title: Identification of new lens protease(s) using peptide substrates having in vivo cleavage sites.
abstract: Bovine lens extracts were incubated in pH 7.5 buffer with succinylated VSREEKPSSAPSS, SGVDAGHS, GKPTSAPSS and GKHNERQD, the peptides having age-dependent in vivo cleavage sites in bovine and human alpha A-crystallin. The reaction products were analyzed to identify the initial protease cleavage sites. The results showed that bovine lens extracts contain protease activity that can cleave. Thr-Ser, Ser-Ala, Ser-Ser bonds in test substrates which correspond to peptide bonds Ser168-Ser169, Ser169-Ala170, Ser172-Ser173 in bovine alpha A-crystallin and Thr-Ser, Ser169-Ala170, Ser172-Ser173 in human alpha A-crystallin. In addition, the same extracts were also capable of hydrolyzing Asp-Ala and Asn-Glu bonds corresponding to Asp151-Ala152 and Asn101-Glu102 in alpha A-crystallin from bovine and human lenses. The cleavage specificity of the newly discovered protease(s) suggests that the in vivo truncation of alpha A-crystallin reported earlier may be due to the action of proteases. The newly discovered lens protease(s) were resistant to inactivation by E-64 and DFP. However, prior treatment of the lens extracts with leupeptin and chymostatin resulted in partial loss of Asn-Glu hydrolytic activity. N-ethylmaleimide treatment completely abolished in the Asn-Glu hydrolyzing activity.
authors.affiliation: Mason Institute of Ophthalmology, University of Missouri, Columbia 65212, USA.
authors.forename: K K
authors.initials: KK
authors.lastname: Sharma
authors.forename: K
authors.initials: K
authors.lastname: Kester
authors.forename: N
authors.initials: N
authors.lastname: Elser
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013386:Succinates
chemical_list: D003459:Crystallins; D009842:Oligopeptides; D013386:Succinates; D010450:Endopeptidases