scholarly journals Inhibition of chicken calpain II by proteins of the cystatin superfamily and α2-macroglobulin

1987 ◽  
Vol 248 (2) ◽  
pp. 589-594 ◽  
Author(s):  
C Crawford
Keyword(s):  
Rate Constant ◽  
Cystatin C ◽  
Human Liver ◽  
Initial Rate ◽  
Peptide Substrate ◽  
Α2 Macroglobulin ◽  
Human Cystatin C ◽  
Calpain Ii ◽  
Human Cystatin ◽  
Chicken Cystatin

Inhibition of chicken calpain II by proteins of the cystatin superfamily and alpha 2-macroglobulin was investigated. Human liver cystatins A and B, human cystatin C, chicken cystatin and rat T-kininogen were found not to be inhibitory. Inhibition was, however, observed for bovine and rat kininogens, with Ki (inhibition constant) values of 0.8 nM and 30 nM respectively. alpha 2-Macroglobulin inhibits calpain with an initial rate constant of the order of 3 X 10(4) M-1.S-1. Calpain complexed with alpha 2-macroglobulin showed only limited reactivity towards azocasein, but reacted readily with the peptide substrate Suc-Leu-Tyr-4-methyl-7-coumarylamide and with L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64). The calpain in the complexes was at least partially protected from loss of activity due to autolysis. The calpain-alpha 2-macroglobulin complexes contained both the calpain subunits.

scholarly journals Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes

1994 ◽  
Vol 299 (1) ◽  
pp. 219-225 ◽  
Author(s):  
I Björk ◽  
E Pol ◽  
E Raub-Segall ◽  
M Abrahamson ◽  
A D Rowan ◽  
...  
Keyword(s):  
Rate Constant ◽  
Cystatin C ◽  
Rate Constants ◽  
Dissociation Rate ◽  
Dissociation Rate Constant ◽  
Association Rate Constant ◽  
Association Rate ◽  
Human Cystatin C ◽  
Human Cystatin ◽  
Chicken Cystatin

The importance of the N-terminal region of human cystatin C or chicken cystatin for the kinetics of interactions of the inhibitors with four cysteine proteinases was characterized. The association rate constants for the binding of recombinant human cystatin C to papain, ficin, actinidin and recombinant rat cathepsin B were 1.1 x 10(7), 7.0 x 10(6), 2.4 x 10(6) and 1.4 x 10(6) M-1.s-1, whereas the corresponding dissociation rate constants were 1.3 x 10(-7), 9.2 x 10(-6), 4.6 x 10(-2) and 3.5 x 10(-4) s-1. N-Terminal truncation of the first ten residues of the inhibitor negligibly affected the association rate constant with papain or ficin, but increased the dissociation rate constant approx. 3 x 10(4)- to 2 x 10(6)-fold. In contrast, such truncation decreased the association rate constant with cathepsin B approx. 60-fold, while minimally affecting the dissociation rate constant. With actinidin, the truncated cystatin C had both an approx. 15-fold lower association rate constant and an approx. 15-fold higher dissociation rate constant than the intact inhibitor. Similar results were obtained for intact and N-terminally truncated chicken cystatin. The decreased affinity of human cystatin C or chicken cystatin for cysteine proteinases after removal of the N-terminal region is thus due to either a decreased association rate constant or an increased dissociation rate constant, or both, depending on the enzyme. This behaviour indicates that the contribution of the N-terminal segment of the two inhibitors to the interaction mechanism varies with the target proteinase as a result of structural differences in the active-site region of the enzyme.

scholarly journals Interaction of recombinant human cystatin C with the cysteine proteinases papain and actinidin

1992 ◽  
Vol 281 (1) ◽  
pp. 49-55 ◽  
Author(s):  
P Lindahl ◽  
M Abrahamson ◽  
I Björk
Keyword(s):  
Cystatin C ◽  
Rate Constants ◽  
Proteinase Inhibitors ◽  
Equilibrium Constants ◽  
Fluorescence Emission ◽  
Cysteine Proteinases ◽  
Human Cystatin C ◽  
Kinetics Of ◽  
Human Cystatin ◽  
Chicken Cystatin

The interaction between recombinant human cystatin C and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The absorption, near-u.v.c.d. and fluorescence-emission difference spectra for the cystatin C-proteinase interactions were all found to be similar to the corresponding spectra for chicken cystatin. The kinetics of binding of cystatin C to the two enzymes were best described by a simple reversible one-step bimolecular mechanism, like the kinetics of the reaction of chicken cystatin with several cysteine proteinases. Moreover, the second-order association rate constants at 25 degrees C, pH 7.4 and I0.15, of 1.1 x 10(7) and 2.4 x 10(6) M-1.s-1 for the reactions of cystatin C with papain and actinidin respectively, were similar to the corresponding rate constants for the chicken inhibitor and close to the value expected for a diffusion-controlled rate. The dissociation equilibrium constants, approx. 11 fM and approx. 19 nM for the binding of cystatin C to papain and actinidin respectively, were also comparable with the dissociation constants for chicken cystatin. The affinity between cystatin C and several inactivated papains or actinidins decreased with increasing size of the inactivating group in a manner similar to that in earlier studies with the chicken inhibitor. Together, these results strongly indicate that the mechanisms of the reactions of cystatin C and chicken cystatin with cysteine proteinases are identical or highly similar, but differ from that of reactions between serine-proteinase inhibitors and their target enzymes. The model for the proteinase-inhibitor interaction, based on the X-ray structure of chicken cystatin, therefore should be largely applicable also to human cystatin C.

The disulphide bridges of human cystatin C (γ-trace) and chicken cystatin

FEBS Letters ◽  
1984 ◽  
Vol 170 (2) ◽  
pp. 370-374 ◽  
Author(s):  
Anders Grubb ◽  
Helge Löfberg ◽  
Alan J. Barrett
Keyword(s):  
Cystatin C ◽  
Human Cystatin C ◽  
Human Cystatin ◽  
Chicken Cystatin

scholarly journals Papain labelled with fluorescent thiol-specific reagents as a probe for characterization of interactions between cysteine proteinases and their protein inhibitors by competitive titrations

1991 ◽  
Vol 276 (2) ◽  
pp. 387-394 ◽  
Author(s):  
P Lindahl ◽  
E Raub-Segall ◽  
S T Olson ◽  
I Björk
Keyword(s):  
Cystatin C ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Equilibrium Constants ◽  
Sulphonic Acid ◽  
Cysteine Proteinases ◽  
Human Cystatin C ◽  
Human Cystatin ◽  
Chicken Cystatin

Papain was labelled by attachment of the fluorescent groups 2-(4′-acetamidoanilino)naphthalene-6-sulphonic acid (AANS) or N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid (AEDANS) to the active-site cysteine residue, with the aim of using the labelled papains as probes in competitive titrations of unlabelled cysteine proteinases with their inhibitors. The interaction between the labelled papains and cystatins was accompanied by an increase in fluorescence emission of up to 38-fold for AANS-papain and approximately 3.5-fold for AEDANS-papain. Fluorescence titrations gave dissociation equilibrium constants of 3.1 and 0.6 microM for the binding of chicken cystatin and recombinant human cystatin C respectively to AANS-papain and of 11.9 microM for the binding of chicken cystatin to AEDANS-papain. The kinetics of interaction of chicken cystatin with AANS-papain showed an unusual biphasic dependence of the observed pseudo-first-order rate constant on inhibitor concentration, consistent with the reaction occurring via both pathways of a general two-step binding mechanism. AANS-papain was selected as the most suitable probe for competitive titrations of unlabelled active or inactivated cysteine proteinases with inhibitors. This technique, which provides stoichiometries and dissociation constants for the interaction between unlabelled enzyme and inhibitor, allows monitoring of the interactions by a large fluorescent signal in a wavelength region where the interacting proteins do not contribute to the observed fluorescence. Such competitive titrations of active papain or actinidin with chicken cystatin or recombinant human cystatin C all gave inhibitor/enzyme stoichiometries of close to 1.0. A dissociation constant of 1.8 microM for the reaction of chicken cystatin with a papain derivative, S-[N-(3-carboxypropyl)succinimidyl]-papain, was also determined by the same technique. These results show the usefulness of the fluorescent papains for the characterization of interactions between cysteine-proteinase inhibitors and their target enzymes.

Affinity Purification and Elimination of Methionine Oxidation in Recombinant Human Cystatin C

1997 ◽  
Vol 11 (1) ◽  
pp. 111-118 ◽  
Author(s):  
Paul J. Berti ◽  
Irena Ekiel ◽  
Peter Lindahl ◽  
Magnus Abrahamson ◽  
Andrew C. Storer
Keyword(s):  
Cystatin C ◽  
Affinity Purification ◽  
Methionine Oxidation ◽  
Human Cystatin C ◽  
Human Cystatin

scholarly journals Application of amide hydrogen/deuterium exchange mass spectrometry for epitope mapping in human cystatin C

Amino Acids ◽  
2016 ◽  
Vol 48 (12) ◽  
pp. 2809-2820 ◽  
Author(s):  
Martyna Prądzińska ◽  
Izabela Behrendt ◽  
Juan Astorga-Wells ◽  
Aleksandr Manoilov ◽  
Roman A. Zubarev ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cystatin C ◽  
Epitope Mapping ◽  
Hydrogen Deuterium Exchange ◽  
Amide Hydrogen ◽  
Exchange Mass Spectrometry ◽  
Human Cystatin C ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Human Cystatin
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