| 1. |
- Abrahamson, Magnus, et al.
(författare)
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Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin
- 1987
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 262:20, s. 9688-9694
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
- Abrahamson, Magnus, et al.
(författare)
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Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids
- 1986
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 261:24, s. 11282-11289
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Tidskriftsartikel (refereegranskat)abstract
- Six cysteine proteinase inhibitors were isolated from human urine by affinity chromatography on insolubilized carboxymethylpapain followed by ion-exchange chromatography and immunosorption. Physicochemical and immunochemical measurements identified one as cystatin A, one as cystatin B, one as cystatin C, one as cystatin S, and one as low molecular weight kininogen. The sixth inhibitor displayed immunochemical cross-reactivity with salivary cystatin S but had a different pI (6.85 versus 4.68) and a different (blocked) N-terminal amino acid. This inhibitor was tentatively designated cystatin SU. The isolated inhibitors accounted for nearly all of the cysteine proteinase inhibitory activity of the urinary pool used as starting material. The enzyme inhibitory properties of the inhibitors were investigated by measuring inhibition and rate constants for their interactions with papain and human cathepsin B. Antisera raised against the inhibitors were used in immunochemical determinations of their concentrations in several biological fluids. The combined enzyme kinetic and concentration data showed that several of the inhibitors have the capacity to play physiologically important roles as cysteine proteinase inhibitors in many biological fluids. Cystatin C had the highest molar concentration of the inhibitors in seminal plasma, cerebrospinal fluid, and milk; cystatin S in saliva and tears; and kininogen in blood plasma, synovial fluid, and amniotic fluid.
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| 3. |
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| 4. |
- Buttle, David, et al.
(författare)
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The biochemistry of the action of chymopapain in relief of sciatica
- 1986
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Ingår i: Spine. - 0362-2436. ; 11:7, s. 688-694
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Tidskriftsartikel (refereegranskat)abstract
- A study has been made of the mechanism of action of intradiscal injections of preparations of chymopapain in the treatment of sciatica. Such preparations were found to contain at least four distinct proteins, but enzymatically active chymopapain was the component mainly responsible for releasing glycosaminoglycan from cartilaginous tissue. Previous suggestions that an electrostatic interaction between chymopapain and glycosaminoglycan is important to the action of injected enzyme were not supported by the finding that both positively and negatively charged forms of chymopapain efficiently released glycosaminoglycan from cartilaginous tissue. In contrast, cysteine alone did not cause release of glycosaminoglycan. Chymopapain was found to be inhibited efficiently by the protein inhibitors, cystatin C and low molecular weight kininogen in vitro, and the possible relevance of this finding to the efficacy and safety of chemonucleolysis is discussed.
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| 5. |
- Salvesen, Guy, et al.
(författare)
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Human low-Mr kininogen contains three copies of a cystatin sequence that are divergent in structure and in inhibitory activity for cysteine proteinases.
- 1986
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Ingår i: Biochemical Journal. - 0264-6021. ; 234:2, s. 429-434
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Tidskriftsartikel (refereegranskat)abstract
- We point out that human low-Mr kininogen contains three cystatin-like sequences, rather than two, as had previously been thought. The protein was purified by affinity chromatography on carboxymethyl-papain-Sepharose, and subjected to limited proteolysis by trypsin and chymotrypsin. Fragments were isolated, and three corresponding to the individual cystatin-like domains were identified. By comparison with the known amino acid sequence of the protein they were numbered 1 to 3 from the N-terminus. Domain 1 was not found to have any inhibitory activity for cysteine proteinases, which is consistent with the absence of residues that are highly conserved in inhibitors of the cystatin superfamily, and have previously been suggested to be essential for activity. Domain 2 was a good inhibitor of chicken calpain, and also papain and cathepsin L. Domain 3 showed negligible inhibition of calpain, but inhibited papain and cathepsin L strongly. The probable arrangement of disulphide bonds in the heavy chain of low-Mr kininogen is deduced from the homology with the cystatins and other evidence contained in the present paper.
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