Bovine Trypsinogen Research Articles

Activity Assay for Endothia parasitica Protease Using Trypsinogen as Substrate

A sensitive activity assay method for Endothia parasitica protease based on activation of trypsinogen has been developed. Activation of trypsinogen was performed at pH 4.0 and 30.0C using 3.0 mg/ml of bovine trypsinogen and 1.25 μ/ml of purified Endothia parasitica protease in .04M sodium formate −.3M KCl buffer. At intervals of 1, 2, 3, 5, and 7 minutes the amounts of trypsin formed were determined on .05ml aliquots with 1.95ml of 1% casein in .2M Tris −.01M CaCl2 buffer, pH 8.0 and 30C for 10min. The trichloroacetic acid-soluble products were determined at 280nm. Enzyme activity was determined from the initial rate of trypsinogen activation. The pH optimum for activation of trypsinogen by Endothia parasitica protease was 3.5 to 3.6, Vmax, 2,950±177 micromoles trypsinogen activated per minute per micromole Endothia parasitica protease, and Km was 8.32±.30×10−5 M. Sensitivity of the trypsinogen activation assay was 11,400 times that of direct determination of Endothia parasitica protease activity on casein at pH 3.0 and 30C and 600 times more sensitive than a milk clotting assay at pH 5.1 and 30C.

A new fluorescent probe for protein and nucleoprotein conformation. Binding of 7-(p-methoxybenzylamino)-4-nitrobenzoxadiazole to bovine trypsinogen and bacterial ribosomes.

A new fluorescent probe for protein and nucleoprotein conformation. Binding of 7-(p-methoxybenzylamino)-4-nitrobenzoxadiazole to bovine trypsinogen and bacterial ribosomes.

Purification and Specificity of Porcine Enterokinase

Enterokinase (enteropeptidase, EC 3.4.4.8) has been purified from porcine duodenal extracts by chromatography on DEAE-cellulose, carboxymethyl cellulose, Sephadex G-100, and Sephadex G-200. The final product was found to be homogeneous by a number of tests. Its specific activity against trypsinogen (nanomoles of trypsinogen activated per 30 min per mg) was 6550, which represented a 650-fold purification over the first clear extract. The preparation was still contaminated, however, by traces of aminopeptidase activity. Porcine enterokinase activates bovine trypsinogen much better than does bovine trypsin (Km 6 times lower and kcat 2000 times higher). This property arises from the fact that the specificity site of enterokinase recognizes in trypsinogen not merely the basic residue of the -Lys-Ile-bond (residues 6 and 7) which is split during activation of the zymogen but also recognizes the sequence -Asp4-Lys (residues 2 to 6) which is present in all of the trypsinogens so far studied. By contrast, aspartyl residues are known to slow down the action of trypsin on the zymogen (autoactivation). The sequence -Asp4-Lys appears to be essential for the interaction of enterokinase with peptide or protein substrates and inhibitors. For example, the enzyme does not cleave the peptide Val-Ala2-Lys-Ile-Val-Gly and it does not activate chymotrypsinogen A. It does not hydrolyze the fully denatured S-carboxymethyl derivative of chymotrypsinogen and it cleaves exclusively the -Lys-Ile-bond (residues 6 and 7) in S-carboxymethylated bovine trypsinogen. Moreover, enterokinase is not inhibited by relatively high concentrations of soybean trypsin inhibitor or the two pancreatic inhibitors described by Kunitz and Kazal. On the other hand, enterokinase is similar to trypsin in several respects. For example, it is inhibited by α-tosylly-sine chloromethyl ketone and by diisopropylphosphorofluoridate, which suggests that an active serine and a histidine residue participate in the catalytic action of enterokinase. Moreover, enterokinase cleaves the synthetic trypsin substrates benzoylarginine ethyl ester and tosylarginine methyl ester. This probably means that the specificity site of enterokinase is composed of several subsites. One, possibly similar to that of trypsin, is responsible for the binding of low molecular weight basic substrates and inhibitors. The others, responsible for the interactions with aspartyl side chains, participate in the selective union of the enzyme with peptides or protein substrates and inhibitors. The properties of enterokinase suggest that it initiates the activation of trypsinogen which occurs in the duodenum. Therefore, it appears to be a key enzyme of digestion since trypsin is responsible for the activation of all of the other pancreatic zymogens.

The isolation of chymotrypsin-like enzymes by affinity chromatography using sepharose-4-phenylbutylamine.

Affinity chromatography of chymotrypsin-like proteases on a column of Sepharose–4-phenylbutylamine (PBA) has been developed. Sepharose–PBA (Sepharose–NH∙[CH2]4∙C6H5) has been shown to selectively adsorb chymotrypsin α and B from weakly alkaline solutions and to allow to pass through unretarded porcine trypsin, bovine trypsinogen, and chymotrypsin α modified with active-site-directed irreversible inhibitors. Chymotrypsinogen A and bovine trypsin were only slightly retarded whereas a preparation of subtilisin was markedly retarded and separated into two distinct peaks. Sepharose–PBA has been utilized successfully for the selective isolation of chymotryps in-like proteases from extracts of moose pancreas (Alces alces).

Kinetics of structural transitions induced by pH changes. Experiments on pancreatic proteins.

Bovine trypsinogen, trypsin and chymotrypsinogen undergo an isomerization in acidic medium, like many other proteins. We have studied the kinetics of these isomerizations by means of pH changes in a stopped‐flow apparatus. The order of magnitude of the rate constants is 20–100 sec‐1. The model used to interpret the effect of pH on kinetics leads to an unequivocal identification of at least one carboxylate group buried in the native form of these proteins. The effects of temperature and ionic strength on the kinetics are described.

Amino-acid composition of crayfish trypsin.

THE homology in primary structure of mammalian pancreatic serine proteases is well established1,2. Progress has now been made in the elucidation of the evolutionary history of this group of proteases. Neurath and his co-workers3 studied the amino-acid composition of trypsinogen isolated from the pancreas of the spiny Pacific dogfish and presented a preliminary examination of the primary structure of this enzyme. From these data a high degree of homology as compared with bovine trypsinogen can be inferred. It is now of interest what will be found beyond the gap that separates vertebrate and invertebrate animals. Invertebrate proteases are difficult to investigate because of the small amounts of material obtainable from these animals. In the crayfish Astacus leptodactylus we found an enzyme resembling mammalian trypsin in many respects4,5. It exhibits a distinct tryptic cleavage specificity when tried on the oxidized B-chain of insulin and is inhibited by all natural and synthetic trypsin inhibitors including TLCK. It is a serine protease and possesses the sequence –Asp–Ser–Gly– around the active centre serine residue6. In addition, extended immunological studies have been carried out on the crayfish trypsins of three species which revealed that they do occur as iso-enzymes varying in number and electrophoretic mobility7.

Synthesis of N-Terminal Nonapeptide of Trypsinogen and Its Hydrolysis by Trypsin

Abstract An N-terminal nonapeptide fragment of bovine trypsinogen, H-l-Val-l-Asp-l-Asp-l-Asp-l-Asp-l-Lys-l-Ile-l-Val-Gly-OH, was synthesized via two routes and its susceptibility with trypsin was investigated. Protected derivatives, t-butyloxycarbonyl protected nonapeptide t-butyl ester and benzyloxycarbonyl protected nonapeptide benzyl ester, were prepared by stepwise elongation. Removal of the protecting groups was carried out by treatment with trifluoroacetic acid and hydro-genation respectively. The naked peptide was purified with chromatography on ECTEOLA cellulose column using dilute acetic acid as a solvent. Upon lyophilization the desired nonapeptide was obtained as colorless powder, a portion of the product being digested with trypsin. Rate of tryptic hydrolysis of the peptide was found to be unexpectedly low in the absence of calcium ion, while considerable acceleration of the hydrolysis by calcium ion was noted.

Aspartic acid residue no. 177 of bovine trypsinogen

FEBS LettersVolume 8, Issue 4 p. 176-177 Full-length articleFree Access Aspartic acid residue no. 177 of bovine trypsinogen V. Tomášek, V. Tomášek Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, CzechoslovakiaSearch for more papers by this authorJ. Strmeň, J. Strmeň Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, CzechoslovakiaSearch for more papers by this authorF. Šorm, F. Šorm Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, CzechoslovakiaSearch for more papers by this author V. Tomášek, V. Tomášek Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, CzechoslovakiaSearch for more papers by this authorJ. Strmeň, J. Strmeň Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, CzechoslovakiaSearch for more papers by this authorF. Šorm, F. Šorm Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, CzechoslovakiaSearch for more papers by this author First published: June 08, 1970 https://doi.org/10.1016/0014-5793(70)80257-5Citations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume8, Issue4June 08, 1970Pages 176-177 ReferencesRelatedInformation

Comparison of the partial amino-acid sequence of dogfish trypsinogen with bovine trypsinogen.

Trypsinogen molecules from two species of different evolutionary periods are compared. Homology has been established by the sequencing of over half the amino-acids of the dogfish enzyme.

Immunochemical studies on bovine trypsin and trypsinogen derivatives

Immunochemical studies on bovine trypsin and trypsinogen derivatives

The promotion of activation of bovine trypsinogen by specific modification of aspartyl residues.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPromotion of activation of bovine trypsinogen by specific modification of aspartyl residuesT. M. Radhakrishnan, Kenneth A. Walsh, and Hans NeurathCite this: Biochemistry 1969, 8, 10, 4020–4027Publication Date (Print):October 1, 1969Publication History Published online1 May 2002Published inissue 1 October 1969https://doi.org/10.1021/bi00838a019RIGHTS & PERMISSIONSArticle Views36Altmetric-Citations36LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (835 KB) Get e-Alerts Get e-Alerts

Differential titration of trypsin-like enzymes

The radius of gyration (Rg) of bovine trypsinogen and β-trypsin was measured by an energy-dispersive X-ray technique as a function of Ca2+ or SO42− concentration; these results have been supplemented with measurements of association equilibrium constants of Ca2+ to its binding site(s) on both serine proteases and some of their adducts (with an effector and/or an inhibitor). As a whole, all information reported in the present work demonstrates that: (i) the strains exerted by different ions on these proteases produce diverse structural modifications; and (ii) at least in the case of Ca2+, the changes in Rg can be ascribed to the direct interaction of the binding site(s) on the protein matrix with the cation.

The mechanism of activation of trypsinogen. The role of the four N-terminal aspartyl residues.

1 This paper examines in detail the activation of bovine and porcine trypsinogens and of bovine chymotrypsinogens A and B by trypsin and aspergillopeptidase A. Kinetic data have also been obtained (Km and kcat) for the hydrolysis catalyzed by these proteases of several model peptides with sequences related to the N-terminal sequence of bovine trypsinogen. 2 The N-terminal sequence of (aspartyl)4 residues is not necessary for the recognition of the strategic Lys-Ile bond of trypsinogen. 3 We have shown previously that there are two binding sites for Ca2+ on trypsinogen. One of these sites is constituted by the 2 aspartyl residues which are the nearest neighbours of the important Lys-Ile bond. The saturation of the site by Ca2+ improves the formation of the trypsinogen-trypsin complex; Ca2+ has no effect on the rate of decomposition of this complex. 4 The comparison of the kinetic data for the activation of trypsinogens and chymotrypsinogens A and B on one hand and the comparison of kcat and Km of different model peptides, among them the synthetic nonapeptide Val-(Asp)4-Lys-Ile-Val-Gly, on the other hand, implies that the 4 aspartyl residues confer no special reactivity to the Lys-Ile bond but conversely have a very negative effect. For example kcat= 1.6 sec−1 for chymotrypsinogen B but only 2.5 and 5.5 × 10−3 sec−1 for bovine and porcine trypsinogens. The values of Km are similar. For the peptide Val-(Ala)2-Lys-Ile-Val-Gly kcat= 7.0 sec−1 but kcat between 10−4 and 10−2 sec−1 has been found for the peptide Val-(Asp)4-Lys-Ile-Val-Gly. This very negative effect is observed only with trypsin. In the activations by aspergillopeptidase A, trypsinogen is a much better substrate than chymotrypsinogen. The implications of this exceptionally slow hydrolysis of the Lys-Ile bond are discussed. The problem of the formation of inert proteins in particular appears to be closely related to the very poor quality of this bond as a substrate for trypsin. A mechanism is given for the formation of inert proteins; a similar mechanism also explains the degradative autolysis of trypsin.

The structural properties of trypsinogen and trypsin Alkylation and oxidation of methionines

The side chains of methionines 92 and 166 are buried in the hydrophobic interior of several of the molecular forms (I, I′, III and III′ (refs. 1 and 2)) of bovine trypsinogen and trypsin; they cannot react with H 2O 2, iodoacetamide and iodoacetate. Reversible denaturation by heat or by urea unmasks both thioether functions which are then readily alkylated or oxidized. The kinetics of these modifications are discussed. For example the rate constant for the alkylation of trypsin in 6 M urea (pH 2.2) and 40° is similar to that found by Stark and Stein for the alkylation of N-acetyl methionine under the same conditions. Dialkylated trypsin is devoid of catalytic activity; it has the physical properties of a denatured form. Disulfoxide trypsin is an active enzyme with peptidase, amidase and esterase activities. However, oxidation decreases the thermal stability of the enzyme, lowers its affinity somewhat for benzamidine, and decreases the rates of acylation and deacylation of the strategic serine in the active center. Disulfoxide trypsinogen remains autoactivable in the presence of Ca 2+; when the cation is omitted inert proteins are formed as for unsubstituted trypsinogen. The specific binding site for Ca 2+ is preserved in disulfoxide trypsinogen.

On proteins. CVI. Covalent structure of bovine trypsinogen

On proteins. CVI. Covalent structure of bovine trypsinogen

Covalent structure of bovine trypsinogen. The position of the remaining amides

Our recent paper concerning bovine trypsinogen 1,2 presented a description of the primary structure of this protein, including the disulfide bridges. However, at that time an unequivocal assignment of all amino acid residues had not been made for several amino acid residues (positions 121, 177, 180). Thus it was not known whether these residues occurred in the form of acids or amides. The aspartic acid residue in position 151 has now been correctly determined as asparagine. The aim of the present communication was to remove all these uncertainties and to complete the investigation of the covalent structure of this protein. A preliminary report of this work was presented at the Third Federation Meeting of European Biochemical Societies 3.

Sur la réactivité des fonctions ionisables des trypsinogènes de bœuf et de proc

Summary The reactivity of ionisable side-chain groups, as measured by titrimetric techniques, has been determined for bovine and porcine trypsinogens. The number of α, β and γ-carboxyl, α- et e-amino, phenolic and guanidyl groups agrees well with data obtained by amino acid analysis. Normal pK values are found for the e-amino groups. However, the abnormal titration behavior of the imidazole, phenolic and carboxyl groups underlines their unique character in these proteins. 1. The imidazole groups (3 in bovine and 4 in porcine trypsinogen) are found to fall into three classes: Two imidazoles in porcine trypsinogen and one in bovine trypsinogen have normal pKs (6.5 and 6.8 respectively). One imidazole in each trypsinogen has an abnormally low pit (5.7). One imidazole in each trypsinogen had an abnormally high pit (8.3 or 7.8). 2. The value of the electrostatic factor ω, empirically calculated from the titration curve of the carboxyl groups, is abnormally high in both proteins when compared with the values obtained by theoretical calculation, titration of trypsinogen at alkaline pH or titration of bovine chymotrypsinogen A at any pH. Several of the carboxyl groups may have pit's lower than the normal value (4.4–4.5). 3. The spectrophotometric titration of the tyrosine residues revealed 4 of the 8 in porcine trypsinogen and 6 of the 10 in bovine trypsinogen to be masked. The unmasking of these groups occurs in several consecutive first-order reactions and is complete only at pH values above 13 or in 6 M urea. Ca2+ stabilize the conformation of the molecule, as revealed by an antagonistic effect on tyrosine unmasking; the trypsins formed by trypsinogen autoactivation are more stable than their precursors towards alkaline denaturation. The autoactivation of porcine trypsinogen does not change the number of masked tyrosines. The autoactivation of bovine trypsinogen involves the unmasking of one residue of tyrosine.

Sur le trypsinogène et la trypsine de porc

A chromatographic procedure on carboxymethyl cellulose at pH 5.0 is described for the simultaneous purification of porcine trypsinogen and one of the porcine chymotrypsinogens. The yield calculated from the first extract of the gland in dilute sulfuric acid is 45% with a 2–3 fold purification. 1 kg of fresh pancreas gives at least 2.8 g of pure product.The amino acid compositions of bovine and porcine trypsinogens reveal some similarities. The molecular weight of both proteins is of the same order. But, a series of sizable differences of composition suggest that large regions of the molecules have in fact quite different structures.The diisopropylphosphoryl derivative of the trypsin formed by autoactivation of porcine trypsinogen can be purified by chromatography on carboxymethyl cellulose at pH 5.0. When the same technique is applied to active trypsin, a highly active, but slightly autolyzed, product is obtained.Autoactivation of porcine and bovine trypsinogens proceeds at about the same rate to give about the same maximal specific activity. Calcium ions have the same effect. In both cases, the activation process involves the specific splitting of a Lys-Ileu bond belonging to the N-terminal sequence of the chain and having on its left 4 aspartic acid residues. However, this bond is the 8th of the sequence in the case of porcine trypsinogen. The peptide set free is the octapeptide Phe-Pro-Thr-(Asp)4-Lys. Thus, the apparently essential region having the same structure in both trypsinogens is very narrowly restricted. Beyond it, structural modifications reflecting deep changes in the genetical information can be noted.Porcine trypsinogen and porcine trypsin have the same C-terminal sequence: Thr-(Ileu,Glu(NH2))-Ala-Asp(NH2).This identity is the first direct proof that trypsinogen activation does not involve any covalent modification in the C-terminal region of the chain.