Multiple intermediates in the reaction of bovine beta-trypsin with bovine pancreatic trypsin inhibitor (Kunitz)

The kinetics of the formation of the complex between bovine β-trypsin and the bovine basic pancreatic trypsin inhibitor (BPTI) was investigated using three different signals: the displacement of proflavine, the optical density changes in the UV region, and the loss of the enzymatic activity. For the three different signals, with inhibitor in excess over bovine β-trypsin ([BPTI] ≥ 5 × [bovine β-trypsin]), the time course of the reaction corresponds to a pseudo-first-order process. The concentration dependence of the rate is second order at low BPTI concentrations and tends to first order at high inhibitor concentrations. This behavior may be explained by relatively rapid preequilibria followed by limiting first-order processes according to The values of K_i, k_+i, and k_(on)i ( = k_+i/K_i) have been determined for the different reactions at three pH values: 6.80, 4.80, and 3.50. The kinetic parameters differ widely for the processes reflected by the various signals; the difference increases upon lowering pH. The results indicate that the formation of the bovine β-trypsin–BPTI complex is not an all-or-nothing process, but involves several intermediates corresponding to discrete reaction steps, which are differently affected by ionization processes.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) to human and bovine factor Xa. A thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human and bovine factor Xa (Stuart-Prower factor; EC 3.4.21.6) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to human and bovine factor Xa are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 degrees C) for BPTI binding to human and bovine factor Xa decrease, thus reflecting the acidic pK shift of the His57 catalytic residue from 7.1, in the free enzyme, to 5.2, in the proteinase-inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human and bovine factor Xa are: Ka = 2.1 x 10(5)M-1 (at 21.0 degrees C), delta G degree = -29.7 kJ/mol (at 21.0 degrees C), delta S degree = +161 entropy units (at 21.0 degrees C), and delta H degree = +17.6 kJ/mol (temperature-independent over the explored range, from 5.0 degrees C to 45.0 degrees C). Thermodynamics of BPTI binding to human and bovine factor Xa have been analysed in parallel with those of related serine (pro)enzyme/Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human and bovine factor Xa was related to the inferred stereochemistry of the proteinase/inhibitor contact region.     

On the reaction of acetamidomethanol with native and reduced bovine pancreatic trypsin inhibtor (Kunitz inhibitor).

The stability of native and reduced bovine pancreatic trypsin inhibitor (Kunitz inhibitor) in anhydrous hydrogen fluoride and their reaction with acetamidomethanol, in the same solvent, have been investigated. The bovine Kunitz inhibitor appears to be stable in liquid hydrogen fluoride but the reduced molecule loses about 50% of its ability to regain inhibitory power, upon air oxidation, by exposure to this solvent. Tyrosine residues appear to be affected by acetamidomethylation of the native protein to give a modified inhibitor which is still highly active in inhibiting trypsin. The extent of correct refolding, upon reoxidation, of the reduced tyrosine modified-inhibitor is greatly diminished. Tyrosine modification can be prevented by carrying out the acetamidomethylation reaction in the presence of excess anisole. The stability constants and the standard free energies of binding of the complexes between trypsin and the native and the tyrosine modified-inhibitor have been determined.     

Heterogeneity of the basic pancreatic inhibitor (Kunitz) in various bovine organs

Four protein protease inhibitors (I, II, III, IV) having low molecular weights (10 600-6500) and basic isoelectric points were isolated by affinity chromatography from bovine spleen. Inhibitor IV was identified as the basic pancreatic trypsin inhibitor (Kunitz inhibitor); the presence and distribution of components I, II and III vary in the different bovine organs. Spleen inhibitors I, II, III and IV were purified by ion-exchange chromatography; they form 1:1 complexes with trypsin and inhibit enzymatic activity of trypsin, chymotrypsin and kallikrein. Inhibitors I, II and III contain carbohydrate moieties (7-4%) covalently bound to the polypeptide chain. Specific basic pancreatic trypsin inhibitor antiserum has shown the complete identity between inhibitor IV and the basic pancreatic trypsin inhibitor, while partial cross-reactivity between the basic pancreatic trypsin inhibitor and inhibitors I, II and III can be seen from a double immunodiffusion test.     

Binding of porcine pancreatic secretory trypsin inhibitor to bovine beta-trypsin: a kinetic study

The kinetics of the formation of the complex between bovine β-trypsin and the porcine pancreatic secretory trypsin inhibitor (PSTI; Kazal-type inhibitor) was investigated following the spectral changes associated with the displacement of proflavine from the enzyme, upon inhibitor binding, between pH 3.5 and 8.0 (I = 0.1M) at 21 ± 0.5°C. With inhibitor in excess over the enzyme ([PSTI] ≥ 5 × [bovine β-trypsin]), the time course of the reaction corresponds to a pseudo-first-order process. Over the whole pH range explored, the concentration dependence of the rate is second order at low PSTI concentrations but tends to first order at high inhibitor concentrations. This behavior may be explained by a relatively fast pre-equilibrium followed by a limiting first-order process. Values of kinetic parameters for PSTI binding to bovine β-trypsin depend, between pH 3.5 and 8.0, on the acid–base equilibrium of a single ionizing group (probably His-57 of bovine β-trypsin) that undergoes an acidic pK_a shift from 7.0 in the free bovine β-trypsin to 5.5 in the enzyme:PSTI complex. Kinetics of the bovine β-trypsin:PSTI adduct formation has been analyzed and compared with that of other (pro)enzyme:inhibitor reactions. Considering the known molecular structures of free serine (pro)enzymes, of Kazal- and Kunitz-type inhibitors, as well as of their complexes, the binding behavior of PSTI to bovine β-trypsin has been related to the inferred stereochemistry of the proteinase:inhibitor contact region.     

The inhibition of human leukocyte elastase by basic pancreatic trypsin inhibitor

The effects of 30-min intravenous infusions of ethanol (about 50 mm blood concentration), acetaldehyde (about 100 μm blood concentration), and acetate (equimolar dose to acetaldehyde) were studied in normal and adrenalectomized rats. Blood glucose, plasma free fatty acids (FFA), plasma immunoreactive insulin, and glucagon and hepatic glycogen concentrations were measured. Ethanol itself in the presence of 4-methylpyrazole (4-MP) produced no marked changes in the parameters measured. Its infusion without 4-MP reduced plasma insulin by 35% in the normal rats, but not in the adrenalectomized rats, with no simultaneous changes in blood glucose. Acetaldehyde infusion produced hyperglycemia and relatively slight hyperinsulinemia in the normal rats, but not in the adrenalectomized rats. Equimolar acetate was not as potent a stimulator of glycogenolysis as acetaldehyde. Plasma FFA concentrations were markedly reduced by ethanol (without 4-MP), acetaldehyde and acetate both in the normal and adrenalectomized rats, but in the presence of 4-MP ethanol was without effect. The results indicate that metabolites of ethanol (mostly acetaldehyde) produced during ethanol oxidation in vivo are responsible for the stimulation of glycogenolysis through the release of catecholamines from the adrenal glands. The ethanol-induced decrease in plasma FFA is also attributable to the metabolites of ethanol, acetaldehyde having a more potent depressing action than acetate. The mode of inhibition of lipolysis is not related to hormonal factors.     

Influence of various fluorescent and non-fluorescent labels on the kinetics of the complex formation of alpha-chymotrypsin with basic pancreatic trypsin inhibitor (Kunitz).

The association of alpha-chymotrypsin with basic pancreatic trypsin inhibitor was studied using extrinsic signals produced by fluorescent and nonfluorescent labels. The reactive dyes were covalently bound to the proteins in the complexed state, in which the binding region was protected. The signals were sufficiently large to measure the complex formation at protein concentrations of 10(-9)M by fluorescence and down to 10(-6)M by absorption. Therefore, the association and dissociation could be followed over a broad range of concentration. Good correspondence was observed between data which were obtained with different labels and with published values for the unlabeled proteins. Existing differences could be explained by different buffer conditions used by the different authors. Also the pH dependence of the dissociation rate constants was essentially unaltered by the introduction of the labels. The large signals allowed a direct measurement of the equilibrium constants of dissociation, even at high pH, at which they are in the range of 10(-8)M. The experimentally determined binding constants were in agreement with those calculated from the rate constants. The temperature dependence of the binding constants revealed a small positive and pH-dependent enthalpy change [deltaHo = 4.0 kcal/mol (16.7 kJ) at H 7.0[. The results prove that the labeling can be performed in such a way that the equilibrium and kinetic parameters of the system studied are not significantly influenced.     

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.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to the 33,000 Mr and 54,000 Mr species of human urokinase: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to the 33,000 Mr and 54,000 Mr species of human urokinase (EC 3.4.21.31) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to the 33,000 Mr and 54,000 Mr species of human urokinase are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 degrees C) for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) decrease thus reflecting the acidic pK-shift of the His-57 catalytic residue from 6.9, in the free enzyme, to 5.1, in the proteinase:inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) are: Ka = 4.9 x 10(4) M-1, delta G degree = -6.3 kcal/mol, and delta S degree = -37 entropy units (all at 21.0 degrees C); and delta H degree = +4.6 kcal/mol (temperature independent over the explored range, from 5.0 degrees C to 45.0 degrees C). Thermodynamics of BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) have been analyzed in parallel with those of related serine (pro)enzyme Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human urokinase (33,000 Mr and 54,000 Mr species) was related to the inferred stereochemistry of the proteinase/inhibitor contact region.     

A circular dichroism study of the cyanogen bromide fragments of soybean trypsin inhibitor (Kunitz).

Glutathione reductase (NAD(P)h:oxidized glutathione oxidoreductase, EC 1.6.4.2) has been purified 1000-fold from the cytoplasmic fraction of human platelets. Salts, including the heretofore unreported effect of sodium citrate, activate the NADPH-dependent reduction of oxidized glutathione. Sodium citrate and monovalent salt activation appears to involve multiple sites having different binding affinities. At sub-saturating sodium phosphate, non-linear double reciprocal plots indicative of substrate activation by oxidized glutathione were observed. Initial velocity double reciprocal plots at sub-saturating and saturating concentrations of phosphate generate a family of converging lines. NADP+ is a partial inhibitor, indicating that the reduction of oxidized glutathione can proceed by more than one pathway. FMN, FAD, and riboflavin inhibit platelet glutathione reductase by influencing only the V while nitrofurantoin inhibition is associated with an increase Koxidized glutathione and a decreased V.     

Conformational aspects of beta-trypsin interaction with substrates and pancreatic trypsin inhibitor. III. Catalytic act of trypsin and its inhibition

Basing on the results of the theoretical conformational analysis of the nonbonded and valence complexes of trypsin with substrate molecules, the catalytical act of the enzyme is described in details as a spontaneous process. Conformational aspects of interactions of trypsin with pancreatic trypsin inhibitor are analysed. The complete inhibition process and the geometry of the enzyme-inhibitor complex are described in details. The point amino acid replacements, which will provide for an exclusion of BPTI inhibition and will radically change the specificity of the enzyme are proposed.     

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.