Bovine Trypsinogen Research Articles

Trypsin activation. Effect of the Ile-Val dipeptide concentration on Kazal inhibitor binding to bovine trypsinogen

The effect of Ile-Val concentration (up to 2.0 M) on the thermodynamic parameters for the binding of the porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor) to trypsinogen has been investigated at pH 5.5 between 7°C and 42°C. Thermodynamic parameters for Kazal inhibitor binding to the Ile-Val:zymogen adduct are more favorable than those observed for inhibitor association to the free proenzyme, but less so than those reported for β-trypsin: Kazal inhibitor adduct formation (even under saturating dipeptide concentrations), suggesting that the effector dipeptide does not induce a complete rigidification of the proenzyme's activation domain. Considering the dependence of the association equilibrium constant for Kazal inhibitor binding to trypsinogen from Ile-Val concentration, thermodynamic parameters for the effector dipeptide binding to the free proenzyme and to its binary complex with Kazal inhibitor have been obtained. Differences in affinity for Ile-Val binding to the free zymogen and its binary complexes with inhibitors and substrates are indicative of the presence of different activation levels of the proenzyme, none of them exactly coincident with that of β-trypsin. Such different discrete states should correspond to those involved in the zymogen-to-active-enzyme transition which should not be considered as an all-or-nothing process, but as a multistep event.

Activating effect of the Ile-Val dipeptide on the catalytic properties of bovine trypsinogen

The dependence of pre-steady-state and steady-state kinetics for the trypsinogen-catalyzed hydrolysis of ZArgONp on the concentration (up to 2.0 M) of the Ile-Val effector dipeptide has been investigated at pH 8.0 and 21 ± 0.5°C. Kinetic parameters for the hydrolysis of ZArgONp catalyzed by the Ile-Val: zymogen adduct are more favorable than those observed for the free proenzyme but lower than those reported for β-trypsin; these data indicate that the effector dipeptide induces only a partial activation of the zymogen even under saturating Ile-Val concentrations. From the dependence of kinetic parameters of proenzyme catalysis on the effector dipeptide concentration, values of the equilibrium constants for binding of Ile-Val to the free trypsinogen, to the reversible zymogen-ZArgONp complex and to the proenzyme-ZArg acyl intermediate have been obtained. Thermodynamics of binding of Ile-Val to trypsinogen, in the presence and absence of substrates and inhibitors, are indicative of the presence of different activation levels of the proenzyme, none of which is superimposable on that of β-trypsin. On this basis, it is suggested that some of these different states could correspond to those involved in the zymogen-to-active-enzyme transition, which should be considered as a multistep process, rather than all-or-nothing event.

The identification of neotrypsinogens in samples of bovine trypsinogen

Isoelectric focusing of commercial samples of bovine trypsinogen detected a component with a lower isoelectric pH than that of trypsinogen. The isoelectric pH was 8.75 compared to 9.3 for trypsinogen, and the amount of the component varied from 16 to 41% of the total protein. The protein (24,000 Da) was converted to fragments of 13,800 and 10,500 Da on reduction with dithioerythritol, showing that the component was a modified form of trypsinogen containing a cleaved peptide bond. The cleavage site was established from the study of four polypeptide fragments which were isolated from the fully reduced and S-carboxymethylated trypsinogen. The molecular weights, amino acid compositions, and amino-terminal sequences of these fragments identified a cleavage of Lys 131-Ser 132, namely from a Ser-neotrypsinogen, or at Arg 105-Val 106, from a Val-neotrypsinogen. Val-neotrypsinogen was the more abundant of the two and was approximately 71% of the total neotrypsinogen in the trypsinogen sample. Both neotrypsinogens were converted to active trypsin molecules in high yields, showing that the zymogens closely resembled the conformation of intact trypsinogen. Presumably, the neotrypsinogens were produced during the isolation of the zymogen when pancreatic tissue was partly autolyzed and active trypsin was present.

The refined 2.2‐Å (0.22‐nm) X‐ray crystal structure of the ternary complex formed by bovine trypsinogen, valine‐valine and the Arg15 analogue of bovine pancreatic trypsin inhibitor

Large orthorhombic crystals of the complex formed by bovine trypsinogen and a semisynthetic homologous bovine pancreatic trypsin inhibitor with the reactive-site lysine residue replaced by an arginine residue [( Arg15]PTI) have been obtained which are isomorphous with the crystals of PTI-trypsinogen [Bode, W., Schwager, P. and Huber, R. (1978) J. Mol. Biol. 118, 99-112]. The X-ray crystal structure of the ternary complex of trypsinogen-[Arg15]PTI with the dipeptide Val-Val has been determined by X-ray data to 2.2-A (0.22-nm) resolution by means of difference Fourier methods and has been crystallographically refined to a final R-value of 0.17. Replacement of the reactive-site Lys15 by an arginine residue is accompanied in the complex by small movements of polar side groups of trypsin and enclosed solvent molecules within the specificity pocket. Only solvent molecule 414 OH which mediates the hydrogen bond interactions between Lys15 NZ and Asp189 carboxylate is expelled, thus allowing the bulkier guanidyl group to approach this carboxylate. The dipeptide Val-Val binds in the pocket accepting the Ile-Val N-terminus in trypsin. The cavity left by the CD-methyl group of Ile16 upon replacement by a valine residue is only partially filled by slight rearrangements of neighbouring peptide side chains. Part of the positive free energy change observed upon replacement of Ile-Val may allow for the maintenance of this cavity.

Electrophoretic analysis of pancreatic proteases and zymogen-activating factors in the mouse.

Mouse pancreatic proteases were analyzed by one- and two-dimensional electrophoresis. Active proteases that existed in the luminal fluid were separated into at least eight bands in 8% polyacrylamide gel. Pancreatic proteases activated by intestinal extract were separated into at least seven bands. The mobilities of these bands were exactly the same as those of proteases in the luminal fluid except for those of the most cathodal band. Two kinds of trypsin (Try-I group and Try-II) and one kind of chymotrypsin (Chy-I) were determined by specific and nonspecific protease staining. Try-I group and Try-II were derived from different trypsinogens (Try G-I group and Try G-II), whereas Chy-I was derived from a single chymotrypsinogen (Chy G). Although Try G-II was activated by both intestinal extract and by bovine trypsin, Try G-I group activated only by intestinal extract. Intestinal-activating factors were analyzed by two-dimensional electrophoresis. Mouse enterokinase (enteropeptidase EC 3.4.4.8), which can activate bovine trypsinogen, had a slow mobility. In the intestine of the mouse there are several activating factors in addition to enterokinase. Although it is unclear what intestinal-activating factors can activate Chy G, there is a factor that can convert chymotrypsinogen into chymotrypsin directly. These data suggest that intestinal-activating factors play an important role in the activating mechanisms of mouse pancreatic zymogens.

Interaction between serine (pro)enzymes, and kazal and kunitz inhibitors

Equilibrium constants (Kd) for PSTI (porcine pancreatic secretory trypsin inhibitor: Kazal inhibitor) and BPTI (bovine basic pancreatic trypsin inhibitor: Kunitz inhibitor) binding to bovine β-trypsin, bovine trypsinogen, bovine α-chymotrypsin, human urinary kallikrein and porcine pancreatic kallikrein have been obtained at pH 8. Kd values have been determined by measuring the loss of the enzymatic activity and the spectral changes accompanying the formation of the PSTI: (pro)enzyme and BPTI: (pro)enzyme adducts. PSTI is characterized by systematically lower affinities than BPTI and does not inhibit either kallikrein up to inhibitor concentrations of 3 m m . As in the case of BPTI, the affinity of PSTI for bovine trypsinogen increases in the presence of 0·02 m -H-Ile-Val-OH, which mimics, in the proenzyme, the N-terminal segment of the activated bovine β-trypsin. BPTI: bovine trypsinogen: H-Ile-Val-OH and PSTI: bovine trypsinogen: H-Ile-Val-OH complexes show absorption spectra similar to those of BPTI: bovine β-trypsin and PSTI: bovine β-trypsin adducts. The pH dependence of the equilibrium constants for PSTI and BPTI binding to bovine β-trypsin, bovine trypsinogen and bovine α-chymotrypsin is roughly the same for both the inhibitors with the following scale of apparent affinities: β-trypsin ≫ α-chymotrypsin > trypsinogen. The simplest model that describes the experimental data, between pH 5 and 9·5, takes into account a single ionizable group with a pKUNL of approximately 7·2 in the free (pro)enzymes, which appears to decrease by about two units upon PSTI and BTPI binding. The most probable ionizing group to account for such a behaviour is His57, present at the active site of (pro)enzymes. Comparison of the three-dimensional structures of PSTI and BPTI adducts shows that the respective reactive sites, despite structural identity around the scissile peptide bond, differ in their geometry of subsites further away from it. In particular, the wider reactive site polypeptide loop of PSTI as compared to that of BPTI may be responsible, at least in part, for the different binding behaviour of the two inhibitors.

Three-dimensional structure of the complex between pancreatic secretory trypsin inhibitor (Kazal type) and trypsinogen at 1.8 Å resolution: Structure solution, crystallographic refinement and preliminary structural interpretation

The three-dimensional structure of the proteic complex formed by bovine trypsinogen and the porcine pancreatic secretory trypsin inhibitor (Kazal type) has been solved by means of Patterson search techniques, using a predicted model of the trypsin-ovomucoid complex ( Papamokos et al., 1982 ). The structure of the complex, including 162 solvent molecules, has been refined at 1.8 Å resolution (26,341 unique reflections) to a conventional crystallographic R factor of 0.195. The inhibitor molecule binds to trypsinogen via hydrogen bonds and/or apolar interactions at sites P9, P7, P6, P5, P3, P1, P1′, P2′ and P3′ of the contact area. The structure of the inhibitor itself resembles closely that of the third domain of Japanese quail ovomucoid inhibitor, recently reported by Weber et al. (1981) . The trypsinogen part of the complex resembles trypsin, as is the case in the trypsinogen-basic pancreatic trypsin inhibitor complex, but two segments of the activation domain adopt a different conformation. Most notably in the N-terminal region the Ile16-Gly19 loop, which is disordered in free trypsinogen and in the trypsinogen-basic pancreatic trypsin inhibitor complex ( Huber & Bode, 1978), assumes a regular structure and the polypeptide chain can be traced as far as residue Asp14. This new and fixed structure allows the formation of a buried salt link between the side-chains of Lys156 and Asp194. Conformations differing from those of trypsin are also found for residues 20 to 28 and residues 141 to 155. Some structural perturbation is observed in other parts of the molecule, including the calcium loop.

Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from alpha-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity.

We report a novel assay method for enterokinase capable of detecting approx. 1 fmol of enzyme. The method depends on quantification of the release of specifically radiolabelled activation peptides from bovine trypsinogen and is unaffected by trypsin inhibitors. The assay is applicable to biological fluids such as serum. The substrate was produced by selective epsilon-amidination of bovine trypsinogen followed by acetylation with [3H]acetic anhydride and deprotection. The assay has been used to study the effects of pH, Ca2+, ionic strength abd glycodeoxycholate on enterokinase activity.

Interaction of chymotrypsinogens with alpha 1-protease inhibitor.

In a previous report [Largman, C., Brodrick, J.W., Geokas, M.C., Sischo, W.M., & Johnson, J.H. (1979) J. Biol. Chem. 254, 8516-8523] it was demonstrated that human proelastase 2 and alpha 1-protease inhibitor react slowly to form a complex that is stable to denaturation with sodium dodecyl sulfate and beta-mercaptoethanol and that the zymogen can be recovered from the isolated complex following dissociation by hydroxylamine. The present report demonstrates that bovine chymotrypsinogen A reacts with human alpha 1-protease inhibitor in a very similar manner. The rate of complex formation was measured by two methods. In the first, the reaction was followed by determining the loss of the inhibitory activity of alpha 1-protease inhibitor as a function of time. A second-order rate constant for complex formation formation (pH 7.6, 36 degrees C) of 12.9 +/- 2.4 M-1s-1 was obtained. In the second procedure, the reaction of fluorescein isothiocyanate labeled chymotrypsinogen A with alpha 1-protease inhibitor was measured by fluorescence polarization. A second-order rate constant (pH 7.6, 37 degrees C) of 13.9 +/- 2.1 M-1s-1 was obtained. The rate of complex formation is approximately 10(-5) of that measured for the reaction of bovine chymotrypsin with alpha 1-protease inhibitor. Dissociation of the complex was not observed after dilution or the addition of excess bovine alpha-chymotrypsin. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments, human chymotrypsinogens I and II react with alpha 1-protease inhibitor at rates that are approximatley equivalent to that determined for bovine chymotrypsinogen A. In contrast, bovine trypsinogen reacts very slowly with alpha 1-protease inhibitor, at a rate that is at most 10(-2) of that of bovine chymotrypsinogen A. These results suggest that zymogens react with alpha 1-protease inhibitor by virtue of partially formed active sites and that the potential active-site specificity of the zymogen in part determines the rate of complex formation.

Trypsinogen-kinase from Aspergillus fumigatus.

The activation of bovine trypsinogen by an extracellular acid proteinase from A. fumigatus is described. The enzyme activates trypsinogen optimally at pH 3.5 and 32 degrees C. The effect of substrate and enzyme concentrations on the activation has been studied and the Km-value has been determined.

Low-temperature protein crystallography. Effect on flexibility, temperature factor, mosaic spread, extinction and diffuse scattering in two examples: bovine trypsinogen and Fc fragment

Low-temperature protein crystallography. Effect on flexibility, temperature factor, mosaic spread, extinction and diffuse scattering in two examples: bovine trypsinogen and Fc fragment

Conformational transition from trypsinogen to trypsin: 1H nuclear magnetic resonance at 360 MHz and ring current calculations

The 1 H nuclear magnetic resonance spectrum of bovine trypsinogen differs from that of trypsin. The appearance of the high field region of the spectrum is dependent on temperature, pH and the addition of calcium, lanthanides, the basic pancreatic trypsin inhibitor, and H-Ile-Val-OH. In particular, simultaneous addition of trypsin inhibitor and H-Ile-Val-OH to trypsinogen induces the high field spectrum of the latter to resemble closely that of trypsin, and this change is reversible. Resonances of the N-terminal “activation” hexapeptide of trypsinogen are identified by the use of paramagnetic lanthanides and pH titrations. The spectroscopie evidence indicates a flexible “random coil” conformation for this hexapeptide in trypsinogen. Detailed ring current analyses are described, which are based on ten sets of crystal co-ordinates for trypsin and trypsinogen, and six sets for the trypsin inhibitor, together with the Johnson-Bovey ring current equation calibrated previously for this application. These show that the high field signals of the proteinase and its zymogen in both the free form and its complex with trypsin inhibitor arise from about 16 methyl groups found in the five-sixths of the proteinase not involved in the “activation domain”. Tentative assignments of the two most high field shifted signals are proposed. Combination with the spectral results shows that trypsinogen activation involves several subtle differences of conformation and flexibility in localized regions of the protein that were previously thought to be similar within the precision of the crystallographic analyses.

Effect of manganese(II) ion on trypsinogen activation

The effect of Mn 2+ and Ca 2+ on the kinetics of the tryptic activation of bovine trypsinogen was studied at pH 7.3 and 36.5°C. For comparison, the rate constants of autolysis and esterolytic activity of trypsin were also determined. It can be concluded that Mn 2+ increases the conversion rate of trypsinogen into trypsin in a 25–40% larger extent than Ca 2+. The manganese(II) ion bond to trypsinogen is supposed to keep the N-terminal part of the zymogen in a better conformation for binding at the primary and secondary binding sites of trypsin.

An assay for human enterokinase based on detecting the activation peptide of bovine trypsinogen [proceedings

An assay for human enterokinase based on detecting the activation peptide of bovine trypsinogen [proceedings

Comparative studies on the mechanism of activation of the two human trypsinogens

The activation of human trypsinogens 1 and 2 by porcine enterokinase at pH 5.6 shows that the two human zymogens are equivalent substrates for this enzyme and that both proteins are activated faster than the cationic bovine trypsinogen. At pH 8.0 and in the presence of 20 mM calcium the two human trypsinogens are activated by either human trypsin at the same rate but the affinity of both trypsins is higher for trypsinogen 1 than for trypsinogen 2. Two Ca 2+ binding sites are identified in the two human zymogens and their p K(Ca 2+) values determined. For trypsinogen 1 the values are respectively of 2.8 and 3.3 for the primary and secondary Ca 2+ binding sites, and for trypsinogen 2 of 3.4 and 2.7. These values are markedly different from those obtained for bovine cationic trypsinogen, especially in the case of trypsinogen 1. These results point out a different degree of saturation of the calcium binding sites of the 2 human zymogens that must exist in physiological conditions, suggesting different biological activities of the two trypsinogens.

Kinetics and mechanism for the conformational transition in p-guanidinobenzoate bovine trypsinogen induced by the isoleucine-valine dipeptide.

The interaction between p-guanidinobenzoate-trypsinogen and the isoleucine-valine dipeptide has been investigated by temperature-jump relaxation spectrometry. Using the absorbance at 281 nm the concentration dependence of the relaxation parameters is consistent with the conventional induced-fit model: rapid ligand binding coupled to a slower intramolecular change; some alternative mechanisms can be excluded. At 296 K, 0.1 M Tris HCl, pH = 7.4, the dissociation equilibrium constant for the overall process is K = 5.1(+/- 0.2) X 10(-5) M; for the binding step K1 = 2.3(+/- 0.3) X 10(-3) M and the rate constants for the structural change are k2 = 26(+/-6)s-1 and k-2 = 0.61(+/- 0.04)s-1; the overall dissociation reaction enthalpy is delta H0 = 26(+/-6)KJmol-1 and the reactiom entropy is delta S0 = 4(+/- 20) kJ-1 mol-1. In combination with CD and X-ray crystallographic data, the results of this study suggest that the binding of the dipeptide to a trypsinogen-like, partially disordered conformation induces a transition to a trypsin-like highly ordered structure.

Refolding of bovine trypsinogen with one and two disulfide bonds reduced and carboxymethylated.

Refolding of bovine trypsinogen with one and two disulfide bonds reduced and carboxymethylated.

Refolding of the mixed disulfide of bovine trypsinogen and glutathione.

The mixed disulfide of bovine trypsinogen and glutathione refolded with high yields at protein concentrations of 20 microgram/ml or less, at 4-25 degrees C, pH 8.0 to 8.7, in the presence of 3 to 6 mM cysteine under anaerobic conditions. The regenerated protein behaved as native trypsinogen as judged by gel exclusion chromatography, isoelectric focusing, and activation with bovine enterokinase or trypsin. However, refolded samples that were quenched with iodoacetate and analyzed by disc gel electrophoresis formed two components corresponding to trypsinogen and S-(carboxymethylcysteine)2-(179-203)-trypsinogen. The use of cysteine as a disulfide interchange catalyst caused reduction of the 179 to 203 disulfide bond, and quenching of the refolding mixture with iodoacetate produced the carboxymethylated derivative. The overall yield of the regenerated product was 70% and the half-time at 4 degrees C was 55 min.

The preparation and properties of bovine enterokinase.

Bovine enterokinase was purified from duodenal mucosa. The purification included an initial extraction with 2% deoxycholate, ammonium sulfate fractionations, DEAE-cellulose chromatography, and affinity chromatography on basic pancreatic trypsin inhibitor (Kunitz) (PTI)-Sepharose. The purified enzyme contained 35% carbohydrate; it had a molecular weight of 150,000, with a heavy (115,000) and light (35,000) chain connected by one or more disulfide bonds. Enterokinase hydrolyzed lysine and arginine substrates and slowly reacted with the trypsin active site titrant 4-methylumbelliferyl-p-guanidinobenzoate. The enzyme activated bovine trypsinogen with kinetic parameters similar to those of other preparations of enterokinase. Bovine enterokinase was inhibited by Kunitz pancreatic trypsin inhibitor with a Kassoc of 2 X 10(8) M-1 and only weakly by other proteinase inhibitors. The amino acid composition differed from bovine enterokinase isolated from duodenal contents (Anderson, L.E., Walsh, K.A., and Neurath, H. (1977) Biochemistry 16, 3354-3360). The mucosal enzyme and the duodenal contents enzymes also differed in the size of the heavy and light chains. The mucosal enterokinase more closely resembled the properties of porcine enterokinase (Baratti, J., Maroux, S., Louvard, D., and Desnuelle, P. (1973) Biochim. Biophys. Acta 315, 147-161). The amino acid composition and size of the light chain were also similar to bovine trypsin.

The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding: II. The binding of the pancreatic trypsin inhibitor and of isoleucine-valine and of sequentially related peptides to trypsinogen and to p-guanidinobenzoate-trypsinogen

p-Guanidinobenzoate-trypsinogen is transformed into a trypsin-like conformation upon binding of Ile-Val as evidenced by specific changes in its circular dichroism spectrum. By means of this signal the association constants for the binding of a variety of peptides sequentially analogous to either the bovine trypsin N-terminus or to the N-terminal activation peptide sequences of several trypsinogens have been determined at different Ca 2+ concentrations. Ile-Val and Ile-Val-Gly exhibit the strongest binding affinity of all peptides investigated. Replacement of the first isoleucine or of the second valine residue by other amino acids considerably reduces the peptide affinity. Discussion of these is based on the known spatial arrangement of the Ile16-Val17-Gly18 N-terminus and of the Ile-Val dipeptide in the Ile16 cleft (crystal structures of bovine trypsin and of the trypsinogen-PTI ‡ ‡ Abbreviations used: PTI, pancreatic trypsin inhibitor, Trasylol R (Bayer AG); pGB, p-guanidinobenzoate; NPGB, p-nitrophenole- p′-guanidinobenzoate; Tg, trypsinogen. The amino acid sequence numbers refer to the chymotrypsinogen enumeration (Hartley & Shotton, 1971); residues of PTI and soybean trypsin inhibitor are indicated by (I). -Ile-Val complex; Bode et al., 1978). The free energies of binding of the first and of the second peptide residue are almost additive indicating independency between both subsites. The third residue, glycine, does not significantly contribute to binding. The peptide analogues of various trypsinogen N-termini exhibit no measurable affinity for the Ile 16 cleft. The equilibrium constant for the binding of PTI to trypsinogen and the affinity of Ile-Val for the resulting binary complex have been determined in the presence and absence of Ca 2+, using the competitive PTI-binding to α-chymotrypsin. These competition experiments allow the estimation of the standard free-energy changes due to the conformational transition of trypsinogen into a trypsin-like state (+43 kJ mol −1, 20 °C; stabilization of the “activation domain”; Fehlhammer et al., 1977), due to the binding of the trypsin N-terminus (—55 kJ mol −1) and of the peptide analogues (e.g. Ile-Val; −28 kJ mol −1) into the preformed Ile 16 cleft, and due to the specific burying of the covalently linked pGB group in the fixed specificity pocket (— 39 kJ mol −1). This pocket is co-operatively linked with the Ile 16 cleft according to a free-energy change coupling of —43 kJ mol −1.