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.

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.

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 Characterization by spectroscopic, kinetic and equilibrium methods of the interaction between recombinant human cystatin A (stefin A) and cysteine proteinases

1995 ◽  
Vol 311 (1) ◽  
pp. 275-282 ◽  
Author(s):  
E Pol ◽  
S L Olsson ◽  
S Estrada ◽  
T W Prasthofer ◽  
I Björk
Keyword(s):  
Cystatin C ◽  
Active Site ◽  
Rate Constants ◽  
Equilibrium Constants ◽  
Association Rate ◽  
Dissociation Equilibrium ◽  
Cystatin A ◽  
Association Rate Constants ◽  
Human Cystatin ◽  
Chicken Cystatin

The near-UV spectroscopic changes induced by the binding of recombinant human cystatin A to papain were appreciably different from those induced by cystatin C, reflecting mainly interactions involving the single tryptophan of cystatin C, Trp-106. Cystatin A bound tightly and rapidly to papain and cathepsin L, with dissociation equilibrium constants of approximately 10(-11)-10(-13) M and association rate constants of 3 x 10(6)-5 x 10(6) M-1.s-1. These affinities are at least 50-100-fold higher than previously reported values. The kinetics of binding to papain were consistent with a simple reversible bimolecular reaction mechanism, indicating that cystatin A, like chicken cystatin and cystatin C, binds to papain with no appreciable conformational adaptation of either reacting protein. Cystatin A bound more weakly to actinidin and cathepsins B, C and H, with dissociation equilibrium constants of 10(-8)-10(-9) M. The weaker binding to cathepsin B was largely due to a considerably reduced association rate constant (approximately 4 x 10(4) M-1.s-1), consistent with the ‘occluding loop’ of cathepsin B markedly restricting the access of cystatin A to the active site. The lower affinities for actinidin and cathepsins C and H were due partly to lower association rate constants (2 x 10(5)-6 x 10(5) M-1.s-1) but primarily to higher dissociation rate constants. The mode of binding of cystatin A to inactivated papains indicated that there is appreciably less space around the active-site cysteine of papain in the complex with cystatin A than in the complexes with chicken cystatin and cystatin C. An N-terminally truncated form of cystatin A, lacking the first six residues, had considerably lower affinity for papain than the full-length inhibitor, consistent with an intact N-terminal region being of importance for proteinase binding.

scholarly journals Importance of the evolutionarily conserved glycine residue in the N-terminal region of human cystatin C (Gly-11) for cysteine endopeptidase inhibition

1993 ◽  
Vol 291 (1) ◽  
pp. 123-129 ◽  
Author(s):  
A Hall ◽  
H Dalbøge ◽  
A Grubb ◽  
M Abrahamson
Keyword(s):  
Cystatin C ◽  
Equilibrium Constants ◽  
Terminal Segment ◽  
Site Directed Mutagenesis ◽  
Side Chains ◽  
Wild Type ◽  
Human Cystatin C ◽  
Evolutionarily Conserved ◽  
Cysteine Endopeptidases ◽  
Human Cystatin

Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by site-directed mutagenesis and expression in Escherichia coli. The five variants were isolated and structurally verified. Their inhibitory properties were compared with those of wild-type recombinant cystatin C by determination of the equilibrium constants for dissociation (Ki) of their complexes with the cysteine endopeptidases papain and human cathepsin B and with the cysteine exopeptidase dipeptidyl peptidase I. The Ser-11 and Ala-11 cystatin C variants displayed Ki values for the two endopeptidases that were approx. 20-fold higher than those of wild-type cystatin C, while the corresponding values for the Trp-11. Arg-11 and Glu-11 variants were increased by a factor of about 2000. In contrast, the Ki values for the interactions of all five variants with the exopeptidase differed from that of wild-type cystatin C by a factor of less than 10. Wild-type cystatin C and the Ser-11, Ala-11 and Glu-11 variants were incubated with neutrophil elastase, which in all cases resulted in the rapid hydrolysis of a single peptide bond, between amino acid residues 10 and 11. The Ki values for the interactions with papain of these three N-terminal-decapeptide-lacking cystatin C variants were 20-50 nM, just one order of magnitude higher than the value for N-terminally truncated wild-type cystatin C, which in turn was similar to the corresponding values for the full-length Glu-11, Arg-11 and Trp-11 variants. These data indicate that the crucial feature of the conserved Gly residue in position 11 of wild-type cystatin C is that this residue, devoid of a side-chain, will allow the N-terminal segment of cystatin C to adopt a conformation suitable for interaction with the substrate-binding pockets of cysteine endopeptidases, resulting in high-affinity binding and efficient inhibition. The functional properties of the remaining part of the proteinase contact area, which is built from more C-terminal inhibitor segments, are not significantly affected even when amino acids with bulky or charged side-chains replace the Gly-11 residue of the N-terminal segment.

Sandwich enzyme immunoassay of cystatin C in serum with commercially available antibodies

1993 ◽  
Vol 39 (9) ◽  
pp. 1885-1890 ◽  
Author(s):  
M Pergande ◽  
K Jung
Keyword(s):  
Enzyme Immunoassay ◽  
Cystatin C ◽  
Isotopic Method ◽  
Human Cystatin C ◽  
Beta 2 Microglobulin ◽  
Sandwich Enzyme Immunoassay ◽  
Reduced Kidney Function ◽  
Kinetics Of ◽  
Human Cystatin ◽  
Better Than

Abstract We developed a sandwich enzyme immunoassay for determining cystatin C in serum by using commercially available antibodies. We optimized each assay step (e.g., concentrations of coating rabbit anti-human cystatin C antibodies and horseradish peroxidase-conjugated antibodies) and studied the binding kinetics of antigen and antibodies. The within-assay CV was < 5%, the between-assay CV was 8.8%, the detection limit was 0.9 microgram/L, and the assay can be performed within 2 h. Cystatin C concentrations in sera from men were significantly higher than in women (mean and SD: 2.14 +/- 0.31 vs 1.78 +/- 0.26 mg/L). We studied the cystatin C concentrations in sera of 31 outpatients with suspected kidney damages to characterize the behavior of this low-M(r) protein as a possible indicator for estimating the glomerular filtration rate. The correlation with the values obtained by a standard isotopic method involving 99mTc-diethylenetriaminopentaacetic acid was rs = -0.89. The diagnostic sensitivity of cystatin C was 88.2% of that of the standard isotope clearance method and better than those of the conventional serum indicators of reduced kidney function, beta 2-microglobulin (64.7%) and creatinine (52.9%).

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 Characterization by rapid-kinetic and equilibrium methods of the interaction between N-terminally truncated forms of chicken cystatin and the cysteine proteinases papain and actinidin

1992 ◽  
Vol 286 (1) ◽  
pp. 165-171 ◽  
Author(s):  
P Lindahl ◽  
M Nycander ◽  
K Ylinenjärvi ◽  
E Pol ◽  
I Björk
Keyword(s):  
Rate Constants ◽  
Fluorescence Emission ◽  
Terminal Segment ◽  
Dissociation Rate ◽  
Binding Energies ◽  
Terminal Region ◽  
Cysteine Proteinases ◽  
Difference Spectra ◽  
Chicken Cystatin ◽  
Leu 7

The interaction between five N-terminally truncated forms of chicken cystatin (starting at Leu-7, Leu-8, Gly-9, Ala-10 and Asp-15) and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The u.v. absorption, near-u.v. c.d. and fluorescence emission difference spectra for the interactions with papain were all similar to the corresponding spectra for intact cystatin. The second-order association rate constants at 25 degrees C, pH 7.4, I 0.15, for the binding of the truncated forms to papain varied about 2-fold, from 6 x 10(6) to 1.5 x 10(7) M-1.s-1, and were comparable to the value of 9.9 x 10(6) M-1.s-1 for intact cystatin. In contrast, the rate constants for the dissociation of the complexes with papain increased markedly with increasing extent of truncation, from 7.5 x 10(-6)s-1 for Leu7 cystatin (a truncated form of cystatin having Leu-7 as its N-terminal amino acid) to 1.6s-1 for Ala10-cystatin, whereas the dissociation rate constants for the latter form and Asp15-cystatin were similar. Consequently, the binding affinities between the truncated cystatins and papain decreased in an analogous manner, as was also shown for the interaction with actinidin by equilibrium measurements. Studies of the binding of the truncated cystatins to inactivated papains indicated that small substituents on the active-site cysteine of the enzyme can be accommodated in the complex without any loss of affinity when the N-terminal segment of the inhibitor is removed. Taken together, the results suggest that in the N-terminal region of chicken cystatin only residues preceding Ala-10 participate in the interaction with proteinases. Of these residues, Leu-7 and Leu-8 together account for about two-thirds of the unitary free energy of binding contributed by the N-terminal region, the relative importance of the two residues being dependent on the target proteinase. Both Gly-9 and residues N-terminal of Leu-7 further stabilize the interaction but contribute substantially smaller binding energies than do the two leucine residues.

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.

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