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1.
H Kataoka K Nabeshima N Komada M Koono 《Virchows Archiv. B, Cell pathology including molecular pathology》1989,57(3):157-165
Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human alpha 1-antitrypsin in double immunodiffusion. PI-1 corresponding to alpha 1-antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200-300 Kd) than that of PI-1, and did not cross-react with antisera against human alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, alpha 2-plasmin inhibitor, inter-alpha-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including alpha 1-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors. 相似文献
2.
Novel trypsin inhibitors from the white rot fungus Abortiporus biennis were isolated, partially purified, and char- acterized. The inhibitors were purified by heat treatment, anion-exchange chromatography,
and gel filtration. SDS-PAGE of the purified preparation demonstrated the presence of two proteins with molecular masses of
20 and 21.5 kDa. The A. biennis inhibitors were most active against trypsin, while chymotrypsin α, proteinase K, and Carlsberg subtilisin were inhibited
to a smaller extent. The inhibitors are acidic proteins with remarkably high heat stability.
Published in Russian in Biokhimiya, 2009, Vol. 74, No. 2, pp. 278–283. 相似文献
3.
Futoshi Aranishi 《Marine biotechnology (New York, N.Y.)》1999,1(1):81-88
A serine proteinase inhibitor, termed serpin62, was purified to homogeneity from carp serum with an increase in specific
inhibitory activity of 6.2-fold and a 3% recovery rate after separation from α1-antitrypsin. Specific inhibitory activity of serpin62 against bovine pancreatic trypsin was less than half of the specific
antitryptic activity of α1-antitrypsin. Under both reducing and nonreducing conditions, serpin62 was estimated to have a molecular weight (62,000) apparently
larger than that of α1-antitrypsin (55,000). They both consist of single polypeptide chains, but serpin62 differs from serine proteinase inhibitors
from muscles of carp and white croaker in molecular weight and structure. Antibody raised against serpin62 immunologically
crossreacted with serpin62 and had no crossreactivity with fish serum α1-antitrypsin and muscular analogues. The antibody was susceptible to both serpin62 and its derivatives, which were widely
distributed in carp tissues. Serpin62 is most likely distinct from other fish serine proteinase inhibitors expressing antitryptic
activity physicochemically and immunologically.
Received June 4, 1998; accepted September 10, 1998. 相似文献
4.
Frédéric Perraud Wilfried Dalemans Dalila Ali-Hadji Andrea Pavirani 《Cytotechnology》1992,9(1-3):69-75
We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins,
such as human α1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of
anonc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular
carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype
under specific culture conditions, being capable of producing biologically active and correctly processed human α1-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome
P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug
safety testing. 相似文献
5.
Isolation and primary structures of seven serine proteinase inhibitors from Cyclanthera pedata seeds
Jolanta Kowalska Agnieszka ZabłockaTadeusz Wilusz 《Biochimica et Biophysica Acta (BBA)/General Subjects》2006
Seven new trypsin inhibitors, CyPTI I–VII, were purified from ripe seeds of Cyclanthera pedata by affinity chromatography on immobilized chymotrypsin in the presence of 5 M NaCl followed by preparative native PAGE at pH 8.9. The CyPTIs (Cyclanthera pedata trypsin inhibitors) belong to a well-known squash inhibitor family. They contain 28–30 amino acids and have molecular weights from 3031 to 3367 Da. All the isolated inhibitors strongly inhibit bovine β-trypsin (Ka > 1011 M− 1) and, more weakly, bovine α-chymotrypsin (Ka ≈ 104–106 M− 1). In the presence of 3 M NaCl the association constants of CyPTIs with α-chymotrypsin increased a few hundred fold. Taking advantage of this phenomenon, a high concentration of NaCl was used to isolate the inhibitors by affinity chromatography on immobilized chymotrypsin. It was found that although one of them, CyPTI IV, had split the Asn25–Gly26 peptide bond, its inhibitory activity remained unchanged. The hydrolyzed bond is located downstream of the reactive site. Presumably, the inhibitor is a naturally occurring, double-chain protein arising during posttranslational modifications. 相似文献
6.
Al. V. Konarev I. N. Anisimova V. A. Gavrilova V. T. Rozhkova R. Fido A. S. Tatham P. R. Shewry 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(1):82-88
A highly sensitive gelatin overlay procedure was used to identify inhibitors of serine proteinases and of the cysteine proteinase
ficin in seeds and leaves of sunflower. One major and two minor groups of trypsin inhibitors were identified in seeds, the
former having a high pI (@10) and also inhibiting chymotrypsin. Three groups of trypsin/subtilisin inhibitors were also present
in seeds, together with three inhibitors of ficin. All groups showed polymorphism between lines of Helianthus annuus, while the trypsin and trypsin/subtilisin inhibitors also varied between wild species of Helianthus, with no apparent relationship to growth type (annual or perennial), genome constitution or ploidy level. Genetic analysis
showed that the major trypsin inhibitor and three groups of trypsin/subtilisin inhibitors are each controlled by single Mendelian
loci, with the three loci for trypsin/subtilisin inhibitors showing recombination values of 0.23–0.40. Purification by RP-HPLC
allowed the M
r
of two trypsin inhibitors to be determined by SDS-PAGE to be about 1,500 and 2,500, while the three trypsin/subtilisin inhibitors
varied in M
r
from about 1,500 to 6,000.
Received: 7 March 1999 / Accepted: 18 March 1999 相似文献
7.
Extracts ofAscophyllum nodosum, Fucus serratus, F. vesiculosus andPelvetia canaliculata contain inhibitors of α-amylase, lipase and trypsin. The inhibitors were isolated and identified by1H NMR spectroscopy as polyphenols which have apparent molecular weights in the range from 30 000 to 100 000 daltons, as determined
by ultra-filtration with Amicon membranes. These polyphenols account for the whole of the inhibitory activity in crude algal
extracts. The compounds inhibit α-amylase and trypsin in an apparently non-competitive manner, when preincubated with the
enzymes, and the inhibition is directly proportional to the concentration of the inhibitor. Starch protects α-amylase when
added to the enzyme together with the inhibitors. Under this condition the effectiveness of the inhibitors is reduced ten-fold. 相似文献
8.
Previous studies have shown that the domestic mites Dermatophagoides pteronyssinus and D. farinae contain allergens with serine protease activity. These proteolytic allergens include trypsin, chymotrypsin, elastase, kallikrein,
and C3/C5 convertase. However, it is not known whether the domestic mite Blomia tropicalis shares with other mite species the serine protease activities. The enzymatic activity present in extracts obtained from food-free
B. tropicalis was investigated using specific substrates and inhibitors. Based upon the concentration response and inhibition profiles,
and the digestion of specific substrates our data demonstrate that extracts from B. tropicalis exhibit several serine-protease-like activities. The enzyme activities detected in the B. tropicalis extracts are trypsin, elastase, chymotrypsin, kallikrein, C3/C5 convertase, and mast cell protease. Our results also demonstrate
that kallikrein and C3/C5 convertase-like activities were not significantly affected by the α1-antiprotease, a naturally occurring serine protease inhibitor which protects lung mucosa from the enzymatic action. These
data strongly suggest that the Echymyopodidae mite B. tropicalis shares at least five serine proteases with members of other mite families, the Glycyphagidae and Pyroglyphidae. In addition,
our data demonstrate the potential use of biochemical methods to detect serine proteases for evaluation of mite growth in vitro, or to detect environmental exposures to these enzymes.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
9.
J. Hejgaard S. E. Bjørn G. Nielsen 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1984,68(1-2):127-130
Summary Wheat-barley chromosome addition lines were compared by isoelectric focusing of protein extracts to identify chromosomes carrying loci for the major immunochemically distinct protease inhibitors of barley grains. Structural genes for the following inhibitors were localized: an inhibitor of both endogenous -amylase 2 and subtilisin (ASI) on chromosome 2, two chymotrypsin/subtilisin inhibitors (CI-1 and CI-2) on chromosome 5 (long arm) and the major trypsin inhibitor (TI-1) on chromosome 3. 相似文献
10.
Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L 总被引:2,自引:0,他引:2
A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M
r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K
a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 107 M–1 and 3.1 × 107 M–1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively. 相似文献
11.
Barbara Chan Panayotis Kalabalikis Nigel Klein Robert Heyderman Michael Levin 《Biotherapy》1996,9(4):221-228
A wide range of immunomodulating agents are now available which may be of benefit in reducing inflammatory cell activation
in meningococcal sepsis. In order to facilitate selection of candidate anti-inflammatory agents for clinical trials, we have
used an in vitro whole blood model to evaluate the effects on meningococcal induced neutrophil and monocyte activation, of
dexamethasone, prostacyclin, pentoxifylline and a human IgM anti-lipid A monoclonal antibody (HA-1A).
Known concentrations of heat and penicillin killed meningococci were added to whole blood and the time course of cellular
activation was determined. Using elastase-α
1-antitrypsin (elastase-α
1-AT) and TNFα production as markers of neutrophil and monocyte activation respectively, plasma levels of elastase-α
1-AT and TNFα were found to increase in a dose-dependant manner. Elastase-α
1-AT was detected early, with most release occurring between 15–30 min whereas TNFα was detected later, between 120–180 min.
Dexamethasone, prostacyclin and pentoxifylline caused a dose dependant inhibition of TNFα release but had no effect on elastase release. HA-1A had no effect on either TNFα or elastase release.
This model may be useful in determining the sequence of inflammatory cell activation and in selecting candidate anti-inflammatory
agents for evaluation in clinical trials. 相似文献
12.
A specific protein—an inhibitor of Colletotrichum lindemuthianum protease—was isolated from kidney bean seeds in a homogeneous form. The purification procedure included gel filtration, isoelectric focusing and affinity chromatography on trypsin-Sepharose column. The latter was introduced to separate the fungal protease inhibitor from closely related trypsin and chymotrypsin inhibitors present in kidney bean seeds. 相似文献
13.
Marta Grófová Erik Larsson Allan Bengtsson Jozef Bizik Bengt Westermark Jan Pontén 《In vitro cellular & developmental biology. Plant》1988,24(5):369-372
Summary The presence ofα
2-macroglobulin was detected with the avidin-biotin technique in more than 20-yr-old paraffin blocks from human sarcomas.α
2M was found mainly in the cytoplasm of the tumor cells, and almost all tumor cells were positive. This serum glycoprotein,
which is a major plasma proteinase inhibitor with a wide specificity, was also shown to be synthesized and secreted by all
three cell lines derived from primary sarcomas but was not detected in cultures of the autologous skin fibroblasts. For the
detection ofα
2M in situ and in vitro an antiserum to tumor-associatedα
2-macroglobulin was used.
Our work was supported by grant no. 55-B86-21XB, from the Swedish Cancer Society. 相似文献
14.
F. A. Carvalho J. P. Lopes de Almeida T. Freitas-Santos C. Saldanha 《The Journal of membrane biology》2009,228(2):89-97
Circulating acetylcholine, substrate of membrane acetylcholinesterase (AChE), is known to enhance the band 3 protein degree
of phosphorylation. The purpose of this study was to verify whether the band 3 phosphorylation status is associated with a
G protein and whether it is an influent factor on AChE enzyme activity. From blood samples of healthy donors, erythrocyte
suspensions were prepared and incubated with AChE substrate (acetylcholine) and inhibitor (velnacrine), along with protein
tyrosine kinase (PTK) and tyrosine phosphatase (PTP) inhibitors. AChE activity was determined by spectrophotometry and extract
samples were analyzed by western blotting using primary antibodies to different G protein subunits. Our results with phosphorylated
band 3 (PTP inhibitor) show an increase in erythrocyte AChE (p < 0.0001). A dephosphorylated band 3 state (PTK inhibitor) shows a significant decrease. We identified a potential linkage
of protein subunits Gαi1/2 and Gβ with band 3 protein. Gαi1/2 and Gβ may be linked to the band 3 C-terminal site. Gαi1/2 is associated with the band 3 N-terminal domain, except for the control and ACh aliquots. Gβ is associated with both phosphorylated and dephosphorylated band 3 in the presence of velnacrine. We conclude that an erythrocyte
G protein with subunits Gαi1/2 and Gβ is associated with band 3. AChE depends on the degree of band 3 phosphorylation and its association with Gαi1/2 and Gβ. 相似文献
15.
Protease inhibitory activity in jackfruit seed (Artocarpus integrifolia) could be separated into 5 fractions by chromatography on DEAE-cellulose at pH 7.6. A minor fraction (I) that did not bind
to the matrix, had antitryptic, antichymotryptic and antielastase activity in the ratio 24:1.9:1.0. Fraction II bound least
tightly to the ion exchanger eluting with 0.05 M NaCl and could be resolved into an elastase/chymotrypsin inhibitor and a
chymotrypsin/trypsin inhibitor by chromatography on either immobilized trypsin or phenyl Sepharose CL-4B. Fractions III and
IV eluted successively with 0.10 M NaCl and 0.15 M NaCl from DEAE-cellulose, inhibited elastase, chymotrypsin and trypsin
in the ratio 1.0: 0.53:0.55 and 1.0:8.9:9.8 respectively. Fraction V, most strongly bound to the matrix eluting with 0.3 M
NaCl and was a trypsin/chymotrypsin inhibitor accounting for 74% of total antitryptic activity. This inhibitor was purified
further. The inhibitor with a molecular weight of 26 kd was found to be a glycoprotein. Galactose, glucose, mannose, fucose,
xylose, glucosamine and uronic acid were identified as constitutent units of the inhibitor. Dansylation and electrophoresis
in the presence of mercaptoethanol indicated that the inhibitor is made up of more than one polypeptide chain. The inhibitor
combined with bovine trypsin and bovine α-chymotrypsin in a stoichiometric manner as indicated by gel chromatography. It had
very poor action on subtilisin BPN′, porcine elastase, pronase,Streptomyces caespitosus protease andAspergillus oryzae protease. It powerfully inhibited the caseinolytic activities of rabbit and horse pancreatic preparations and was least effective
on human and pig pancreatic extracts. Modification of amino groups, guanido groups and sulphydryl groups of the inhibitor
resulted in loss of inhibitory activity. Reduction of disulphide bridges, reduction with sodium borohydride and periodate
oxidation also decreased the inhibitory activity. 相似文献
16.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1994,1204(2):189-194
Giant taro (Alocasiamacrorrhiza) contains a protein which inhibits both trypsin and chymotrypsin. This trypsin/chymotrypsin inhibitor exists as a dimer of two identical monomers each with slight polymorphism and is an attractive candidate for conferring insect resistance in transgenic plants. The 184 amino-acid sequence (molecular mass of 19774 Da for the Met-24, Glu-50 form) has been determined and is compared with those of other Kunitz-type trypsin, chymotrypsin and subtilisin inhibitors. There appears to be greater ‘homology’ between the giant taro inhibitor and those inhibitors from other monocotyledons than inhibitors from dicotyledons. The P1 loop region is different from that of other Kunitz-type inhibitors and contains a sequence Leu-Ala-Phe-Phe-Pro at residues 56–60. This section of sequence differs only by a Leu/Ile replacement to a tight binding inhibitor of neutrophil elastase, Recently produced by genetic engineering. The most likely candidate for the P1 residue in the giant taro trypsin/chymotrypsin inhibitor is Leu-56. 相似文献
17.
The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit. 相似文献
18.
Nimi Osamu Norimoto Yu Nomi Ryosaku 《Bioscience, biotechnology, and biochemistry》2013,77(11):1819-1821
The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1:1, while that of chymotrypsin inhibitor with chymotrypsin was 1:2, judging from the titration patterns of their inhibitory activities.The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have Mr 19,242 and Mr 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45%) with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues. 相似文献
19.
Cell death in rice roots due to zinc (Zn) toxicity was investigated using inhibitors of signal molecules known to regulate
programmed cell death in plants. Zn (5.0– 25.0 mM) induced cell death in a dose- and time-dependent manner. Sodium benzoate,
a scavenger of reactive oxygen species (ROS), increased the cell viability under toxic Zn level (25.0 mM), suggesting a role
of ROS in Zn-induced cell death. The protective role of rotenone in cell death indicated the involvement of mitochondrial
electron transport chain in this Zn-induced ROS generation. Cantharidin and endothall, two serine/threonine phosphatase inhibitors,
and sodium orthovanadate (Na3VO4) and phenylarsine oxide (PAO), two protein tyrosine phosphatase inhibitors, blocked Zn-induced root cell death. Conversely,
K252-a, a serine/threonine kinase inhibitor, increased Zn-induced cell death. Furthermore, the phosphatidylinositol 3-Kinase
(PI-3K) inhibitors, LY 294002 and wortmannin inhibited Zn-induced root cell death. These results suggest that the ROS, protein
phosphatase and PI-3K may function in the Zn-induced cellular toxicity in rice roots. 相似文献
20.
Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases 总被引:7,自引:0,他引:7
G Pearce L Sy C Russell C A Ryan G M Hass 《Archives of biochemistry and biophysics》1982,213(2):456-462
Two polypeptides, isolated to electrophoretic homogeneity from Russet Burbank potato tubers, are powerful inhibitors of pancreatic serine proteinases. One of the inhibitors, called polypeptide trypsin inhibitor, PTI, has a molecular weight of 5100, and inhibits bovine trypsin. The inhibitor is devoid of methionine, histidine, and tryptophan and contains eight half-cystine residues as four disulfide bridges. The second inhibitor, polypeptide chymotrypsin inhibitor II, PCI-II, has a molecular weight of 5700 and powerfully inhibits chymotrypsin. This inhibitor is also devoid of methionine and tryptophan but it contains only six of half-cystines as three disulflde bonds. Both polypeptides strongly inhibit pancreatic elastase. In immunological double diffusion assays, polypeptide trypsin inhibitor and polypeptide chymotrypsin inhibitor II exhibit a high degree of immunological identity (a) with each other, (b) with a polypeptide chymotrypsin inhibitor (PCI-I, Mr 5400) previously isolated from potato tubers, and (c) with inhibitor II, a larger (monomer Mr ~ 12,000) inhibitor of both trypsin and chymotrypsin which has also been previously isolated from potato tubers. The four polypeptide proteinase inhibitors now isolated from Russet Burbank potato tubers cumulatively inhibit all five major intestinal digestive endo- and exoproteinases of animals. The inhibitors are thought to be antinutrients that are present as part of the natural chemical defense mechanisms of potato tubers against attacking pests. 相似文献