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1.
The somatic extract of mature T. pisiformis has been demonstrated to contain a potent inhibitor capable of inactivating the esterolysis of N-α-benzoyl-L-arginine ethyl ester and N-benzoyl-L-tyrosine ethyl ester by trypsin and chymotrypsin, respectively, of bovine, dog and rabbit origin, but not affecting the caseinolytic activity of subtilisin and elastase. The protease inhibitor, partially purified by trichloroacetic acid treatment, Sephadex G-100 column chromatography and affinity chromatography on CNBr-activated Sepharose 4B-bovine chymotrypsin conjugate, was soluble in 5% trichloroacetic acid, stable to heating at 100°C for up to 30 min, tolerated the pH range of 1.5–9.0, and was unaffected by 8 m-urea or 0.2 M-2-mercaptoethanol. The molecular weight of the inhibitor was estimated to be 7000–7200 by Sephadex G-100 chromatography. Activity determinations on crystalline bovine trypsin and chymotrypsin revealed that both inhibitory actions are located on the same or closely adjacent sites of the inhibitor molecule. Complex formation between the inhibitor and mammalian trypsin and chymotrypsin required 3–4 min for completion.  相似文献   

2.
Bovine pancreatic ribonuclease has been modified by exposure to acidic conditions, addition of indole propionic acid and crosslinking with glutaraldehyde. The ‘acid-esterase’ generated was purified up to 100-fold by ammonium sulphate fractionation and gel filtration on Biogel P-30. The partially purified acid-esterase hydrolysed tryptophan ethyl ester (TrEE) and N-benzoyl-l-arginine ethyl ester (BAEE) effectively at pH 6.0–6.3, but it had very little activity towards glycine ethyl ester and lysine ethyl ester. Hydrolysis of TrEE was competitively inhibited by tryptophan. The acid-esterase exhibited amidase activity towards benzoyl-l-arginine p-nitroanilide.  相似文献   

3.
A proteinase inhibitor(s) was found in extracts of the excretory gland cells, intestines, esophagi, reproductive organs, and body walls from Stephanurus dentatus adults. The specific activity of the inhibitor(s) in the excretory gland cell extract was 45–175 times greater than in the other tissues. It is heat stable at pH 5.0 and inhibits the esterolytic activity of trypsin and chymotrypsin using p-toluenesulfonyl-l-arginine methyl ester hydrochloride (TAME) and benzoyl-l-tyrosine ethyl ester (BTEE) as the substrates, respectively, and also the proteolytic activity of both chymotrypsin and trypsin using casein as the substrate. S. dentatus adults maintained in NCTC 109 medium, secreted a trypsin inhibitor.  相似文献   

4.
Condensation of 3,5-di-O-benzoyl-β-d-ribofuranosyl chloride severally with 3-acetyl-5-alkylpyridines, 5-alkyl-3-methoxycarbonylpyridines (alkyl = Me, Et, Pr, and iPr), 5-isopropylnicotinamide, and 3,5-diacetylpyridine bis(ethylene acetal) in acetonitrile at ?5° gave the corresponding 1-(3,5-di-O-benzoyl-β-d-ribofuranosyl))-3,5-disubstituted pyridinium chlorides in excellent yield (90%). From the reaction of a series of 2,3-O-isopropylidene-β-d-ribofuranosyl halides with 3-acetyl-5-methyl-pyridine at room temperature, the α-nucleosides were obtained.  相似文献   

5.
1. Two chymotrypsins with isoelectric points pI 6.2 and 5.8 were purified from the pyloric caeca of Atlantic cod using a phenyl-Sepharose column and chromatofocusing chromatography. The apparent molecular weight was 26,000 as judged by SDS-polyacrylamide gel electrophoresis and gel filtration. 2. The cod enzymes differed from bovine chymotrypsin in having a slightly higher molecular weight and more acidic pI points. N-terminal amino acid sequence analysis of cod chymotrypsin B showed considerable similarity with bovine chymotrypsin. 3. Heat stability and stability towards acidic pH were reduced in the cod enzymes. Generally, the cod and bovine chymotrypsins responded similarly to various protease inhibitors. However, the cod chymotrypsins were less sensitive to aprotinin inhibition but more sensitive towards soybean trypsin inhibitor and cysteine. 4. Kinetic properties were examined and the cod enzymes found to be more active towards both ester (N-benzoyl-tyrosine ethyl ester) and amide (N-benzoyl-tyrosine-p-nitroanilide) substrates. The observed differences in kinetic properties are indicative of an adaptive response towards the low temperature environment in which the cod lives.  相似文献   

6.
Portions of crude homogenates of etiolated wheat seedlings incubated with Mg-protoporphyrin IX and S-adenosyl-L-methionine and then added to other portions of the same crude homogenates that were pretreated with [1-3H]ethanol and yeast alcohol dehydrogenase provided, after a short reaction period, 3H-labeled Mg-protoporphyrin IX monomethyl ester. The 3H-labeled Mg-protoporphyrin IX monomethyl ester thus obtained was shown to contain the 3H in one reduced (to ethyl) vinyl side-chain. Subsequently, 3H-labeled Mg-monoethyl-(monodivinyl)-protoporphyrin IX monomethyl ester was obtained when Mg-protoporphyrin IX monomethyl ester and [3H]NADH were added to dialyzed crude homogenates of etiolated wheat seedlings. Insignificant amounts of 3H were incorporated into poprhyrin substrates when Mg-2,4-divinylpheoporphyrin a5 or [3H]NADPH were substituted in reaction mixtures for Mg-protoporphyrin IX monomethyl ester or [3H]NADPH, respectively. The results of these and further experiments suggest that an NADPH-dependent enzyme in the crude homogenates of etiolated wheat seedlings was capable of catalyzing the reduction to ethyl of one vinyl side-chain of Mg-protoporphyrin IX monomethyl ester. These findings suggest that the 4-vinyl side-chain reductive reaction likely occurs after the biosynthesis IX monomethyl ester, but before isocyclic ring formation in the pathway to chlorophyll a.  相似文献   

7.
A trypsin and chymotrypsin inhibitor was partially purified from Bauhenia purpurea seeds and separated from a second inhibitor by Ecteola cellulose chromatography. The factor inhibited bovine trypsin and chymotrypsin as well as pronase trypsin and elastase. It formed a complex with trypsin and with chymotrypsin, but a ternary complex could not be detected. Differences were detected in the effect on trypsin and on chymotrypsin, although one enzyme interfered with the inhibition of the other. The results obtained point to two active centers on the inhibitor for the trypsin and chymotrypsin inhibition such that the one cannot complex with the inhibitor after this inhibitor had complexed with the other.  相似文献   

8.
A glycopeptide derived from bovine nasal septum by sequential treatment with trypsin, chymotrypsin, 0.05N HCl in dry methanol (desulfation), testicular hyaluronidase and β-glucuronidase, was coupled to Sepharose-4B in the presence of cyanogen bromide. β-N-Acetylhexosaminidase A was selectively retarded when crude extracts of human skin fibroblasts or liver were applied to the affinity column and was subsequently eluted with 0.1% Triton X-100 in 0.1M citrate-phosphate buffer pH 4.4, providing a simple method for purification.  相似文献   

9.
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   

10.
Bovine and porcine pancreatic residue, remaining after the extraction of insulin, has been used to prepare a proteinase powder. This powder was used as a source of trypsin and chymo-trypsin. The individual enzymes were isolated and purified by chromatography on sulfopropyl (SP)-Sephadex C-25 and affinity chromatography on soybean trypsin inhibitor (STI)-Sepharose. The bovine proteinase powder contained a-chymotrypsin, trypsin and chymotrypsin B in the ratio 5:2:1. The porcine powder contained cationic trypsin, anionic trypsin and cationic chymotrypsin in the ratio 5 : 1. 4 : 3. The isolated enzymes were characterized and found to be identical with enzymes isolated from fresh tissue with the exception of porcine chymotrypsin. Porcine cationic chymotrypsin was isolated as two distinct forms, A-l and A-2, which appear to be different activation products of porcine chymotrypsinogen A. Both forms resemble bovine a-chymotrypsin, a three chain structure, rather than porcine chymo-trypsin A, a two chain structure. Furthermore, the B-chain appears to be cleaved, possibly at residues Phe89-Lys90.  相似文献   

11.
A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC50 (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca2+ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.  相似文献   

12.
Sensitive methods for the determination of rat mast cell protease I, rat mast cell protease II, human skin chymotrypsinlike enzyme, dog skin chymotrypsinlike enzyme, human leukocyte cathepsin G, and bovine chymotrypsin Aα with peptide thiobenzyl ester substrates are reported. Kinetic constants as well as the maximum sensitivity for the hydrolysis of the peptide substrates succinyl-phenylalanyl-leucyl-phenylalanine thiobenzyl ester and succinyl-alanyl-alanyl-prolyl-phenylalanine thiobenzyl ester were determined. Hydrolysis rates were followed spectrophotometrically at 324 nm by the formation of 4-thiopyridone (? = 19,800 m?1 cm?1), the product of the reaction between benzylthiol, released during hydrolysis of the peptide thiobenzyl esters, and 4,4′-dithiodipyridine present in the assay mixture. Peptide thiobenzyl ester substrates were shown to be very sensitive substrates, predominantly because of the large extinction coefficient of 4-thiopyridone and the high kcatKm values for these compounds.  相似文献   

13.
Rogers W. P. 1982. Enzymes in the exsheathing fluid of nematodes and their biological significance. International Journal for Parasitology12: 495–502. The characteristics of an enzyme which hydrolysed denatured collagen and a lipase in exsheathing (ecdysal) fluid are described. A highly purified collagenase from Clostridium histolytica attacked isolated sheaths and reacted to additives in the same way as exsheathing fluid. However, relative to their activities with Azocoll or p-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg as substrates, the enzyme of exsheathing fluid was >400 times as active as the bacterial collagenase in its action on isolated sheaths.It is suggested that the lipase in exsheathing and hatching fluids may, in association with the pseudocollagenase (and sometimes with chitinase also) have a function in the hatching of eggs. The pseudocollagenase alone may serve as the exsheathing enzyme. The leucine aminopeptidase in hatching and exsheathing fluids may be concerned in the breakdown of the excretory cell and the release of the fluids.  相似文献   

14.
A low-molecular-weight interferon-mediated ribosome-associated inhibitor of reovirus mRNA translation was purified from the 0.5 M KCl ribosomal salt-wash fraction of mouse L929 cells. The inhibitor possessed nucleolytic activity with reovirus [3H]mRNA as a substrate. Loss of translational inhibitory activity correlated with the thermal inactivation of the nuclease. A low-molecular-weight (<10K) component present in the Bio-Gel P150 chromatography fractions which contained the interferon-mediated nucleolytic activity was labeled in vivo with [14C]valine; a smaller component present in the same fractions was phosphorylated in vitro with [γ-32P]ATP. The <10K components were resolved from ~50K, ~30K and ~20K phosphorylatable proteins associated with ribosomes that possess the interferon-mediated inhibitor(s) of viral mRNA translation.  相似文献   

15.
A procedure for synthesizing 14C-labeled N-benzoyl-l-tyrosine ethyl ester with the label in the benzoyl group has been described. Using this substrate, which is specific for chymotrypsin, and employing trypsin activation of chymotrypsinogen in an incubation medium containing radiolabeled BTEE, we have presented a method which permits assay of nanogram quantities of chymotrypsin activity in organ-cultured tissue. The radiolabeled product of enzyme hydrolysis, N-benzoyltyrosine, is readily separated by paper chromatography from the unreacted labeled substrate and measured by radioactivity counting.  相似文献   

16.
17.
Qualitative differences in the active center of rat trypsins 1 and 2 resulted in different ratios of Kcat for N1-tosyl-l-arginine methyl ester vs Kcat for N1-benzoyl-l-arginine ethyl ester. These ratios were 2.5 for trypsin 1 and 1.2 for trypsin 2.Substrate activation with N1-tosyl-l-arginine methyl ester enhanced the catalytic rate constant of rat trypsin 1 2.5-fold and that of rat trypsin 2 only 1.5-fold. The increase in the catalytic rate constant found with N1-benzoyl-l-arginine ethyl ester was the same (1.5-fold) for both trypsins. Consequently, at 20 mm substrate concentration, trypsin 1 catalyzed the esterolysis of N1-tosyl-l-arginine methyl ester 4.5 times faster than that of N1-benzoyl-l-arginine ethyl ester, while trypsin 2 was only 1.3 times more efficient with the first substrate.Furthermore, the activation of both rat enzymes by N-acetyl-l-tyrosine ethyl ester was even more effective than that obtained with the two cationic esters; the maximum rates of hydrolysis of this neutral substrate by trypsins 1 and 2 were enhanced 120- and 50-fold, respectively, by high concentrations of N-acetyl-l-tyrosine ethyl ester.  相似文献   

18.
Partial acid hydrolyzates of the extracellular polysaccharide from Porphyridiunm cruentum yield three disaccharides and two uronic acids. These constitute all of the uronic acid in the polymer. The novel disaccharides are 3-O-(α-D-glucopyranosyl- uronic acid)-L-galactose, 3-O-(2-O-methyl-ca-glucopyranosyluronic acid)-D- galactose, and 3-0-(2-0-methyl-a-D-glucopyranosyluronic acid)-D-glucose. The polyanion of high molecular weight contains D- and L-galactose, xylose, D-glucose, D-glucuronic acid and 2-O-methyl-D-glucuronic acid, and sulfate in molar ratio (relative to D-glucose) of 2.12:2.42:1.00:1.22:2.61. Preliminary periodate-oxidation studies suggest that the hexose and uronic acids are joined to other residues by ( 1→3) glycosidic linkages. About one-half of the xylose residues are (1→3)-linked.  相似文献   

19.
  • 1.1. In human plasma, an enzyme is present which hydrolyzes 4-methylumbelliferyl-tetra-N-acetylchitotetraoside. The function of this enzyme is unknown.
  • 2.2. We have examined whether hyaluronidase, neutral endoglucosaminidase, N-acetyl-β-d-hexosaminidase, aspartylglucosaminidase, β-d-glucosidase, and chitobiase could hydrolyze MU-TACT. The results obtained are detailed below.
  • 3.3. A purified commercial preparation of hyaluronidase does not hydrolyze MU-TACT.
  • 4.4. Substrate specificity requirements, pH optimum and subcellular localization indicate that neutral endoglucosaminidase is distinguishable from MU-TACT hydrolase. Also commercial neutral endoglucosaminidase D and H have no affinity towards MU-TACT.
  • 5.5. N-Acetyl-β-d-hexosaminidase is different from MU-TACT hydrolase for the following reasons: (a) a purified enzyme preparation does not hydrolyze MU-TACT; (b) there is no correlation in the activity of the enzymes; (c) MU-TACT hydrolase is not deficient in cells of a patient with a deficiency of total N-acetyl-β-d-glucosaminidase; and (d) the 2 enzymes have very different Chromatographic characteristics and Con A binding properties.
  • 6.6. Enzyme characteristics, substrate structural requirements and a lack of correlation with MU-TACT hydrolase activity suggest that aspartylglucosaminidase, β-d-glucosidase, and chitobiase are not involved in the hydrolysis of MU-TACT.
  • 7.7. None of the enzymes which we have considered corresponds to MU-TACT hydrolase. The exact nature and the function of the enzyme remains an enigma.
  相似文献   

20.
Enzymic hydrolysis of triacylglycerol has been studied with very low density lipoproteins from an individual with a genetically determined absence of apoC-II, the activator apoprotein for lipoprotein lipase. Normal rates of ester cleavage by purified bovine milk lipoprotein lipase can be achieved invitro with native apoC-II and by three shorter synthetic peptides, apoC-II(55–78), apoC-II(50–78) and apoC-II(43–78), which contain part of the carboxyl terminal third of the native apoprotein. At 0.5 μM concentration, all peptides produced a 7-fold activation. ApoC-II(43–78), but not apoC-II(50–78) or apoC-II(55–78), could bind VLDL as shown by separation of unbound 125I peptides and the lipoproteins. Thus, residues 43–50 of apoC-II are part of a lipid binding region. High affinity binding of apoC-II peptides to the lipoprotein substrate is not obligatory for activation of lipoprotein lipase.  相似文献   

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