Bovine pancreatic trypsin inhibitor and related isoinhibitors in bovine liver

Summary A Kunitz-type inhibitor family has been biochemically and histochemically characterized in bovine liver. This family includes the well-known pancreatic trypsin inhibitor (BPTI) and three BPTI-related molecular forms (isoinhibitors I, II and III). The purification of the inhibitors was performed by affinity chromatography on immobilized trypsin followed by fast protein liquid chromatography. The inhibitors were identical to those identified previously in bovine spleen and lung. Light immunohistochemical experiments were done by a streptavidin-biotin-peroxidase method using two different immunoglobulin preparations, which selectively discriminated between BPTI and the other isoinhibitors. BPTI-related immunoreactivity was found exclusively at the level of isolated cells, of which many were identified as mast cells by toluidine blue staining. By contrast, isoinhibitor-related immunoreactivity showed a more widespread distribution, including hepatocytes, mast cells and biliary duct epithelial cells. Finally, specific immunoreactivity was also present in plasma. These results suggest that: i) BPTI and related isoinhibitors may be involved in the regulation of the activity of some mast cell proteases, as it happens in other bovine organs (Businaro et al. 1987, 1988); ii) BPTI isoinhibitors, but not BPTI itself, may also control proteolytic activities in hepatic specific structures (hepatocytes and biliary duct epithelial cells).     

Vascular localization of bovine pancreatic trypsin-inhibitor-related molecular forms in bovine spleen

Bovine spleen proteic inhibitors of serine proteases, belonging to the bovine pancreatic trypsin inhibitor (BPTI or aprotinin) family, have been localized, using immunocytochemical techniques, in the smooth muscle cells of some bovine spleen blood vessels. This vascular localization also occurs in a variety of bovine organs and differs from that of BPTI itself which is found exclusively in bovine mast cells, in agreement with previous reports. These data would be in favour of a possible involvement of one or more BPTI-type inhibitors in vascular processes by acting at the level of the smooth muscle cells, the tissue responsible for vasodilation/vasoconstriction events.     

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.     

Flexibility of bovine pancreatic trypsin inhibitor

The native conformation of a protein may be expressed in terms of the dihedral angles, phi's and psi's for the backbone, and kappa's for the side chains, for a given geometry (bond lengths and bond angles). We have developed a method to obtain the dihedral angles for a low-energy structure of a protein, starting with the X-ray structure; it is applied here to examine the degree of flexibility of bovine pancreatic trypsin inhibitor. Minimization of the total energy of the inhibitor (including nonbonded, electrostatic, torsional, hydrogen bonding, and disulfide loop energies) yields a conformation having a total energy of -221 kcal/mol and a root mean square deviation between all atoms of the computed and experimental structures of 0.63 A. The optimal conformation is not unique, however, there being at least two other conformations of low-energy (-222 and -220 kcal/mol), which resemble the experimental one (root mean square deviations of 0.66 and 0.64 A, respectively). These three conformations are located in different positions in phi, psi space, i.e., with a total deviation of 81 degrees, 100 degrees and 55 degrees from each other (with a root mean square deviation of several degrees per dihedral angle from each other). The nonbonded energies of the backbones, calculated along lines in phi, psi space connecting these three conformations, are all negative, without any intervening energy barriers (on an energy contour map in the phi, psi plane). Side chains were attached at several representative positions in this plane, and the total energy was minimized by varying the kappa's. The energies were of approximately the same magnitude as the previous ones, indicating that the conformation of low energy is flexible to some extent in a restricted region of phi, psi space. Interestingly, the difference delta phi i+1 in phi i+1 for the (i + 1)th residue from one conformation to another is approximately the same as -delta psi i for the ith residue; i.e., the plane of the peptide group between the ith and (i + 1)th residues re-orient without significant changes in the positions of the other atoms. The flexibility of the orientations of the planes of the peptide groups is probably coupled in a cooperative manner to the flexibility of the positions of the backbone and side-chain atoms.     

Identification of a molecular switch that selects between two crystals forms of bovine pancreatic trypsin inhibitor.

Two crystals forms of bovine pancreatic trypsin inhibitor are produced between pH 8.39 and 10.13 when crystals are grown at room temperature from solutions of 1.5 M potassium phosphate. Lower pH values favor the form II crystals, whereas higher pH values favor the form III. The transition from one crystal form to the other occurs at pH 9.35. We examined the crystal lattice contacts in both crystal forms and identified an unusual interaction we believe explains these observations. Spanning the crystallographic 2-fold axis in form III crystals, the Lys 41 side-chain amino nitrogens from 2 symmetry-related molecules are only 2.72 A apart, implying they are hydrogen bonded to one another. In form II crystals, the Lys 41 side-chain amino group is protonated and forms a salt bridge with a solvent-derived phosphate group. For the Lys 41 side-chain amino groups to hydrogen bond in form III crystals, at least 1 member of the pair must be deprotonated. The transition that occurs at pH 9.35 marks the pKa for deprotonation. In solution, the pKa for the Lys 41 side chain is around 10.8. The pKa for one of the interacting Lys 41 side chains in form III crystals is therefore shifted downward by about 1.5 pH units. The energy for lowering the pKa value comes from the many additional intermolecular hydrogen bonds that are present in form III crystals: 19 compared to only 8 in form II crystals.     

The structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor

The structure of the complex between anhydro-trypsin and pancreatic trypsin inhibitor has been determined by difference Fourier techniques using phases obtained from the native complex (Huber et al., 1974). It was refined independently by constrained crystallographic refinement at 1.9 å resolution. The anhydro-complex has Ser 195 converted to dehydro-alanine. There were no other significant structural changes. In particular, the high degree of pyramidalization of the C atom of Lys 15 (I) of the inhibitor component observed in the native complex is maintained in the anhydro-species.     

Renaturation of the reduced bovine pancreatic trypsin inhibitor

Refolding of the reduced pancreatic trypsin inhibitor has been investigated using thiol-disulphide exchange with various disulphide reagents to regenerate the three disulphide bonds. Essentially quantitative renaturation was routinely achieved. The refolded inhibitor was indistinguishable from the original protein in interaction with trypsin and chymotrypsin, electrophoretic mobility, and nature of disulphide bonds.The kinetics of refolding using oxidized dithiothreitol to form the disulphide bonds have been studied in some detail. The renaturation reaction is usually of second-order, being first-order in both inhibitor and disulphide reagent concentrations. A short lag period in the appearance of inhibitor activity and the inhibition of the rate, but not the extent, of renaturation by low levels of reduced dithiothreitol suggest the accumulation of metastable intermediates. In addition, heterogeneity of the refolding reaction is apparent at high concentrations of disulphide reagent, with a fraction of the material being only slowly renatured.     

Bovine pancreatic trypsin inhibitor and related isoinhibitors in bovine liver. A biochemical and histochemical study

A Kunitz-type inhibitor family has been biochemically and histochemically characterized in bovine liver. This family includes the well-known pancreatic trypsin inhibitor (BPTI) and three BPTI-related molecular forms (isoinhibitors I, II and III). The purification of the inhibitors was performed by affinity chromatography on immobilized trypsin followed by fast protein liquid chromatography. The inhibitors were identical to those identified previously in bovine spleen and lung. Light immunohistochemical experiments were done by a streptavidin-biotin-peroxidase method using two different immunoglobulin preparations, which selectively discriminated between BPTI and the other isoinhibitors. BPTI-related immunoreactivity was found exclusively at the level of isolated cells, of which many were identified as mast cells by toluidine blue staining. By contrast, isoinhibitor-related immunoreactivity showed a more widespread distribution, including hepatocytes, mast cells and biliary duct epithelial cells. Finally, specific immunoreactivity was also present in plasma. These results suggest that: i) BPTI and related isoinhibitors may be involved in the regulation of the activity of some mast cell proteases, as it happens in other bovine organs (Businaro et al. 1987, 1988); ii) BPTI isoinhibitors, but not BPTI itself, may also control proteolytic activities in hepatic specific structures (hepatocytes and biliary duct epithelial cells).     

Folding of the twisted beta-sheet in bovine pancreatic trypsin inhibitor

The dominant role of local interactions has been demonstrated for the formation of the strongly twisted antiparallel beta-sheet structure consisting of residues 18-35 in bovine pancreatic trypsin inhibitor. Conformational energy minimization has indicated that this beta-sheet has a strong twist even in the absence of the rest of the protein molecule. The twist is maintained essentially unchanged when energy minimization is carried out by starting from the native conformation. By starting from a nontwisted beta-sheet conformation of residues 18-35, a strongly twisted structure (higher in energy than the native) is obtained. The high twist of the native-like beta-sheet is a consequence of its amino acid sequence, but it is enhanced strongly by interchain interactions that operate within the beta-sheet. The existence of the twisted beta-sheet structure does not require the presence of a disulfide bond between residue 14 and residue 38. It actually may facilitate the formation of this bond. Therefore, it is likely that the beta-sheet structure forms during an earlier stage of folding than the formation of this disulfide bond. This study provides an example of the manner in which conformational energy calculations can be used to provide information about the probable pathway of the folding of a protein.     

Identification and immunohistochemical localization of various bovine pancreatic trypsin inhibitor-isoforms in bovine pituitary gland

Summary Three isoinhibitors of bovine pancreatic trypsin inhibitor (BPTI) have been identified and isolated from bovine pituitary gland. The results of the purification process by affinity chromatography on immobilized trypsin, the electrophoretic mobility in non-denaturing conditions, the antiproteolytic activity and the immunochemical reactions indicate that these inhibitors correspond to those previously isolated from bovine spleen and lung. In addition, immunohistochemical experiments show that the isoinhibitors and BPTI are exclusively localized in the mast cells, and not in the endocrine cells, of the pars intermedia and posterior lobe (neurohypophysis) of the pituitary gland. The physiological implications of these findings are discussed.     

Reduced bovine pancreatic trypsin inhibitor has a compact structure

The conformation of reduced bovine pancreatic trypsin inhibitor (R-BPTI) under reducing conditions was monitored by measurements of nonradiative excitation energy-transfer efficiencies (E) between a donor probe attached to the N-terminal Arg1 residue and an acceptor attached to one of the lysine residues (15, 26, 41, or 46) [Amir, D., & Haas, E. (1987) Biochemistry 26, 2162-2175]. High-excitation energy-transfer efficiencies that approach those found in the native state were obtained for the reduced labeled BPTI derivatives in 0.5 M guanidine hydrochloride (Gdn.HCl) and 4 mM DTT. Unlike the dependence expected for a random coil chain, E does not decrease as a function of the number of residues between the labeled sites. The efficiency of energy transfer between probes attached to residues 1 and 15 in the reduced state is higher than that found for the same pair of sites in the native state or reduced unfolded (in 6 M Gdn.HCl) state. This segment also shows high dynamic flexibility. These results indicate that the overall structure of reduced BPTI under folding (but still reducing) conditions shows a high population of conformers with interprobe distances similar to those of the native state. Reduced BPTI seems to be in a molten globule state characterized by a flexible, compact structure, which probably reorganizes into the native structure when the folding is allowed to proceed under oxidizing conditions.     

Structure of form III crystals of bovine pancreatic trypsin inhibitor

The structure of bovine pancreatic trypsin inhibitor has been solved in a new crystal form III. The crystals belong to space group P2(1)2(1)2 with a = 55.2 A, b = 38.2 A, c = 24.05 A. The structure was solved on the basis of co-ordinates of forms I and II of the inhibitor by molecular replacement, and the X-ray data extending to 1.7 A were used in a restrained least-squares refinement. The final R factor was 0.16, and the deviation of bonded distances from ideality was 0.020 A. Root-mean-square discrepancy between C alpha co-ordinates of forms III and I are 0.47 A, whilst between forms II and III the discrepancy is 0.39 A. These deviations are about a factor of 3 larger than the expected experimental errors, showing that true differences exist between the three crystal forms. Two residues (Arg39 and Asp50) were modeled with two positions for their side-chains. The final model includes 73 water molecules and one phosphate group bound to the protein. Sixteen water molecules occupy approximately the same positions in all three crystal forms studied to date, indicating their close association with the protein molecule. Temperature factors also show a high degree of correlation between the three crystal forms.     

Immunohistochemical localization of pancreatic secretory trypsin inhibitor in fetal and adult pancreatic and extrapancreatic tissues

Pancreatic secretory trypsin inhibitor (PSTI) has been thought to be only a secretory trypsin inhibitor of human pancreas, but the serum content of immunoreactive PSTI is elevated without pancreatic disease. Using the peroxidase-antiperoxidase method, immunoreactive cells for PSTI were found in human pancreas, stomach, duodenum, appendix, colon and urinary tract of both fetus and adult, adult gall bladder, and fetal lung. PSTI-immunoreactive cells were identified in fetal pancreas at the tenth gestational week, and in extrapancreatic tissues at the sixteenth (gastrointestinal and urinary tract) and twentieth weeks (lung). PSTI-immunoreactive cells of fetal lung were present in neuroepithelial bodies. Strongly positive cells in fetal duodenum were argyrophilic and resembled endocrine cells. Immunohistochemical study was also performed on tissues associated with inflammatory diseases of gastrointestinal tract. The distribution pattern of immunoreactive cells in the stomach varied in accordance with chronic gastritis. Immunoreactive cells were also found in endocrine micro-nests and in a carcinoid tumor associated with fundic gastritis. These results suggest that PSTI may play some physiological role other than secretory trypsin inhibition of the pancreas.     

Production and characterization of monoclonal antibodies against bovine pancreatic trypsin inhibitor

Two monoclonal antibodies (MAbs) have been produced, without the use of a supporting carrier, against bovine basic pancreatic trypsin inhibitor (BPTI or aprotinin), a mini-protein composed of 58 amino acids. Both MAbs obtained were found to be IgM. One of them was purified and further characterized. This MAb (ICI) binds to the immunogen with an association constant of 1.6 X 10(6)M-1 at pH 7.4. Competition experiments with trypsin or inactivated trypsin demonstrate that ICI MAb interacts with BPTI at, or near, the proteinase-binding site. ICI MAb binds, with a much lower association constant (approximately 200M-1), to an isoinhibitor (spleen inhibitor II) which differs from BPTI in seven amino-acids; three of these substitutions are at the active site, in the contact area with the proteinase.     

Prediction of a low-frequency mode in bovine pancreatic trypsin inhibitor molecule

One of the frontiers today in molecular biology is to measure, identify and go further to predict the low-frequency internal motion of biological macromolecules, which is crucially important for understanding the dynamic mechanism of various biological functions occurring in such molecules. Based on the theory of continuity model developed recently for dealing with the internal low-frequency motion of a biological macromolecule, it is predicted that the low-frequency phonons with wave number of about 23 cm^?1 might be excited in BPTI molecule.     

Stability studies on derivatives of the bovine pancreatic trypsin inhibitor

Gibbs energy, enthalpy, and entropy data were determined for two selectively modified analogues of bovine pancreatic trypsin inhibitor (BPTI) to provide a model free set of thermodynamic parameters that characterize (a) the energetic and entropic contributions of the 14-38 disulfide bridge and (b) the variation of the overall stability resulting from the introduction of two negative charges into the positions 14 and 38. The two BPTI analogues studied were BPTI having Cys-14 and Cys-38 carboxymethylated (BPTI-RCOM) and BPTI having Cys-14 and Cys-38 carboxamidomethylated (BPTI-RCAM). They were obtained from native BPTI by reduction, followed by modification of the sulfhydryl groups with iodoacetic acid or iodoacetamide, respectively. The temperature dependence of all thermodynamic parameters of BPTI is drastically altered in the absence of the third disulfide bridge. Even the apparently minute difference of two dissociable carboxyl groups instead of uncharged amide groups in positions 14 and 38 has surprisingly large effects on the temperature dependence of the stabilization enthalpy. The Gibbs energy of BPTI at pH 2, 25 degrees C, decreases by approximately 70% when the 14-38 disulfide bond is cleaved. BPTI-RCOM is more stable than BPTI-RCAM in the whole pH range studied. The difference of -4 kJ/mol at pH 2, 25 degrees C, is reduced to -2.7 kJ/mol at pH 5, 25 degrees C. This finding demonstrates that the presence of two negative charges reduces the higher stability of BPTI-RCOM slightly; however, the overall effect of the two charges is still a stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR-detected order in core residues of denatured bovine pancreatic trypsin inhibitor

The NMR characteristics of [14-38]Abu, a synthetic variant of BPTI that is partially folded in aqueous buffer near neutral pH, support a model of early folding events which begin with stabilization of the nativelike, slow exchange core [Barbar, E., Hare, M., Daragan, V., Barany, G., and Woodward, C. (1998) Biochemistry 37, 7822-7833 (1)]. In partially folded [14-38]Abu, urea denaturation profiles for representative amide protons show that global unfolding is non-two-state and that core residues require a higher concentration of urea to unfold. Dynamic properties of pH-denatured [14-38]Abu and fully reduced and unfolded BPTI analogue were determined from heteronuclear NMR relaxation measurements at similar solution conditions. Differences at various sites in the polypeptide chain were evaluated from spectral density functions determined from T1, T2, and steady-state heteronuclear NOE data. Although denatured [14-38]Abu contains no persistent secondary structure, its most ordered residues are those that, in native BPTI, fold into the slow exchange core. The fully reduced analogue is significantly more mobile and shows less heterogeneous dynamics, but at 1 degree C, restricted motion is observed for residues in the central segments of the polypeptide chain. These observations indicate that there is a developing core or cores even in highly unfolded species. Apparently the effect of 14-38 disulfide on unfolded     

Fluorescence depolarization and rotational modes of tyrosine in bovine pancreatic trypsin inhibitor

The fluorescence of bovine pancreatic trypsin inhibitor (BPTI) is due to one or more of its four tyrosine residues. Observations of the stationary polarization of the fluorescence over a large range of temperatures and viscosities permit the demonstration of at least three modes of tyrosine rotation, and perhaps an ultrafast fourth one. The slowest mode is one of motion of the whole molecule; the second, a much faster motion limited to an amplitude of 11 degrees, is not changed by quenching of the fluorescence through addition of citrate and is therefore ascribed to the motion of internal tyrosines of BPTI. The third mode of motion is faster still; it has an amplitude similar to that of the second and, being sensitive to citrate quenching, is attributed to the rotation of the external tyrosine residue. A residual depolarization corresponding to a rotational amplitude of 22 degrees is deduced by comparison of the polarizations of BPTI and tyrosine dissolved in 80% glycerol-water at -40 degrees C. It is in accord in amplitude with the picosecond tyrosine rotations predicted by Karplus and collaborators from molecular dynamics computer simulations, but it could also originate, in whole or in part, from electronic energy transfer among the tyrosines.