Alpha Crystallin Fraction from Human Lenses Contains a Proteinase

Abstract

Abstract: : Purpose:The purpose of the study was to characterize a proteinase associated with the α-crystallin fraction of human lenses. Method: The α-crystallin fraction was isolated from human lenses of different ages by a size-exclusion column and the proteinase activity determined by either a colorimetric or a fluorometric method using an Arg substrate. The enzyme activity and the proteolysis of the crystallin were determined prior to and after treatments with neutral, anionic or cationic detergents. The proteinase was also purified by a four-step procedure, which included size-exclusion chromatography, preparative non-denaturing PAGE, preparative IFE and ion-exchange chromatography. The molecular properties of the purified proteinase were characterized. Results: No proteinase activity was observed in the α-crystallin fraction until the fraction was treated with a detergent, which also resulted in the proteolysis of the crystallin. Among various detergents, sodium deoxycholate provided an optimal release of a 38 kDa enzyme species from the α-crystallin fraction during size-exclusion chromatography. The four-step procedure provides over 1090-fold purification with an increase in the specific acitivity of about 500X. The enzyme was found to be a serine-type, and was labeled with [₃H]-diisopropylfluoro phosphate (DFP). Further, the proteinase showed inhibition by α-crystallin but only by a crystallin preparation that was first denatured with urea and renatured on urea removal. This step inactivated the α-crystallin-associated proteinase, which interfered during the inhibitor assay. The proteinase activity was reduced by 50% at the α-crystallin concentration of 500 µg/ml at the ratio 1:6 (proteinase:alpha). Conclusion: A proteinase that exists in an inactive state in the α-crystallin fraction could be released by detergent treatment. The proteinase may exist in an inactive state in vivo with α-crystallin because of its inhibition by the crystallin.

Keywords: 378 crystallins • 530 proteolysis • 527 protein structure/function