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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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Proteinase activity in four fractions (WS proteins, α-crystallin, βH-crystallin, and membrane fractions) following treatment with sodium deoxycholate and size-exclusion Agarose A1.5 m chromatography. WS proteins (A),   α-crystallin fraction (B), βH-crystallin fraction (C), and membrane fraction (D). Each fraction was first isolated from a homogenate of six pooled lenses of donors 30–40 years old by Agarose A1.5 m chromatography. Next, each fraction was treated with the detergent and fractionated by the size-exclusion chromatography, and the column fractions were examined for their absorbance at 280 nm (red line) and proteinase activity (green line) with BAPNA as a substrate. Note that the proteinase activity eluted in the identical fractions during chromatography from each of the four fractions.
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f8: Proteinase activity in four fractions (WS proteins, α-crystallin, βH-crystallin, and membrane fractions) following treatment with sodium deoxycholate and size-exclusion Agarose A1.5 m chromatography. WS proteins (A), α-crystallin fraction (B), βH-crystallin fraction (C), and membrane fraction (D). Each fraction was first isolated from a homogenate of six pooled lenses of donors 30–40 years old by Agarose A1.5 m chromatography. Next, each fraction was treated with the detergent and fractionated by the size-exclusion chromatography, and the column fractions were examined for their absorbance at 280 nm (red line) and proteinase activity (green line) with BAPNA as a substrate. Note that the proteinase activity eluted in the identical fractions during chromatography from each of the four fractions.

Mentions: Following size-exclusion Agarose A1.5 m chromatograph of either the WS protein, α-crystallin, or βH-crystallin fractions, very little proteinase activity was observed in the column fractions with either the colorimetric or fluorometric substrates. However, after treatment with sodium deoxycholate and fractionation by a size-exclusion Agarose A1.5 m column, each fraction showed enzyme activity. The enzyme from each of the four fractions eluted in fractions 35-50 (in the βH-crystallin region), suggesting their identical molecular weights (Figure 8A-D).


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

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

Proteinase activity in four fractions (WS proteins, α-crystallin, βH-crystallin, and membrane fractions) following treatment with sodium deoxycholate and size-exclusion Agarose A1.5 m chromatography. WS proteins (A),   α-crystallin fraction (B), βH-crystallin fraction (C), and membrane fraction (D). Each fraction was first isolated from a homogenate of six pooled lenses of donors 30–40 years old by Agarose A1.5 m chromatography. Next, each fraction was treated with the detergent and fractionated by the size-exclusion chromatography, and the column fractions were examined for their absorbance at 280 nm (red line) and proteinase activity (green line) with BAPNA as a substrate. Note that the proteinase activity eluted in the identical fractions during chromatography from each of the four fractions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Proteinase activity in four fractions (WS proteins, α-crystallin, βH-crystallin, and membrane fractions) following treatment with sodium deoxycholate and size-exclusion Agarose A1.5 m chromatography. WS proteins (A), α-crystallin fraction (B), βH-crystallin fraction (C), and membrane fraction (D). Each fraction was first isolated from a homogenate of six pooled lenses of donors 30–40 years old by Agarose A1.5 m chromatography. Next, each fraction was treated with the detergent and fractionated by the size-exclusion chromatography, and the column fractions were examined for their absorbance at 280 nm (red line) and proteinase activity (green line) with BAPNA as a substrate. Note that the proteinase activity eluted in the identical fractions during chromatography from each of the four fractions.
Mentions: Following size-exclusion Agarose A1.5 m chromatograph of either the WS protein, α-crystallin, or βH-crystallin fractions, very little proteinase activity was observed in the column fractions with either the colorimetric or fluorometric substrates. However, after treatment with sodium deoxycholate and fractionation by a size-exclusion Agarose A1.5 m column, each fraction showed enzyme activity. The enzyme from each of the four fractions eluted in fractions 35-50 (in the βH-crystallin region), suggesting their identical molecular weights (Figure 8A-D).

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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