Abstract
When an excess of human cystatin C or chicken cystatin was mixed with papain, an enzyme-inhibitor complex was formed immediately. The residual free cystatin was then progressively converted to a form with different electrophoretic mobility and chromatographic properties. The modified cystatins were isolated and sequenced, showing that there had been cleavage of a single peptide bond in each molecule: Gly11-Gly12 in cystatin C, and Gly9-Ala10 in chicken cystatin. The residues Gly11 (cystatin C) and Gly9 (chicken cystatin) are among only three residues conserved in all known sequences of inhibitory cystatins. The modified cystatins were at least 1000-fold weaker inhibitors of papain than the native cystatins. An 18-residue synthetic peptide corresponding to residues 4-21 of cystatin C did not inhibit papain but was cleaved at the same Gly-Gly bond as cystatin C. When iodoacetate or L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane was added to the mixtures of either cystatin with papain, modification of the excess cystatin was blocked. Papain-cystatin complexes were stable to prolonged incubation, even in the presence of excess papain. We conclude that the peptidyl bond of the conserved glycine residue in human cystatin C and chicken cystatin probably is part of a substrate-like inhibitory reactive site of these cysteine proteinase inhibitors of the cystatin superfamily and that this may be true also for other inhibitors of this superfamily. We also propose that human cystatin C and chicken cystatin, and probably other cystatins as well, inhibit cysteine proteinases by the simultaneous interactions with such proteinases of the inhibitory reactive sites and other, so far not identified, areas of the cystatins. The cleavage of the inhibitory reactive site glycyl bond in mixtures of papain with excess quantities of cystatins is apparently due to the activity of a small percentage of atypical cysteine proteinase molecules in the papain preparation that form only very loose complexes with cystatins under the conditions employed and degrade the free cystatin molecules.
Highlights
When iodoac- and the kininogens (Type 3 cystatins), which contain three etateor ~-3-carboxy-trans-2,3-epoxypropionyl-leud-ivergent copies of the Type 2 sequence but with even more cylamido-(4-guanidino)butanewas added to the mix- disulfide loops [1].The cystatins used in the present study, tures of either cystatin with papain, modification of chicken cystatin and human cystatCin, are Type 2 cystatins, the excess cystatin wabslocked
Plexes were stable to prolonged incubation, even in1)t.he presence of excess papain.We conclude that thepepti- The general characteristics of the tight-binding, reversible dyl bond of the conserved glycine residue in human inhibition of cysteine proteinases by cystatins are similar to cystatin C and chicken cystatin probably is part of a those of the inhibitionof serine proteinasesby such inhibitors substrate-like inhibitory reactive sitoef these cysteine as soybean trypsin inhibitor or aprotinin [1,3], but it is not proteinase inhibitors of the cystatin superfamily and known to what extent the mechanismsof interaction are the that this may be true for other inhibitorosf this superfamily
We propose that human cystatin C
Summary
When equimolar amounts of cystatin C and papain were mixed in 100 mM phosphate buffer, pH 6.5, with 1mM dithiothreitol and 2 mM EDTA and incubated a t 22 "C for various periods up to 6 h, agarose gel electrophoresis showed the rapid formation of a stable 1:l complex with an Methods-Concentrations of proteins and peptides were deter- electrophoretic mobility intermediate between those of free mined by amino acid analysis and, for papain and chicken cystatin, by measuring Am
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
