benzoyl-L-tyrosine Ethyl Ester Research Articles

Synthesis of a monolithic, micro-immobilised enzyme reactor via click-chemistry

An immobilised enzyme reactor (IMER) in the form of capillary monolith was developed for a micro-liquid chromatography system. The plain monolith was obtained by in situ thermal copolymerisation of glycidyl methacrylate and ethylene dimethacrylate in a fused silica capillary (200 × 0.53 mm ID) by using n-propanol/1,4-butanediol as porogen. The enzyme, α-chymotrypsin (CT), was covalently attached onto the monolith via triazole ring formation by click-chemistry. For this purpose, the monolithic support was treated with sodium azide and reacted with the alkyne carrying enzyme derivative. CT was covalently linked to the monolith by triazole-ring formation. The activity behaviour of monolithic IMER was investigated in a micro-liquid chromatography system by using benzoyl-L-tyrosine ethyl ester (BTEE) as synthetic substrate. The effects of mobile-phase flow rate and substrate feed concentration on the final BTEE conversion were investigated under steady-state conditions. In the case of monolithic IMER, the final substrate conversion increased with increasing feed flow rate and increasing substrate feed concentration. Unusual behaviour was explained by the presence of convective diffusion in the macropores of monolith. The results indicated that the monolithic-capillary IMER proposed for micro-liquid chromatography had significant advantages with respect to particle-based conventional high-performance liquid chromatography-IMERs.

Identification of Serine Esterases in Tissue Homogenates

Serine esterases react with [3H]diisopropylphosphofluoridate ([3H]DFP) to produce radioactive adducts that can be resolved by denaturing slab gel electrophoresis. To identify an esterase or its catalytic subunit, a potential substrate was included in the reaction mixture with the expectation that it would suppress the enzyme's reaction with [3H]DFP. The nature of the enzyme could be inferred from the character of the substrates that suppress labeling. The validity of this analytical method was tested with two serine proteases, trypsin and α-chymotrypsin, and two serine esterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and several of their natural or model substrates or inhibitors. Application of the method to complex biological systems was tested with chicken embryo brain microsomes. Trypsin labeling with [3H]DFP was suppressed by α-N-benzoyl-l-arginine ethyl ester (BAEE) and poly-l-lysine but not by benzoyl-l-tyrosine ethyl ester (BTEE). [3H]DFP labeling of chymotrypsin was suppressed by both BAEE and BTEE. Labeling of AChE and BuChE was suppressed by their natural and some related substrates and inhibitors. [3H]DFP reacted with brain microsomes to produce nine distinct radioactive bands. When the relevant substrates and inhibitors of AChE were included in the reaction mixtures, labeling of only the 95-kDa band was suppressed, implicating it as AChE. Labeling of the 85- and 79-kDa bands was inhibited by butyrylcholine, suggesting that these proteins have BuChE activity.

A preliminary characterization of alkaline digestive proteases in the posterior midgut of the face fly, Musca autumnalis De Geer

Alkaline proteases in the posterior midgut of the face fly, Musca autumnalis De Geer, were identified using specific synthetic substrates and nonspecific substrate azocasein. Identification was confirmed with potential protease activators and inhibitors. Optimal azocasein hydrolysis occurred at pH 8.0 and substrate hydrolysis was inhibited by EGTA (ethyleneglycol-bis-(2-aminoethyl ether)-N,N-tetraacetic acid), tosyl-L-lysine chloromethyl ketone (TLCK), and soybean trypsin inhibitor (STI). Tryptic proteolysis was identified by maximal hydrolysis, at pH 8.0, of trypsin specific substrates BAPNA (benzoyl-DL-arginine-p-nitroanilide) and TAME (tosyl-L-arginine methyl ester). TAME hydrolysis was inhibited by PMSF (phenylmethylsulfonyl fluoride), STI, pepstatin A, and dithiothreitol (DTT), but BAPNA hydrolysis was inhibited only by STI and DTT. Chymotrypsin was demonstrated by maximal hydrolysis of BTEE (benzoyl-L-tyrosine ethyl ester) at pH 8.0 and DTT, PMSF, and STI inhibited hydrolysis of this substrate. Aminopeptidase activity was demonstrated by maximal hydrolysis of leucine-p-nitro-anilide at pH 8.0 and the peptidase was inhibited by o-phenanthroline. Carboxypeptidase A-like enzyme hydrolyzed hippuryl-DL-phenyllactic acid maximally at pH 7.5 and carboxypeptidase B showed maximum hydrolysis of hippuryl-L-arginine at pH 7.0. Both carboxypeptidases were inhibited by EDTA (ethylene diamine tetraacetic acid), EGTA, and DTT and carboxypeptidase A was also inhibited by PMSF.

A preliminary characterization of digestive proteases in the posterior midgut of the stable fly Stomoxys calcitrans (L.) (Diptera:Muscidae)

Proteinases contained in the posterior midgut of the stable fly, Stomoxys calcitrans (L.), have been tentatively identified by posterior midgut hydrolysis of synthetic substrates. Trypsin was identified by maximal hydrolysis of benzoyl- dl-arginine -p- nitroanilide (BAPNA) and tosyl- l-arginine methyl ester (TAME) at pH 8.0 and chymotrypsin, by maximal hydrolysis of benzoyl- l-tyrosine ethyl ester (BTEE) at pH 8.0. Carboxypeptidase A and B were identified by their maximal hydrolysis of hippuryl- dl-phenyllactic acid and hippuryl- l-arginine at pH 8.0 and 7.5 respectively and aminopeptidase was identified by maximal hydrolysis of leucine- p-nitroanilide at pH 7.5. Molecular weights, determined using gel exclusion chromatography, for proteinases were 19,500 for trypsin, 27,500 and 64,000 for chymotrypsin. Hydrolytic activity for the protein substrate haemoglobin occurred as two peaks with molecular weights of 19,500 and 27,500. Molecular weights for the peptidases were; carboxypeptidase A 26,000, carboxypeptidase B, 28,500 and greater than 1,500,000 for aminopeptidase. These results indicate that the posterior midgut of the stable fly contains both proteinases and peptidases necessary for proteolytic breakdown of the ingested blood meal.

Purification and characterization of benzoyl-L-tyrosine ethyl ester hydrolase from the yolk sac membrane of chicken egg.

An enzyme which hydrolyzes benzoyl-L-tyrosine ethyl ester (BTEE) was purified from yolk sac membranes of day-18 chick embryos. The purified BTEE hydrolase has a molecular weight of 110,000, being composed of 70,000 and 40,000 subunits, and preferred synthetic substrates for chymotrypsin to those for trypsin. The optimum pH and temperature of this enzyme were 6.5-7.0 and 40 degrees C, respectively. The Km value for BTEE of the enzyme was 16 mM at pH 6.5 and 30 degrees C. The enzyme was inhibited markedly by some chymotrypsin inhibitors but scarcely inhibited by trypsin inhibitors. Magnesium ion acted as potent activator, depending on the enzyme purity and its concentration, whereas p-chloromercuribenzoate and zinc ion inactivated the activity markedly. The BTEE hydrolase was found to hydrolyze proteins such as casein and hemoglobin. These data indicated that the enzyme is a proteinase similar to chymotrypsin. This proteinase could act on yolk proteins, suggesting that it plays an important role in the metabolism of yolk at the yolk sac membrane layer.

Identification and partial characterization of a major gut proteinase from larvae of the hessian fly, Mayetiola destructor (say) (Diptera: Cecidomyiidae)

A major digestive proteinase in the guts of larvae of biotype D of Mayetiola destructor is a chymotrypsin-like serine proteinase which can hydrolyze [ 3H]alpha-casein and benzoyl- l-tyrosine ethyl ester (BTEE). Hydrolysis of protein and ester by the gut proteinase were blocked by di isopropyl fluorophosphate (DFP) and inhibited by l-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK). Optimum pH for hydrolysis of BTEE by gut proteinase is 7.8. Hydrolysis of [ 3H]alpha-casein by gut proteinase reaches a maximum at pH 8.4 and then maintains a plateau until pH 10.8. Proteolytic activity is located extracellularly in the lumen contents of the gut. The nature of proteolytic activity appears to be consistent in all larval stages examined. The Bowman-Birk inhibitor (BBI) from soybeans is a strong inhibitor of the proteolytic and esterolytic activities of the gut proteinase. These results are discussed in the context of the potential application of naturally occurring proteinase inhibitors as engineered host plant defences against larvae of the Hessian fly.

Endoproteinase activity in the posterior midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Tests with homogenates of digestive midgut of Rhodnius prolixus Stål showed that hydrolysis of N- benzoyl- dl- arginine naphthylamine (BANA), N- benzoyl- dl- arginine-p- nitroanilide (BAPNA) and casein was activated by EDTA and thiol reagents (cysteine, glutathione, mercaptoethanol, and dithiothreitol) but the hydrolysis of N- benzoyl- l-tyrosine ethyl ester (BTEE) was inhibited. Iodoacetamide and tosyl- l-lysine chloromethyl ketone inhibited BAPNA and casein hydrolysis but did not affect BTEE hydrolysis. Iodoacetamide also inhibited BANA hydrolysis. Horse serum inhibited hydrolysis of all tested substrates but soybean trypsin inhibitor inhibited only casein and BTEE activities. Metal ions Ca 2+, Mg 2+ and Mn 2+ had no affect on hydrolysis of BTEE, BANA or casein when crude midgut homogenate was used. These results suggest that at least two proteinases (one, cathepsin B-like, and another which is a thiol- and EDTA-sensitive endoproteinase) are present in the posterior midgut of R. prolixus.

Purification and characterization of a sulfhydryl-dependent protease from Rhodnius prolixus midgut

The characterization and partial purification of one protease from Rhodnius prolixus midgut are described. Enzyme preparations show a high degree of instability under several conditions, such as handling, freezing, and thawing, and at 4°C. This spontaneous inactivation of either crude or purified enzyme is completely protected by 10 mM tetrathionate. Sulfhydryl compounds such as β-mercaptoethanol, dithiothreitol, cysteine, thioglycolic acid, and reduced glutathione are able to activate the enzyme, whereas iodoacetate, mercuric chloride, p -aminophenylmercuric chloride, tetrathionate, and 5,5-dithiobis(2-nitrobenzoic acid) inhibit the enzyme. Partial inhibition was obtained by hydrogen perioxide, oxygen bubbling, and performic acid. Besides casein, the enzyme hydrolyzed benzoyl- l -tyrosine ethyl ester (BTEE), but showed no esterolytic activity upon p -tosyl- l -arginine methyl ester and benzoyl- l -arginine ethyl ester. Purifications of 210- and 187-fold with yields of 80 and 71% respectively, were obtained for the caseinolytic and esterolytic activities by using a Sepharose-mercurial column as a thiol-group affinity adsorbent. The molecular weight was estimated as 21,500 by gel filtration. α-Chymotrypsin and the midgut enzyme show similar abilities to hydrolyze casein, however, the midgut protease hydrolyzes casein 2.7-fold faster than trypsin and about 3-fold slower than pepsin. Soybean trypsin inhibitor, black-eyed pea chymotrypsin inhibitor, tosyl- l -lysine chloromethyl ketone, and tosyl-amide-phenylethyl chloromethyl ketone do not inhibit the protease activity, whereas, pepstatin showed a 100% inhibitory effect at an inhibitor/enzyme molar ratio of 0.36. Midgut protease and α-chymotrypsin showed similar K m values for BTEE (1.06 and 1.15 m ×10 −4 , respectively), however, the former showed a 9-fold lower catalytic efficiency ( K cat / K m ). Dialysis did not affect the enzyme, whereas Cu 2+ , Pb 2+ , and Hg 2+ showed an inhibitory effect; a slight activation was obtained in the presence of Ca 2+ .

60] Trypsin isoinhibitors from garden beans (Phaseolus vulgaris)

Trypsin inhibitory activity is assayed spectrophotometrically by measuring the inhibition of the tryptic hydrolysis of benzoyl-L-arginine ethyl ester (BAEE). Chymotrypsin-inhibitory activity is determined according to a modification of the method of HummcP as described by Kress et al., using benzoyl-L-tyrosine ethyl ester (BTEE) as substrate. This chapter describes the assay method for trypsin isoinhibitors. It also explains the following steps for the purification process: Extraction, Ammonium Sulfate Fractiontion, and Trichloroacetic Acid Precipitation, Gel Filtration on a Sephadex G-50 Column, Chromatography on a DEAE-Cellulose Column, Further Purification of Pool II from Step 3 on SP-Sephadex and Further Purification of Pool IV from Step 3 on SP-Sephadex. It also discusses the following properties: Purity, physical properties, inhibitors activities, reactive site residues, amino acid composition, peptide maps and partial sequence.

The secretory nature of the excretory gland cells of Stephanurus dentatus: III. Proteinase inhibitors

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.

Non-pancreatic proteases of the chymotrypsin family I. A chymotrypsin-like protease from rat mast cells

A protease has been purified from the isolated granules of mast cells from the peritoneal cavity of the rat. It resembles pancreatic chymotrypsin in most functional aspects, and has (by gel filtration measurement) the same molecular weight. It is active on tyrosine esters, but not arginine esters, and has a pH optimum and Michaelis constant in the hydrolysis of benzoyl- l-tyrosine ethyl ester (BTEE) that are very similar to those for bovine α-chymotrypsin. It exhibits the active-center-directed reactivities that are associated with the serine and with the histidine residues in chymotrypsin. There is evidence to suggest that it exists in the mast cell not as a zymogen, but as the active enzyme, bound to heparin. This enzyme exhibits an exceptional affinity for Sephadex or Bio-Gel gels, and is strongly retarded in filtration on these in neutral 0.1 M salt solutions. This leads to large errors in molecular weight estimation, which are removed when the filtration is conducted in 1.4 M KCl solution. The origin of such anomalies on the gel columns is discussed.