scholarly journals Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin

1985 ◽  
Vol 225 (3) ◽  
pp. 835-835
Keyword(s):  
Dipeptidyl Peptidase ◽  
Egg White ◽  
Cysteine Proteinases

scholarly journals Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin

1984 ◽  
Vol 223 (1) ◽  
pp. 245-253 ◽  
Author(s):  
M J H Nicklin ◽  
A J Barrett
Keyword(s):  
Cathepsin B ◽  
Bond Cleavage ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dipeptidyl Peptidase ◽  
Egg White ◽  
Reversible Inhibitor ◽  
Cysteine Proteinases ◽  
Peptide Bond Cleavage ◽  
Specific Oxidation

The interactions between egg-white cystatin and the cysteine proteinases papain, human cathepsin B and bovine dipeptidyl peptidase I were studied. Cystatin was shown to be a competitive reversible inhibitor of cathepsin B (Ki 1.7 nM, k-1 about 2.3×10(-3) s-1). The inhibition of dipeptidyl peptidase I was shown to be reversible (Ki(app.) 0.22 nM, k-1 about 2.2×10(-3) s-1). Cystatin bound papain too tightly for Ki to be determined, but an upper limit of 5 pM was estimated. The association was a second-order process, with k+1 1.0×10(7) M-1×s-1. Papain was shown to form equimolar complexes with cystatin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of complexes formed between papain or cathepsin B and an excess of cystatin showed no peptide bond cleavage after incubation for 72 h. The reaction of the active-site thiol group of papain with 5,5′-dithiobis-(2-nitrobenzoic acid) at pH 8 and 2,2′-dithiobispyridine at pH 4 was blocked by complex-formation. Dipeptidyl peptidase I and papain were found to compete for binding to cystatin, contrary to a previous report. The two major isoelectric forms of cystatin were found to have similar specific inhibitory activities for papain, and similar affinities for papain, cathepsin B and dipeptidyl peptidase I. This, together with specific oxidation of the N-terminal serine residue with periodate, showed the N-terminal amino group of cystatin 1 to be unimportant for inhibition. General citraconylation of amino groups resulted in a large decrease in the affinity of cystatin for dipeptidyl peptidase I. It is concluded that the interaction of cystatin with cysteine proteinases has many characteristics similar to those of an inhibitor such as aprotinin with serine proteinases.

scholarly journals Cystatin-like cysteine proteinase inhibitors from human liver

1984 ◽  
Vol 218 (3) ◽  
pp. 939-946 ◽  
Author(s):  
G D J Green ◽  
A A Kembhavi ◽  
M E Davies ◽  
A J Barrett
Keyword(s):  
Human Liver ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Tight Binding ◽  
Cell Types ◽  
Dipeptidyl Peptidase ◽  
Tissue Homogenate ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Acetone Fractionation

Cysteine proteinase inhibitor (CPI) forms from human liver were purified from the tissue homogenate by alkaline denaturation of cysteine proteinases with which they are complexed, acetone fractionation, affinity chromatography on S-carboxymethyl-papain-Sepharose and chromatofocusing. The multiple forms of CPI were shown immunologically to be forms of two proteins, referred to as CPI-A (comprising the forms of relatively acidic pI) and CPI-B (comprising the more basic forms). CPI-A and CPI-B are similar in their Mr of about 12400, considerable stability to pH2, pH11 and 80 degrees C, and tight-binding inhibition of papain, several related cysteine proteinases and dipeptidyl peptidase I. Ki values were determined for papain, human cathepsins B, H and L, and dipeptidyl peptidase I. The affinity of CPI-A for cathepsin B was about 10-fold greater than that of CPI-B, whereas CBI-B showed about 100-fold stronger inhibition of dipeptidyl peptidase I. For all the cysteine proteinases the liver inhibitors were somewhat less tight binding than cystatin. The resemblance of both CPI-A and CPI-B in several respects to egg-white cystatin is discussed. CPI-A seems to correspond to the epithelial inhibitor described previously, and CPI-B to the inhibitor from other cell types [Järvinen & Rinne (1982) Biochim. Biophys. Acta 708, 210-217].

scholarly journals The structural origins of the unusual specificities observed in the isolation of chymopapain M and actinidin by covalent chromatography and the lack of inhibition of chymopapain M by cystatin

1995 ◽  
Vol 306 (1) ◽  
pp. 39-46 ◽  
Author(s):  
M P Thomas ◽  
C Verma ◽  
S M Boyd ◽  
K Brocklehurst
Keyword(s):  
Computer Modelling ◽  
Short Form ◽  
Egg White ◽  
Cysteine Proteinases ◽  
General Technique ◽  
Binding Characteristics ◽  
Modelling Techniques ◽  
Dynamics Simulations ◽  
Active Centres ◽  
Covalent Chromatography

1. The selectivity observed when the potentially general technique for the isolation of fully active forms of cysteine proteinases, covalent chromatography by thiol-disulphide interchange, is applied to chymopapain M and to actinidin was investigated by a combination of experimentation and computer modelling. Neither of these enzymes is able to react with the original Sepharose-GSH-2-dipyridyl disulphide gel, but fully active forms of both enzymes are obtained by using Sepharose-2-hydroxypropyl-2′-dipyridyl disulphide gel, which is both electrically neutral and sterically less demanding than the GSH gel. Electrostatic potential calculations, minimization and molecular-dynamics simulations provide explanations for the unusual, but different, specificities exhibited by actinidin and chymopapain M in the interactions of their active centres with ligands. 2. The unique behaviour of chymopapain M in exerting an almost absolute specificity for substrates with glycine at the P1 position and in resisting inhibition by cystatin was examined by the computer-modelling techniques. A new, modelled, structure of the complete chicken egg-white cystatin molecule based on the crystal structure of a short form of cystatin was deduced as a necessary prerequisite. The results suggest that electrostatic repulsion prevents reaction of actinidin with the GSH gel, whereas a steric ‘cap’ resulting from a unique arginine-65-glutamic acid-23 interaction in chymopapain M prevents reaction of the gel with this enzyme and accounts for the lack of its inhibition by cystatin and its specificity in catalysis. 3. Use of chymopapain M as a structural variant of papain demonstrates the validity of the predictions of Lowe and Yuthavong [Biochem. J. (1971) 124, 107-115] relating to the structural requirements and binding characteristics of the S1 subsite of papain.

scholarly journals Cystatin. Amino acid sequence and possible secondary structure

1984 ◽  
Vol 217 (3) ◽  
pp. 813-817 ◽  
Author(s):  
C Schwabe ◽  
A Anastasi ◽  
H Crow ◽  
J K McDonald ◽  
A J Barrett
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Amino Acid Sequence ◽  
Tight Binding ◽  
Alpha Helix ◽  
Egg White ◽  
Striking Similarity ◽  
Cysteine Proteinases ◽  
Amino Acid Residues ◽  
Beta Structure

The amino acid sequence of cystatin, the protein from chicken egg-white that is a tight-binding inhibitor of many cysteine proteinases, is reported. Cystatin is composed of 116 amino acid residues, and the Mr is calculated to be 13 143. No striking similarity to any other known sequence has been detected. The results of computer analysis of the sequence and c.d. spectrometry indicate that the secondary structure includes relatively little alpha-helix (about 20%) and that the remainder is mainly beta-structure.

scholarly journals Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum

1983 ◽  
Vol 211 (1) ◽  
pp. 129-138 ◽  
Author(s):  
A Anastasi ◽  
M A Brown ◽  
A A Kembhavi ◽  
M J H Nicklin ◽  
C A Sayers ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Cathepsin L ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Thiol Group ◽  
Egg White ◽  
Exchange Chromatography ◽  
Cysteine Proteinases ◽  
Total Recovery ◽  
Free Thiol Group

The protein from chicken egg white that inhibits cysteine proteinases, and has been named ‘cystatin’, was purified by ovomucin precipitation, affinity chromatography on carboxymethylpapain-Sepharose and chromatofocusing. The final purification step separated two major forms of the protein (pI 6.5 and 5.6), with a total recovery of about 20% from egg white. By use of affinity chromatography and immunodiffusion it was shown that the inhibitor is also present at low concentrations in the serum of male and female chickens. Tryptic peptide maps of the separated forms 1 and 2 of egg-white cystatin were closely similar, and each form had the N-terminal sequence Ser-Glx-Asx. The two forms showed complete immunological identity, and neither contained carbohydrate. Ki values for the inhibition of cysteine proteinases were as follows: papain (less than 1 × 10(-11)M), cathepsin B (8 × 10(-10)M), cathepsin H (about 2 × 10(-8)M) and cathepsin L (about 3 × 10(-12)M). Some other cysteine proteinases, and several non-cysteine proteinases, were found not to be significantly inhibited by cystatin. The inhibition of the exopeptidase dipeptidyl peptidase I by cystatin was confirmed and the Ki found to be 2 × 10(-10)M. Inhibitor complexes with active cysteine proteinases and the inactive derivatives formed by treatment with iodoacetate, E-64 [L-trans-epoxysuccinylleucylamido(4-guanidino)butane] and benzyloxycarbonylphenylalanylalanyldiazomethane were demonstrated by isoelectric focusing and cation-exchange chromatography. The complexes dissociated in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with or without reduction) with no sign of fragmentation of the inhibitor. Cystatin was found not to contain a free thiol group, and there was no indication that disulphide exchange plays any part in the mechanism of inhibition.

Protein Inhibitors of Cysteine Proteinases. III. Amino-Acid Sequence of Cystatin from Chicken Egg White

1983 ◽  
Vol 364 (2) ◽  
pp. 1487-1496 ◽  
Author(s):  
Vito TURK ◽  
Jože BRZIN ◽  
Mira LONGER ◽  
Anka RITONJA ◽  
Mihael EROPKIN ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Egg White ◽  
Cysteine Proteinases ◽  
Protein Inhibitors ◽  
Chicken Egg

The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases.

1988 ◽  
Vol 7 (8) ◽  
pp. 2593-2599 ◽  
Author(s):  
W. Bode ◽  
R. Engh ◽  
D. Musil ◽  
U. Thiele ◽  
R. Huber ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Egg White ◽  
Cysteine Proteinases ◽  
X Ray ◽  
Chicken Egg

Inactivation of hen egg-white lysozyme. The azide radical

1997 ◽  
Vol 94 ◽  
pp. 356-364 ◽  
Author(s):  
M Faraggi ◽  
E Bettelheim ◽  
M Weinstein
Keyword(s):  
Egg White ◽  
Hen Egg White Lysozyme ◽  
Egg White Lysozyme ◽  
Hen Egg White ◽  
Hen Egg
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