Cross-linking and proteolysis in Ca2(+)-treated lens homogenates.
It was previously shown (Lorand et al. (1985) Biochemistry 24, 1525) that treatment of lens homogenate with Ca2+ produces two sets of changes which are catalyzed by intrinsic enzymes of the lens and which can be readily seen by alterations in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of proteins. With the aid of differential inhibitors of the two reactions (e.g., dansylcadaverine and leupeptin) it was possible to distinguish the transglutaminase-dependent cross-linking of proteins from the proteolytic degradative phenomena. We have now shown that the proteins which are affected by the two processes can be compartmentalized differentially by centrifuging the lens homogenate after exposure to Ca2+. The dimeric and oligomeric beta-crystallin products of transglutaminase-mediated cross-linking are most clearly visible in the soluble supernatant, whereas the proteolytically susceptible proteins--possibly structural in nature, including vimentin--are predominantly present in the pellet. We have found a compound, 2-[3-(diallylamino)propionyl]benzothiophene, which, by virtue of acting as a noncompetitive inhibitor of transglutaminase as well as of calpains I and II, effectively blocked both the cross-linking seen in the supernatant and the proteolysis seen in the pellet fraction, though perhaps with somewhat different sensitivities.
Velasco
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PT
Department of Biochemistry, Molecular and Cell Biology, Northwestern University, Evanston, IL 60208-3500.
Murthy
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P
Goll
D E
DE
Lorand
L
L
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002154:Calpain / Q000037:antagonists & inhibitors*; D003432:Cross-Linking Reagents; D003459:Crystallins / Q000378:metabolism; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology / Q000378:metabolism*; D046911:Macromolecular Substances; D010447:Peptide Hydrolases / Q000378:metabolism*; D011817:Rabbits; D013876:Thiophenes / Q000494:pharmacology; D011503:Transglutaminases / Q000037:antagonists & inhibitors
D003432:Cross-Linking Reagents; D003459:Crystallins; D046911:Macromolecular Substances; D013876:Thiophenes; C051179:2-(3-(diallylamino)propionyl)benzothiophene; D011503:Transglutaminases; D010447:Peptide Hydrolases; D002154:Calpain; D002118:Calcium