but not with βB1- or βB2-crystallin, the recombinant βA3- and βB1-crystallins along with purified α-crystallin proteinase were examined by HPLC using a size-exclusion TSK G-3000 PWXL column. The recombinant βA3/A1-crystallin was purified as previously described [31]. The recombinant protein preparations before and after deoxycholate treatment were HPLC separated using a TSK G-3000 PWXL-column, and the column fractions were examined for proteinase activity. The recombinant βA3-crystallin exhibited proteinase activity, which co-eluted with proteinase purified from the α-crystallin fraction (Figure 5). No such activity was found with βB1- and βB2-crystallins (the βB2-crystallin result is not shown).


Isolation and characterization of betaA3-crystallin associated proteinase from alpha-crystallin fraction of human lenses.

Srivastava OP, Srivastava K, Chaves JM - Mol. Vis. (2008)

Comparison of elution profiles at 280 nm during size-exclusion HPLC  of purified proteinase from the α-crystallin fraction, recombinant βA3/A1-crystallin, and recombinant βB1-crystallin. The recombinant proteins were treated with 1% sodium deoxycholate (w/v) before their chromatography (TSK G3000 PWXL). The proteinase activity was determined by the incubation of column fractions with BAPNA at 37 °C for 10 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2571948&req=5

f5: Comparison of elution profiles at 280 nm during size-exclusion HPLC of purified proteinase from the α-crystallin fraction, recombinant βA3/A1-crystallin, and recombinant βB1-crystallin. The recombinant proteins were treated with 1% sodium deoxycholate (w/v) before their chromatography (TSK G3000 PWXL). The proteinase activity was determined by the incubation of column fractions with BAPNA at 37 °C for 10 h.
Mentions: To determine whether the activity was associated with βA3-crystallin but not with βB1- or βB2-crystallin, the recombinant βA3- and βB1-crystallins along with purified α-crystallin proteinase were examined by HPLC using a size-exclusion TSK G-3000 PWXL column. The recombinant βA3/A1-crystallin was purified as previously described [31]. The recombinant protein preparations before and after deoxycholate treatment were HPLC separated using a TSK G-3000 PWXL-column, and the column fractions were examined for proteinase activity. The recombinant βA3-crystallin exhibited proteinase activity, which co-eluted with proteinase purified from the α-crystallin fraction (Figure 5). No such activity was found with βB1- and βB2-crystallins (the βB2-crystallin result is not shown).

Bottom Line: The purpose was to characterize the properties of a proteinase activity associated with betaA3-crystallin, which was isolated from the alpha-crystallin fraction of human lenses.A serine-type betaA3-crystallin proteinase existed in an inactive state in the alpha-crystallin fraction and was activated by detergents.The enzyme proteolyzed alphaA-, alphaB-, gammaC-, and gammaD-crystallins and was present in three fractions (alpha-crystallin, beta(H)-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses.

View Article: PubMed Central - PubMed

Affiliation: Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA. srivasta@uab.edu

ABSTRACT

Purpose: The purpose was to characterize the properties of a proteinase activity associated with betaA3-crystallin, which was isolated from the alpha-crystallin fraction of human lenses.

Methods: An inactive, Arg-bond hydrolyzing proteinase in the alpha-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 gammaB-, gammaC-, and gammaD-crystallins, and its presence in three different protein fractions of human lenses (i.e., alpha-crystallin, beta(H)-crystallin, and membrane fractions).

Results: An inactive, Arg-bond hydrolyzing proteinase present in the alpha-crystallin fraction showed activity on treatment with detergents such as sodium deoxycholate, Triton X-100, octyl beta-D-glucopyranoside, and CHAPS (3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate). The sodium deoxycholate-activated enzyme was released from the alpha-crystallin fraction since it eluted at a lower molecular weight species than alpha-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 betaA3-, betaB1-, and betaB2-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 betaA3-, betaB1-, or betaB2-crystallins, only the betaA3-crystallin exhibited the proteinase activity following detergent treatment and size-exclusion chromatography. The proteinase also exhibited proteolysis of gammaC- and gammaD- crystallins, and the cleavage of gammaD-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 (alpha-crystallin, beta(H)-crystallin, and membrane fractions).

Conclusions: A serine-type betaA3-crystallin proteinase existed in an inactive state in the alpha-crystallin fraction and was activated by detergents. The enzyme proteolyzed alphaA-, alphaB-, gammaC-, and gammaD-crystallins and was present in three fractions (alpha-crystallin, beta(H)-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses.

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