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1.
Balaban NP Mardanova AM Sharipova MR Gabdrakhmanova LA Sokolova EA Rudenskaya GN Leshchinskaya IB 《Biochemistry. Biokhimii?a》2004,69(4):420-426
A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and kcat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth. 相似文献
2.
Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids 总被引:15,自引:0,他引:15
M Abrahamson A J Barrett G Salvesen A Grubb 《The Journal of biological chemistry》1986,261(24):11282-11289
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. 相似文献
3.
Purification and characterization of a low molecular mass cysteine proteinase inhibitor from human amniotic fluid 总被引:1,自引:0,他引:1
We have purified the human low molecular mass cysteine proteinase inhibitor in good yield from amniotic fluid, using ultrafiltration through 100-kDa and 1-kDa cut-off filters, chromatography on Ultrogel AcA 54, and affinity chromatography on alkylated papain-agarose. Approximately 1-4 mg/l of this inhibitor are present in amniotic fluid. The purified inhibitor had an apparent molecular mass of 10.5-12 kDa, as judged by its electrophoretic behavior. Amino acid analysis showed it to be rich in acidic and aliphatic residues and in cysteine. No carbohydrate side-chains could be demonstrated. The purified inhibitor inhibited papain, ficin, cathepsins B, C, and H, the cathepsin B-like enzyme from B16 melanoma cells, and a bovine chromaffin granule enkephalin-converting activity. No inhibition of Ca2-dependent neutral cysteine proteinase, serine- or metallo-proteinases was seen. Analysis of the purified inhibitor by isoelectric focusing revealed 7 major bands with pI values of 7.95, 7.0, 6.7, 6.55, 6.25, 5.5, and 5.2, all of which inhibited papain. 相似文献
4.
Purification and complex formation analysis of a cysteine proteinase inhibitor (cystatin) from seeds of Wisteria floribunda 总被引:1,自引:0,他引:1
Seeds of Wisteria floribunda contain several kinds of cysteine proteinase inhibitor (cystatin). We purified and characterized one of these inhibitors, named WCPI-3. The molecular weight of WCPI-3 was estimated to be 17,500 and 15,700 by gel filtration and SDS-PAGE, respectively. The isoelectric point was 5.7. WCPI-3 formed an equimolar complex with native papain and the dissociation constant was estimated to be 6.1 nM. Complex formation between WCPI-3 and Cys25-modified papain, such as S-carboxy-methylated or S-carbamoylmethylated papain, could not be observed by gel filtration or native PAGE analysis. A peptide fragment derived from WCPI-3 digested by Achromobacter proteinase (lysyl endopeptidase) had the amino acid sequence of VVAGVNYRFVLK. The VVAG sequence in this fragment corresponds to the conserved sequence QVVAG which is considered to be one of binding regions to cysteine proteinases. The amino acid sequence of the amino-terminal portion (34 residues) of WCPI-3 was highly homologous to that of oryzacystatin from rice seeds. 相似文献
5.
A cysteine proteinase inhibitor (designated as cystatin SA) was isolated from human whole saliva by procedures including chromatography on DE 32 and DEAE-Sepharose CL-6B. The amino acid sequence determined by conventional methods showed sequence homology of 90 and 87% as compared with the sequences of cystatin S and cystatin SN, respectively, both of which are salivary inhibitors characterized previously. The new inhibitor consisted of 117 residues and had a pI value of 4.3. Cystatin SA inhibited ficin and papain more strongly than cystatin S or cystatin SN did. It also exhibited inhibitory activity toward dipeptidyl peptidase I but the activity was much weaker than those toward ficin and papain. 相似文献
6.
Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN-III-ompA system 总被引:1,自引:0,他引:1
E A Auerswald G Genenger R Mentele S Lenzen I Assfalg-Machleidt L Mitschang H Oschkinat H Fritz 《European journal of biochemistry》1991,200(1):131-138
A synthetic gene coding for a chicken egg white cystatin variant was cloned and expressed using the pIN-III-ompA Escherichia coli expression system. After osmotic shock of the E. coli cells, the cysteine proteinase inhibitor was isolated from periplasm and purified by S-carboxymethylpapain affinity chromatography. The resulting inhibitory material was characterized by SDS/PAGE, reversed-phase HPLC, peptide mapping and amino acid sequencing. The recombinant variant chicken AEF-[S1----M, M29----I, M89----L]cystatin shows strong inhibitory activity and displays Ki values in the complex with papain, actinidin and cathepsin B similar to those found for natural chicken cystatin. The purified variant showed a native-chicken-cystatin-like conformational state, as determined by NMR spectroscopy, if the NMR data of 15N-labelled recombinant inhibitor were compared with those of the natural inhibitor. 相似文献
7.
L F Kress T Kurecki S K Chan M Laskowski 《The Journal of biological chemistry》1979,254(12):5317-5320
The inactive 50,000-dalton fragment of human plasma alpha1-proteinase inhibitor resulting from limited proteolysis of the inhibitor by Crotalus adamanteus proteinase II has been isolated and partially characterized. The amino acid composition of the inactivated inhibitor indicates the loss of a peptide fragment from the intact inhibitor. Both intact and inactivated inhibitor contain COOH-terminal lysine. However, the NH2 terminus of the intact inhibitor is Glx, whereas that of inactivated inhibitor is methionine. NH2-terminal analysis of the inactive inhibitor fragment revealed the following sequence: -Met-Phe-Leu-Glu-Ala-Ile-Pro-Met-Ser-Ile-Pro-Pro-Gln-Val-Lys-Phe-Asn. The data show that the venom proteinase has inactivated alpha1- proteinase inhibitor by cleavage of a single bond which differs from that reported for trypsin or papain. 相似文献
8.
Comparative data on the properties of four thiol proteinase inhibitors, and of four serine proteinase inhibitors (two subtilisin and two trypsin inhibitors) isolated from seeds of Vigna are presented. They were similar in their molecular weights (5000–15,000) and dissociation constants (10?8–10?9m). The range of isoelectric points of the thiol proteinase inhibitors was 6.5 to 10.6, and of the serine proteinase inhibitors was 5.0 to 5.9. The amino acid compositions of one papain isoinhibitor, one of subtilisin, and one of trypsin are presented. Papain inhibitor A1 and subtilisin inhibitor 2a were low in cystine. All of the inhibitors were stable upon heating to 80 °C for 5 min at low pH. The subtilisin inhibitor did not bind to catalytically inactive subtilisin derivatives, whereas the papain inhibitor was stoichiometrically bound to the Hg or thioacetamide derivatives of papain. Incubation of the subtilisin inhibitor with catalytic amounts of subtilisin led to the formation of a modified form with the same inhibitor activity as the native inhibitor but with a different electrophoretic mobility. There was no indication of a similar modification of the papain inhibitor by papain. Separate sites are present on the trypsin-chymotrypsin inhibitors for trypsin and chymotrypsin. The papain inhibitors have the same binding sites for papain and ficin. 相似文献
9.
Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains. 总被引:6,自引:1,他引:5
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The protein crystals found in potato (Solanum tuberosum L.) tuber cells consist of a single 85-kD polypeptide. This polypeptide is an inhibitor of papain and other cysteine proteinases and is capable of binding several proteinase molecules simultaneously (P. Rodis, J.E. Hoff [1984] Plant Physiol 74: 907-911). We have characterized this unusual inhibitor in more detail. Titrations of papain activity with the potato papain inhibitor showed that there are eight papain binding sites per inhibitor molecule. The inhibition constant (Ki) value for papain inhibition was 0.1 nM. Treatment of the inhibitor with trypsin resulted in fragmentation of the 85-kD polypeptide into a 32-kD polypeptide and five 10-kD polypeptides. The 32-kD and 10-kD fragments all retained the ability to potently inhibit papain (Ki values against papain were 0.5 and 0.7 nM, respectively) and the molar stoichiometries of papain binding were 2 to 3:1 and 1:1, respectively. Other nonspecific proteinases such as chymotrypsin, subtilisin Carlsberg, thermolysin, and proteinase K also cleaved the 85-kD inhibitor polypeptide into functional 22-kD and several 10-kD fragments. The fragments obtained by digestion of the potato papain inhibitor with trypsin were purified by reverse-phase high-performance liquid chromatography, and the N-terminal amino acid sequence was obtained for each fragment. Comparison of these sequences showed that the fragments shared a high degree of homology but were not identical. The sequences were homologous to the N termini of members of the cystatin superfamily of cysteine proteinase inhibitors. Therefore, the inhibitor appears to comprise eight tandem cystatin domains linked by preteolytically sensitive junctions. We have called the inhibitor potato multicystatin (PMC). By immunoblot analysis and measurement of papain inhibitory activity, PMC was found at high levels in potato leaves (up to 0.6 microgram/g fresh weight tissue), where it accumulated under conditions that induce the accumulation of other proteinase inhibitors linked to plant defense. PMC may have a similar defensive role, for example in protecting the plant from phytophagous insects that utilize cysteine proteinases for dietary protein digestion. 相似文献
10.
Rat tissue concentrations of branched-chain 2-oxo acid dehydrogenase complex. Re-evaluation by immunoassay and bioassay. 总被引:4,自引:2,他引:2
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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. 相似文献
11.
Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S 总被引:2,自引:0,他引:2
A new cysteine proteinase inhibitor, cystatin SN, was purified from human whole saliva by chromatography with DE32, Sephacryl S200, and CM-Sepharose CL6B. Cystatin SN is immunologically related to cystatin S and both inhibitors have a similar molecular mass of about 13 kDa. The new inhibitor, however, was clearly distinguished from cystatin S by its much higher pI value. These inhibitors showed similar inhibitory activity for ficin, but cystatin SN was a much better inhibitor for papain and dipeptidyl peptidase I. The amino acid sequence of cystatin SN deduced in the light of the known structure of cystatin S indicates that they have 10 different amino acid residues in the sequence comprising in total 113 residues. 相似文献
12.
A papain inhibitor or 22 kDa was isolated from human placenta and shown to be identical to residues Cys246-Leu373 of the third domain of human kininogen. This kininogen domain and recombinant human cystatin C were inactivated by peptide bond cleavages at hydrophobic amino acid residues due to the action of cathepsin D. These results further support the proposed role cathepsin D in the regulation of cysteine proteinase activity. 相似文献
13.
Yoza K Nakamura S Yaguchi M Haraguchi K Ohtsubo K 《Bioscience, biotechnology, and biochemistry》2002,66(10):2287-2291
A lambdaZAP II cDNA library was constructed from mRNA in immature seeds of the grass Job's tears. A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library. The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues. The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn. The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found. A remarkable characteristic of the peptide sequence of Job's-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice. The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The purified recombinant protein inhibited papain. 相似文献
14.
Synthesis of a new cell penetrating calpain inhibitor (calpeptin) 总被引:15,自引:0,他引:15
T Tsujinaka Y Kajiwara J Kambayashi M Sakon N Higuchi T Tanaka T Mori 《Biochemical and biophysical research communications》1988,153(3):1201-1208
N-terminal of Leu-norleucinal or Leu-methioninal was modified to obtain a cell penetrative peptide inhibitor against calpain. Benzyloxycarbonyl (Z) derivatives had less active against papain than phenylbutyryl derivatives and leupeptin. Z-Leu-nLeu-H (calpeptin) was more sensitive to calpain I than Z-Leu-Met-H and leupeptin. Calpeptin was most potent among synthesized inhibitors in terms of preventing the Ca2+-ionophore induced degradation of actin binding protein and P235 in intact platelets. After 30 min incubation with intact platelets, calpeptin completely abolished calpain activity in platelets but no effect was observed in case of leupeptin. Calpeptin also inhibited 20K phosphorylation in platelets stimulated by thrombin, ionomycin or collagen. Thus calpeptin was found to be a useful cell-penetrative calpain inhibitor. 相似文献
15.
Limitations of commonly used spectrophotometric assay methods for phosphoenolypyruvate carboxykinase activity in crude extracts of muscle.
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An extracellular thiol proteinase was produced by the growth of a thermophilic fungus, Humicola lanuginosa, on a medium containing 2% casein, and was purified to virtual homogeneity by affinity chromatography on organomercurial columns. The essential thiol group for activity was confirmed by the inhibition of the enzyme by p-chloromercuribenzoate and mercuric ions. The enzyme, purified 27-fold from the extracellular fluid, exhibited an Mr of 23700 on gel filtration and sedimentation equilibrium. The H. lanuginosa proteinase preferentially cleaves at the C-terminal end of hydrophobic amino acid residues. This proteinase differed from the plant enzyme papain in its interaction with three affinity matrices and its substrate specificity towards synthetic substrates. This enzyme represents a unique example of a thiol proteinase obtained from a fungal source. 相似文献
16.
The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L. 相似文献
17.
Comparison of tryptic peptides from the heavy and light subunits of calpain I and calpain II by high performance liquid chromatography 总被引:1,自引:0,他引:1
Two different forms of Ca2+-dependent cysteine proteinase, low-Ca2+-requiring calpain I and high-Ca2+-requiring calpain II, are known to be heterodimers, each composed of one heavy (called 80K) and one light (called 30K) subunit. The most probable identity of the 30K and the substantial difference between the 80K subunits of porcine calpains I and II were clearly demonstrated by comparing the tryptic peptide maps obtained upon running a high performance liquid chromatography which permitted parallel detection of tryptophan-containing peptides by fluorometry. Comparison of the amino acid compositions of the two 30K and 80K subunits also confirmed this conclusion. The same chromatographical analysis also revealed close structural similarity of the human calpain I 30K subunit, and even some similarity existing between the calpain I 80K subunits of human and porcine origins. 相似文献
18.
S Yamato K Tanaka T Murachi 《Biochemical and biophysical research communications》1983,115(2):715-721
A 34,000-dalton inhibitor of calpain (Ca2+-dependent cysteine proteinase) was found in the cytosol of anemic rat liver. When phenylhydrazine hydrochloride was continuously administered to rats, a 280,000-dalton calpain inhibitor that existed originally in the liver gradually disappeared within two weeks and, concomitantly, a 34,000-dalton inhibitor appeared. The purified 34,000-dalton inhibitor resembles 280,000-dalton inhibitor in that both are heat-stable proteins and do not inhibit papain and trypsin. Unlike the protomers of a 280,000-dalton inhibitor, 34,000-dalton inhibitor does not show any sign of self-association. 相似文献
19.
Strukelj B Lenarcic B Gruden K Pungercar J Rogelj B Turk V Bosch D Jongsma MA 《Biochemical and biophysical research communications》2000,269(3):732-736
A cDNA encoding a precursor of equistatin, a potent cysteine and aspartic proteinase inhibitor, was isolated from the sea anemone Actinia equina. The deduced amino acid sequence of a 199-amino-acid residue mature protein with 20 cysteine residues, forming three structurally similar thyroglobulin type-1 domains, is preceded by a typical eukaryotic signal peptide. The mature protein region and those coding for each of the domains were expressed in the periplasmic space of Escherichia coli, isolated, and characterized. The whole recombinant equistatin and its first domain, but not the second and third domains, inhibited the cysteine proteinase papain (K(i) 0.60 nM) comparably to natural equistatin. Preliminary results on inhibition of cathepsin D, supported by structural comparison, show that the second domain is likely to be involved in activity against aspartic proteinases. 相似文献
20.
Neutrophil chemotactic activity was found in the autodigest of calcium dependent cysteine proteinase (calpain) I purified from human erythrocytes, an active peptide was isolated, and its structure was determined. It was an N-acetyl nonapeptide with the sequence: N-acetyl Ser-Glu-Glu-Ile-Ile-Thr-Pro-Val-Tyr. This peptide was identical with the N-terminal amino acid sequence of the large subunit of calpain I deduced from cDNA sequence, except that the peptide was lacking a methionine residue and was acetylated at the N-terminus. A number of N-acetyl peptides with N-terminal amino acid sequences of large and small subunits of calpains I and II were synthesized and their chemotactic activity was estimated. In addition to the N-acetyl nonapeptide from calpain I large subunit, several peptides of different lengths from the small subunit showed dose-dependent migrations of neutrophils. They include N-acetyl tetra, hepta, octa, nona and larger size peptides. Further, it was also revealed that when calpain was incubated with high molecular weight (HMW) or low molecular weight (LMW) kininogen, kinin liberation occurred with simultaneous inhibition of calpains by kininogens. These data suggest that chemical mediators generated from the calpain-kininogen system may participate in migration and accumulation of neutrophils to the inflammatory locus. 相似文献