The effect of urea on the aggregate state and elastase inhibitor activity of the water-insoluble fraction from bovine and human lens.
Preparations of alpha-crystallin from bovine and human lens exhibited elastase inhibitor activity with a specific activity of 100-250 U mg-1 protein. A washed water-insoluble fraction from bovine, human and cataractous lens nuclei, when solubilized by sonication, gave specific activities of 910, 950 and 1270 U mg-1, respectively. Disaggregation of these water-insoluble fractions in 8.0 M urea, with subsequent reaggregation by urea removal, resulted in a decrease in inhibitor activity. Agarose A-5m gel filtration chromatography after the urea treatment resolved a residual high molecular weight (HMW) fraction and a peak which eluted at the position of water soluble alpha-crystallin. Assays showed that the urea-induced 'alpha-crystallin' peaks from all three preparations had specific activities, equivalent to native alpha-crystallin, whereas the HMW fractions retained their original high specific activity. We conclude that the increased elastase inhibitor activity of the water-insoluble fraction is a property of the aggregate state of the component alpha-crystallin molecules, which is lost upon reaggregation to an 800-kDa alpha-crystallin. Amino acid analysis of the bovine water-insoluble fraction suggested a content of 85-90% alpha-crystallin and 10-15% beta H-crystallin, which was confirmed by SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)
Ortwerth
B J
BJ
Mason Institute of Ophthalmology, Columbia, MO.
Sharma
K K
KK
Olesen
P R
PR
D000328:Adult; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002386:Cataract; D002417:Cattle; D003459:Crystallins / Q000032:analysis / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008875:Middle Aged; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D012995:Solubility; D013010:Sonication; D014508:Urea / Q000494:pharmacology*
D000596:Amino Acids; D003459:Crystallins; D014508:Urea; D010196:Pancreatic Elastase