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121.
Two serine proteinases from the midgut of Helicoverpa armigera have been partially purified and characterized. One proteinase, HGP-1, was capable of hydrolyzing a synthetic substrate of elastase and was inhibited by elastatinal. The second proteinase, HGP-2, was inhibited by a trypsin inhibitor. Molecular weights of HGP-1 and HGP-2 were approximately 26.0 and 29.0kDa, respectively. Both the proteinases exhibited alkaline pH optima in the range of 10-11. Furthermore, interaction of HGP-1 and HGP-2 with proteinase inhibitors (PIs) from host and non-host plants was studied. HGP-1 was not only insensitive to a PI from chickpea (host) but was also able to degrade it. The same PI from chickpea was able to inhibit over 50% activity of HGP-2. On the contrary, PIs from potato (non-host) showed strong inhibition of both, HGP-1 and HGP-2 and also demonstrated protection of chickpea seed proteins from digestion by both the HGPs. These results could provide important clues in designing strategies for sustainable use of plant PIs in developing insect-tolerant transgenic plants. 相似文献
122.
Proteasomes are tubular complexes with proteolytic activities on their lumenal surfaces so that large substrates should be sterically hindered from reaching the catalytic sites. Here we examine effects of substrate size on rates of cleavage by 20S proteasomes of Methanosarcina thermophila. Synthetic chromogenic substrates of variable size were prepared by linking a constant substrate group (Ala-Ala-Phe-p-nitroanilide) to a linear polymer (methoxypolyethylene glycol) with variable chain length. The smallest macromolecular substrates were cleaved more efficiently than free tripeptide substrate, and cleavage of macromolecular substrates was saturable, whereas cleavage of free tripeptide substrate was not, indicating mechanistic differences between the cleavage of large and small substrates. Rates of macromolecular substrate cleavage decreased progressively up to 10-fold as the size of the polymeric component of substrates increased. Macromolecular synthetic substrates appear to be better models of proteasome action on natural protein substrates and demonstrate substrate size selectivity of proteasomes. 相似文献
123.
The mammalian aspartic proteinases procathepsin D and pepsinogen form insoluble inclusion bodies when expressed in bacteria. They become soluble but nonnative when synthesized as fusions to the carboxy terminus of E. coli maltose-binding protein (MBP). Since these nonnative states of the two aspartic proteinases showed no tendency to form insoluble aggregates, their biophysical properties were analyzed. The MBP portions were properly folded as shown by binding to amylose, but the aspartic proteinase moieties failed to bind pepstatin and lacked enzymatic activity, indicating that they were not correctly folded. When treated with proteinase K, only the MBP portion of the fusions was resistant to proteolysis. The fusion between MBP and cathepsin D had increased hydrophobic surface exposure compared to the two unfused partners, as determined by bis-ANS binding. Ultracentrifugal sedimentation analysis of MBP–procathepsin D and MBP–pepsinogen revealed species with very large and heterogeneous sedimentation values. Refolding of the fusions from 8 M urea generated proteins no larger than dimers. Refolded MBP–pepsinogen was proteolytically active, while only a few percent of renatured MBP–procathepsin D was obtained. The results suggest that MBP–aspartic proteinase fusions can provide a source of soluble but nonnative folding states of the mammalian polypeptides in the absence of aggregation. 相似文献
124.
Human plasma fibronectin contains two latent aspartic proteinases, FN-gelatinase and FN-lamininase. Both enzymes can be generated and activated in the presence of Ca2+ from the purified cathepsin D-produced 190-kDa fibronectin fragment. We investigated the proteolytic activity and cleavage specificity of both enzymes in a range of pH from 3.5 to 9.0 using the B chain of oxidized bovine insulin and chromogenic peptides as substrates. The inhibition of the enzymes by several natural inhibitors from human plasma was also tested. The specificities of FN-gelatinase and FN-lamininase are similar to other major acidic proteinases, including pepsin, renin, cathepsin D, and HIV-proteinases. Both enzymes mainly hydrolyze three peptide bonds in the oxidized insulin B chain, namely Glu–Ala (residues 13–14), Tyr–Leu (residues 16–17), and Phe–Phe (residues 24–25). For the peptide substrates H-Pro-Thr-Glu-Phe-p-nitro-Phe-Arg-Leu-OH and H-Phe-Gly-His-p-nitro-Phe-Phe-Val-Leu-OMe that were cleaved the respective values of k
cat/K
M were 105.1 and 11.8 mM–1 sec–1 for cleavage by FN-gelatinase, and 123.2 and 15.5 mM–1 sec–1 for cleavage by FN-lamininase. The maximal activities of both enzymes were observed in a range between pH 5.6 and 6.3 and they became inactivated at a pH value above 8.4. Both FN-gelatinase and FN-lamininase were efficiently inhibited by 2-macroglobulin. 相似文献
125.
A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the fibro-inflammatory microenvironment, consisting of activated pancreatic stellate cells, extracellular matrix proteins, and a variety of inflammatory cells, such as T cells, macrophages, or neutrophils. Tumor-infiltrating immune cells, which are found in nearly all cancers, including PDAC, often fail to eliminate the tumor, but conversely can promote its progression by altering the tumor microenvironment. Pancreatic cancer cells are able to attract polymorphonuclear neutrophils (PMN) via tumor secreted chemokines and in human PDAC, PMN infiltrates can be observed in the vicinity of tumor cells and in the desmoplastic tumor stroma, which correlate with undifferentiated tumor growth and poor prognosis. The behavior of tumor-infiltrating neutrophils in the tumor micromilieu is not yet understood at a mechanistic level. It has been shown that PMN have the potential to kill tumor cells, either directly or by antibody-dependent cell-mediated cytotoxicity, but on the other side various adverse effects of PMN, such as promotion of aggressive tumor growth with epithelial-to-mesenchymal transition and increased metastatic potential, have been described. Recent therapeutic approaches for PDAC focus not only the tumor cell itself, but also elements of the tumor microenvironment. Therefore, the role of PMN and their derived products (e.g. cytokines, proteases) as a new vein for a therapeutic target should be critically evaluated in this context. This review summarizes the current understanding of the interplay between proteases of tumor-infiltrating neutrophils and pancreatic tumor cells and elements of the desmoplastic stroma. 相似文献
126.
Summary Two series of intramolecularly quenched fluorogenic oligopeptide substrates with the general sequences DABCYL-Lys-Phe-Gly-Gly-Ala-Xxx-EDANS and Abz-Lys-Phe-Gly-Gly-Ala-Xxx-Tyr(m-NO2)-NH2 have been used to explore the effect of P3 substituents and donor/acceptor pairs on the kinetic parameters for papain-catalyzed hydrolysis. The steady-state constants are moderately affected by amino acid or fluorophore/quencher replacement. No correlation between the hydrophobicity of the P3 substituent and the kinetic parameters was found.Abbreviations Abz
anthranilic acid or anthranilamide
- Boc
tert-butyloxycarbonyl
- BOP
benzotriazolyloxy tris-(dimethylamino)-phosphonium hexafluorophosphate
- DABCYL
4-(4-dimethylaminophenylazo)benzoic acid
- DCM
dichloro-methane
- DIEA
diisopropylethylamine
- DIPCDI
diisopropylcarbodiimide
- DMF
N,N-dimethylformamide
- DMSO
dimethylsulfoxide
- E-64
trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane
- EDCI
1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide hydrochloride
- EDTA
ethylenediaminetetraacetic acid
- EDANS
5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid
- FABMS
fast atom bombardment mass spectrometry
- Fmoc
9-fluorenylmethyloxycarbonyl
- HOBt
1-hydroxybenzotriazole
- HPLC
high pressure liquid chromatography
- MBHA
4-methylbenzhydrylamine (resin)
- MCA
methylcoumarylamide
- Nle
norleucine
- PAL
tris(alkoxy)benzylamide linker
- Su
succinimide
- TFA
trifluoroacetic acid
- tR
retention time
- Tyr(m-NO2)
meta-nitrotyrosine
- Z
benzyloxycarbonyl
Abbreviations used for amino acids follow the recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature [Eur. J. Biochem., 138 (1984) 9]. Amino acid symbols denote the l-configuration. 相似文献
127.
The presence of endoproteinases in the intercellular washing fluid of the first wheat ( Triticum aestivum ) leaf 13 days after sowing was investigated. Two activities were detected after separation of the intercellular fluid proteins by SDS-PAGE using slab gels containing gelatin as substrate. Their sizes were 70 and 100 kDa. Both enzymes hydrolyzed gelatin and casein. They also seem to be involved in the degradation of a 40 kDa protein component of the intercellular fluid. These results indicate that both enzymes are endoproteinases. They were also obtained from leaves previously sumitted to intercellular fluid extraction. However, the 100 kDa enzyme yield was low, indicating that it is mainly located in the intercellular washing fluid. Assays performed with either specific substrates or inhibitors indicate that both enzymes are serine proteinases. 相似文献
128.
Purified barley carboxypeptidase exhibits high activity against a number of N-substituted amino acid esters, which are commonly used as synthetic substrates for mammalian and microbial proteinases. The proteinases of barley, on the contrary, do not hydrolyse these compounds. Because many other plants contain carboxypeptidases closely resembling the barley enzyme, we conclude that synthetic ester substrates should not be used to detect proteinase activity in extracts of higher plants. Plant carboxypeptidases also liberate C-terminal tryptophan from α-casein. Therefore, casein also is an unreliable substrate for plant proteinases. 相似文献
129.
M. Aulikki Salmia 《Physiologia plantarum》1981,53(1):39-47
Resting seeds of Scots pine contained a moderate amount of acid proteinase activity, about 90% of which was inhibited by pepstatin A and about 10% by p-hydroxymer-curibenzoate. In gel chromatography on Sephacryl S-200 the proteinase activity showed a complex elution pattern with poorly separated peaks at positions corresponding to mol. wts. 100,000 and 30,000 and several shoulders. The results suggested that pine proteinases I and II, which are the main proteinases in the endosperms of germinating seeds (Salmia 1981: Physiol. Plant. 51: 253–258), were not present in the resting seeds.—Seedling extracts showed a low level of acid proteinase activity, which separated into several peaks in chromatography on Sephacryl S-200. As none of the peaks had the catalytic properties of proteinase I or II, it seems that these endospermal enzymes are also lacking in the seedling tissues.—In the endosperms of germinating seeds the activity of the pepstatin-sensitive acid proteinase(s) remained at a constant level throughout the period of reserve protein mobilization (lasting up to the stage when the length of dark-grown seedlings was 60 mm). Proteinases I and II were absent from resting seeds, showed a small increase up to the 20-mm stage, and then increased rapidly up to the 60-mm stage.—Resting embryos contained relatively higher acid proteinase activity than resting endosperms, and again about 90% of it was inhibited by pepstatin A and about 10% by p-hy-droxymercuribenzoate. During germination the former activity decreased, the latter activity remained at approximately the same level, and the activity of the other acid proteinases increased continuously with the growth of the seedling.—It is concluded that the pepstatin-sensitive proteinase(s), which is not affected by endogenous proteinase inhibitors, plays a central role in the initiation of reserve protein mobilization in both the embryo and the endosperm. Proteinases I and II, on the other hand, seem to account for the greater part of reserve protein breakdown in the main protein storage tissue, the endosperm. 相似文献
130.
Oscar Campetellaa Javier Martíneza Juan José Cazzuloa 《FEMS microbiology letters》1990,67(1-2):145-150
Epimastigotes of different stocks of Trypanosoma cruzi contain similar levels of proteinase activity on azocasein; amastigotes and trypomastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some cases resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with a polyclonal rabbit antiserum prepared against purified cysteine proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme previously purified is the major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested. 相似文献