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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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SDS–PAGE analysis of various fractions recovered during purification of α-crystallin-associated fractions. Lane 1: α-Crystallin fraction was recovered from a WS protein fraction of lenses that were 60- to 70-years-old. Lane 2: Concentrated α-crystallin fraction applied to the Agarose A1.5 m column. Lane 3: Proteinase fractions recovered following non-denaturing gel electrophoresis of the proteinase fraction released after Agarose A1.5 m gel chromatography. Lane 4: A proteinase-containing fraction recovered following size-exclusion HPLC using TSK G-4000 PWXL column of the fraction recovered in lane 3.
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f3: SDS–PAGE analysis of various fractions recovered during purification of α-crystallin-associated fractions. Lane 1: α-Crystallin fraction was recovered from a WS protein fraction of lenses that were 60- to 70-years-old. Lane 2: Concentrated α-crystallin fraction applied to the Agarose A1.5 m column. Lane 3: Proteinase fractions recovered following non-denaturing gel electrophoresis of the proteinase fraction released after Agarose A1.5 m gel chromatography. Lane 4: A proteinase-containing fraction recovered following size-exclusion HPLC using TSK G-4000 PWXL column of the fraction recovered in lane 3.

Mentions: The α-crystallin fraction, isolated from 60 to 70-year old human lenses, was used for the dissociation and purification of the enzyme by a three-step procedure (see Methods). Following sodium deoxycholate treatment and size-exclusion Agarose A1.5 m chromatography, the enzyme was released, and the proteinase-containing fractions showed a band of about 38 kDa and two bands of 22–25 kDa (Figure 1). The 38 kDa band was identified as keratin by MALDI-TOF mass spectrometric analysis. Upon further purification of the proteinase by the second step of nondenaturing gel-electrophoresis followed by the third step of size-exclusion HPLC, the 22–25 kDa species were progressively purified (Figure 3). Further, no additional species greater than 90 kDa were present in the purified fraction as determined by using 7% polyacrylamide gel during SDS–PAGE analysis (results 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)

SDS–PAGE analysis of various fractions recovered during purification of α-crystallin-associated fractions. Lane 1: α-Crystallin fraction was recovered from a WS protein fraction of lenses that were 60- to 70-years-old. Lane 2: Concentrated α-crystallin fraction applied to the Agarose A1.5 m column. Lane 3: Proteinase fractions recovered following non-denaturing gel electrophoresis of the proteinase fraction released after Agarose A1.5 m gel chromatography. Lane 4: A proteinase-containing fraction recovered following size-exclusion HPLC using TSK G-4000 PWXL column of the fraction recovered in lane 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: SDS–PAGE analysis of various fractions recovered during purification of α-crystallin-associated fractions. Lane 1: α-Crystallin fraction was recovered from a WS protein fraction of lenses that were 60- to 70-years-old. Lane 2: Concentrated α-crystallin fraction applied to the Agarose A1.5 m column. Lane 3: Proteinase fractions recovered following non-denaturing gel electrophoresis of the proteinase fraction released after Agarose A1.5 m gel chromatography. Lane 4: A proteinase-containing fraction recovered following size-exclusion HPLC using TSK G-4000 PWXL column of the fraction recovered in lane 3.
Mentions: The α-crystallin fraction, isolated from 60 to 70-year old human lenses, was used for the dissociation and purification of the enzyme by a three-step procedure (see Methods). Following sodium deoxycholate treatment and size-exclusion Agarose A1.5 m chromatography, the enzyme was released, and the proteinase-containing fractions showed a band of about 38 kDa and two bands of 22–25 kDa (Figure 1). The 38 kDa band was identified as keratin by MALDI-TOF mass spectrometric analysis. Upon further purification of the proteinase by the second step of nondenaturing gel-electrophoresis followed by the third step of size-exclusion HPLC, the 22–25 kDa species were progressively purified (Figure 3). Further, no additional species greater than 90 kDa were present in the purified fraction as determined by using 7% polyacrylamide gel during SDS–PAGE analysis (results 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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