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J Kos

Publications and source records attributed to J Kos.

At least 73 records · Page 4Linked to original sources
PubMed1992

Isolation and characterisation of chicken L- and H-kininogens and their interaction with chicken cysteine proteinases and papain.

We have isolated L- and H-kininogens from chicken egg white and plasma using affinity chromatography on CM-papain Sepharose, Con-A Sepharose, gel filtration and ion exchange chromatography, in pure form. In chicken plasma mostly HK was present, whereas in chicken egg white both LK and HK were identified. The determined Mr of LK and HK were 69000 and about 96000, respectively. They occurred in multiple forms with pI 4.3-5.2. The inhibitory activity of chicken HK was tested against cysteine proteinases cathepsins B and L, isolated from chicken liver, and papain. The obtained Ki values demonstrated that chicken H-kininogen is a strong inhibitor of chicken cathepsin L and papain, but much weaker inhibitor of chicken cathepsin B.

Amino Acid Sequence↗
PubMed1990

Refolding of chicken cystatin and human stefin A studied by fast protein liquid chromatography (FPLC).

Denaturant gradient gel-chromatography [Endo, S. et al. (1983) Anal. Biochem. 131, 108-120] has been used to follow renaturation of chicken cystatin and human stefin A. Slow gradient of GuHCl was applied to the size-exclusion Superose 12 column. The column was maintained at 5 degrees C by circulating water from a cryostate. When the denatured protein has been injected it started to refold on the column. By the simplified equation, according to [Hanai, R. et al. (1986) Biophys. Chem. 25, 27-36] approximate kinetics of folding was obtained. No intermediate species in folding could be detected from 6M to 2M GuHCl, only the close-to-denatured state and the close-to-native state.

Animals↗
PubMed1989

The cysteine proteinase inhibitor chicken cystatin is a phosphoprotein.

Peptide maps obtained by reversed-phase HPLC of tryptic digests of isoelectric form 1 (pI = 6.5) and 2 (pI = 5.6) of chicken egg white cystatin revealed that the difference was located only in a single peptide (residues Ser-74-Lys-91). Ser-80 of cystatin 2 was subsequently identified as being modified by phosphorylation. Moreover, alkaline phosphatase treatment of a mixture of native cystatin forms 1 and 2 was shown by ion-exchange chromatography to cause the disappearance of isoelectric form 2 with a concomitant increase in form 1. Thus, the existence of two isoelectric forms of chicken cystatin is due to the phosphorylated form 2 and non-phosphorylated form 1.

Amino Acid Sequence↗
PubMed1989

Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation.

A synthetic gene coding for the cysteine proteinase inhibitor (desSer1 Ile29 Leu89) chicken cystatin was cloned and expressed in E. coli. The gene was assembled from 12 oligonucleotides and inserted into vector pUC 8. Expression as fusion protein was performed in a temperature-inducible E. coli system. The expression product was synthesized as 20% of total E. coli protein. The fusion protein was purified, the chicken cystatin homologue was split off with CNBr and the N-terminal sequence confirmed up to position 37. The properties of the purified material correspond to those of natural chicken cystatin. The recombinant cystatin variant binds anti-chicken cystatin IgG, is inhibitorily active and displays Ki values with papain and with cathepsin B similar to those determined for natural chicken cystatin.

Amino Acid Sequence↗
PubMed1989

Mechanism of inhibition of papain by chicken egg white cystatin. Inhibition constants of N-terminally truncated forms and cyanogen bromide fragments of the inhibitor.

N-terminally truncated forms of chicken egg white cystatin and its cyanogen bromide fragments were isolated and assayed for inhibition of papain. Truncated forms beginning with Gly-9 and Ala-10 had a 5000-fold lower affinity for papain than the two isoelectric forms (pI = 6.5 and 5.6) of the full-length inhibitor (Ki = 6 pM and 7 pM) or a truncated form beginning with Leu-7 (Ki = 6 pM), indicating the outstanding importance of one or two residues preceding conserved Gly-9 for binding. A weak inhibition of papain (Ki = 900 nM) was exhibited by the intermediate cyanogen bromide fragment (residues 30-89) containing the chicken cystatin QLVSG variation of the QVVAG segment which is conserved in almost all members of the cystatin superfamily. The obtained affinity data provide independent evidence for the validity of the proposed docking model of a chicken cystatin-papain complex [(1988) EMBO J. 7, 2593-2599].

Amino Acid Sequence↗
PubMed1989

[The state of the articular cartilage and capsule of the shoulder joint in the adult population].

Based on the macroscopic investigation of 156 human shoulder joints from subjects aged 21-90 years taken from post-mortem material of the Anatomical Institute, the authors describe the course of the degenerative process and its localization on both contact surfaces. They describe also changes of the synovial and connective tissue layer of the articular capsule in relation of cartilaginous changes.

Adult↗
PubMed1988

Cathepsin D inactivates cysteine proteinase inhibitors, cystatins.

The formation of inactive complexes in excess molar amounts of human cathepsins H and L with their protein inhibitors human stefin A, human stefin B and chicken cystatin at pH 5.6 has been shown by measurement of enzyme activity coupled with reverse-phase HPLC not to involve covalent cleavage of the inhibitors. Inhibition must be the direct result of binding. On the contrary the interaction of cystatins with aspartic proteinase cathepsin D at pH 3.5 for 60 min followed by HPLC resulted in their inactivation accompanied by peptide bond cleavage at several sites, preferentially those involving hydrophobic amino acid residues. The released peptides do not inhibit papain and cathepsin L. These results explain reported elevated levels of cysteine proteinases and lead to the proposal that cathepsin D exerts an important function, through inactivation of cystatins, in the increased activities of cysteine proteinases in human diseases including muscular distrophy.

Amino Acid Sequence↗
PubMed1988

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

The crystal structure of chicken egg white cystatin has been solved by X-ray diffraction methods using the multiple isomorphous replacement technique. Its structure has been refined to a crystallographic R value of 0.19 using X-ray data between 6 and 2.0A. The molecule consists mainly of a straight five-turn alpha-helix, a five-stranded antiparallel beta-pleated sheet which is twisted and wrapped around the alpha-helix and an appending segment of partially alpha-helical geometry. The 'highly conserved' region from Gln53I to Gly57I implicated with binding to cysteine proteinases folds into a tight beta-hairpin loop which on opposite sides is flanked by the amino-terminal segment and by a second hairpin loop made up of the similarly conserved segment Pro103I - Trp104I. These loops and the amino-terminal Gly9I - Ala10I form a wedge-shaped 'edge' which is quite complementary to the 'active site cleft' of papain. Docking experiments suggest a unique model for the interaction of cystatin and papain: according to it both hairpin loops of cystatin make major binding interactions with the highly conserved residues Gly23, Gln19, Trp177 and Ala136 of papain in the neighbourhood of the reactive site Cys25; the amino-terminal segment Gly9I - Ala10I of bound cystatin is directed towards the substrate subsite S2, but in an inappropriate conformation and too far away to be attacked by the reactive site Cys25. As a consequence, the mechanism of the interaction between cysteine proteinases and their cystatin-like inhibitors seems to be fundamentally different from the 'standard mechanism' defined for serine proteinases and most of their protein inhibitors.

Animals↗
PubMed1988

A new purification procedure of human kidney cathepsin H, its properties and kinetic data.

A purification procedure of cathepsin H from human kidney is presented. It includes gel filtration, ion exchange chromatography, and covalent chromatography on thiol Sepharose as an essential step. Purified cathepsin H emerges in an isoelectric focusing gel at pH 6.1 and 6.3. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate shows a molecular mass of about 28 kDa. Less than 20% of the enzyme preparation can be separated into a heavy (24 kDa) and a light chain (4 kDa) after reduction and gel filtration on Sephacryl S-200. The partial amino-acid sequence of human cathepsin H shows its close similarity to rat cathepsin H. Inhibition constants (Ki) of cathepsins H and B with chicken cystatin, two forms of human stefin A, human stefin B, and two forms of human cystatin C are in the range of 10(-9) to 10(-11)M.

Amino Acid Sequence↗
PubMed1987

Collagenolytic cathepsins of rabbit spleen: a kinetic analysis of collagen degradation and inhibition by chicken cystatin.

We have investigated the steady state kinetics of the degradation of native fibrillar collagen at pH 3.4 by four collagenolytic cathepsins of rabbit spleen. For each enzyme, the dependence of initial velocity on collagen concentration was well described by the Michaelis-Menten mechanism. Km, expressed as the concentration of triple-helical chains, and kcat values were determined for cathepsins B, L, N and S. The ratio of Kcat to Km suggest that cathepsins L and N are far more effective at collagen solubilization than either cathepsins S or B. Ki values were determined for the inhibition of collagenolytic activity at pH 3.4 using cystatin, a naturally-occurring cysteine proteinase inhibitor. All four cysteine proteinases were inhibited by cystatin in this assay system, although it was found to be a tighter binding inhibitor of cathepsin L, than for cathepsins N and S (approximately 5-fold less), or cathepsin B (approximately 500-fold less).

Animals↗