Oscillations of trypsinogen activation

Trypsin activity oscillations are shown by the autocatalytic activation of trypsinogen at 0 degrees C in aqueous solution. The oscillations were observed for 3-4 days and show only slight decrease in enzyme activity. The zymogen has been kept at ice water temperature and pH 8.2 in the presence of Mn2+ ion. The mean periods of around 1.5 hr are about half of those found previously at -10 degrees C in frozen aqueous solution, while the amplitudes related to the mean activity are about one-fourth of that in the frozen experiments. The phenomenon of oscillation is interpreted in terms of coupling between the inhomogeneities of protein and ion concentrations of the unstirred solution and a Mn3+/Mn2+ system, causing synchronous, periodic reduction-oxidation of some cystine bridges in the protein chain. These nonequilibrium conditions, together with synchronous transitions among several conformational states, may produce the observed activity oscillations.     

Evolution of trypsinogen activation peptides

The activation peptide of mammalian trypsinogens contains a highly conserved tetra-aspartate sequence (D19-D20-D21-D22) preceding the K23-I24 scissile peptide bond, which is hydrolyzed as the first step in the activation process. Here, we examined the evolution and function of trypsinogen activation peptides through integrating functional characterization of disease-associated mutations with comparative genomic analysis. Activation properties of three chronic pancreatitis-associated activation peptide mutants (the novel D19A and the previously reported D22G and K23R) were simultaneously analyzed, for the first time, in the context of recombinant human cationic trypsinogen. A dramatic increase in autoactivation of cationic trypsinogen was observed in all three mutants, with D22G and K23R exhibiting the most marked increases. The physiological activator enteropeptidase activated the D19A mutant normally, activated the D22G mutant very poorly, and stimulated activation of the K23R mutant. The biochemical and structural data, taken together with a comprehensive sequence comparison, indicates that the tetra-aspartate sequence in mammalian trypsinogen activation peptides has evolved not only for optimal enteropeptidase recognition in the duodenum but also for efficient inhibition of trypsinogen autoactivation within the pancreas. Moreover, the use of lysine instead of arginine at the P1 position of activation peptides also has an advantageous effect against trypsinogen autoactivation. Finally, fixed substitutions in the key residues of the trypsinogen activation peptide may suggest the evolution of new functions unrelated to digestion, as found in the group III trypsinogens of cold-adapted fishes.     

Thermodynamic equilibria of cholesterol-detergent-water.

Cholesterol monomer is incorporated into alkyl sulfate micelles with a unitary free energy of -10.3 kcal/mol. This experimental free energy is in good agreement with that predicted by our previous determination of the hydrophobicity of the sterol suggesting that the partitioning is primarily hydrophobic with little or no contribution to the free energy from head group interactions in this system. The intrinsic hydrophobicity of cholesterol is shown to be insufficient for effective partitioning of the sterol between micelles (or bilayers) and its own self-associated form. This finding strongly supports a model of phospholipid-cholesterol interaction involving significant free energy contributions from head group effects such as alterations in hydrogen bonds or hydration. Since these head group contributions are not observed in the cholesterol-alkyl sulfate system, one concludes that there is a high degree of specificity of interaction between the sterol OH and polar moieties of other amphiphilic molecules.     

Kinetics of the trypsinogen activation by enterokinase and trypsin

A global kinetic analysis of the mechanisms of the trypsinogen activation by enterokinase and trypsin is presented. The kinetic equations of both the transient-phase and the steady-state of these mechanisms are presented. In addition, we here derive the corresponding kinetic equations for the case in which the condition of rapid equilibrium prevails and we propose a kinetic data analysis. The significance of this approach to the treatment of other zymogen activation processes is discussed.     

Involvement of autophagy in trypsinogen activation within the pancreatic acinar cells

Autophagy is mostly a nonselective bulk degradation system within cells. Recent reports indicate that autophagy can act both as a protector and killer of the cell depending on the stage of the disease or the surrounding cellular environment (for review see Cuervo, A.M. 2004. Trends Cell Biol. 14:70-77). We found that cytoplasmic vacuoles induced in pancreatic acinar cells by experimental pancreatitis were autophagic in origin, as demonstrated by microtubule-associated protein 1 light chain 3 expression and electron microscopy experiments. To analyze the role of macroautophagy in acute pancreatitis, we produced conditional knockout mice lacking the autophagy-related 5 gene in acinar cells. Acute pancreatitis was not observed, except for very mild edema in a restricted area, in conditional knockout mice. Unexpectedly, trypsinogen activation was greatly reduced in the absence of autophagy. These results suggest that autophagy exerts devastating effects in pancreatic acinar cells by activation of trypsinogen to trypsin in the early stage of acute pancreatitis through delivering trypsinogen to the lysosome.     

Thermodynamic aspects of the co-operativity in four-step oxygenation equilibria of haemoglobin

Accurate oxygen equilibrium curves of human haemoglobin (concentration, 600 μm as haem) were determined by an automatic recording method (Imai et al., 1970) under a variety of conditions combining six different temperatures with seven sets of solute conditions, producing wide-ranging structural constraints on haemoglobin. The heat and entropy change of oxygenation for four individual steps (ΔH_tand ΔS_i, i = 1 to 4) were evaluated by a least-squares method directly from each set of six equilibrium curves without knowing the values of the four equilibrium constants k_t. As shown in previous studies with dilute haemoglobin solutions (Imai &; Tyuma, 1973; Imai &; Yonetani, 1975b) ΔH_i depended strongly on i; small amounts of heat were liberated at oxygenation steps involving the release of H⁺ and anions such as Cl^?, 2,3-diphosphoglycerate, and inositol hexaphosphate, while large amounts of heat were liberated on the oxygenation of the R state or highly constrained T state, from which no or few non-haem ligands are released. The observed amounts of heat, when corrected for the heat of H⁺ and anion release associated with oxygenation, became uniform to a good approximation, indicating that the intrinsic heat of haem oxygenation is essentially equal for the four oxygenation steps, and a large part of the non-uniformity of ΔH_i may be ascribed to the oxygen-linked release of the non-haem ligands. ΔS_i exhibited similar behaviour. The relation, k₁ ? k₂ ? k₃ ? k₄ which usually holds under physiological conditions, is a consequence of the presence of an enthalpy-entropy compensation process at the first three steps and its absence at the fourth step. The compensation temperature was around 300 K. The origin of the co-operativity cannot be specified as either an enthalpic or entropic effect. In the presence of 0.1 m-Cl^? and 2 mm-2,3-diphosphoglycerate, the T to R transition at any oxygenation step is an endothermic process and haemoglobin gains entropy on the transition. The deoxy T structure is stabilised by the enthalpy term, while the oxy R structure is stabilised by the entropy term, so that the T to R transition occurs at a stop where the entropy contribution exceeds the enthalpy contribution. The present study shows that the oxygen-linked binding of non-haem ligands is very important in co-operative oxygen binding by haemoglobin, as predicted by Perutz, (1970).     

Evaluation of the kinetic parameters of the activation of trypsinogen by trypsin

Kinetic analysis of the mechanism of trypsinogen activation by trypsin under rapid equilibrium conditions and certain relationships between the rate constants are presented. The kinetic equations are valid from the beginning of the reaction. In addition, we suggest a procedure, based on the above equations, for the evaluation of the kinetic parameters of the reaction. This procedure is applied to a set of experimental data collected during the activation of bovine trypsinogen by trypsin at 30 degrees C (pH 8.1) in 0.01 M CaCl2. In this system, the amount of active enzyme increases exponentially, as expected from an autocatalytic process. The apparent rate constant, delta, governing this increase would vary linearly with the trypsinogen concentration, [Z]0, if no Michaelis complex was detectable. However, the increase in delta with [Z]0 is clearly non-linear and fits a hyperbola (delta = k2[Z]0/(Kz + [Z]0)) well.     

Density-functional study on the equilibria in the ThDP activation

The equilibria among the various ionization and tautomeric states involved in the activation of ThDP is addressed using high level density functional theory calculations, X3LYP/6-311++G(d,p)//X3LYP(PB)/6-31++G(d,p). This study provides the first theoretically derived thermodynamic data for the internal equilibria in the activation of ThDP. The role of the medium polarity on the geometry and thermodynamics of the diverse equilibria of ThDP is addressed. The media chosen are cyclohexane and water, as paradigms of apolar and polar media. The results suggest that all ionization and tautomeric states are accessible during the catalytic cycle, even in the absence of substrate, being APH⁺ the form required to interconvert the AP and IP tautomers; and the generation of the ylide proceeds via the formation of the IP form. Additionally, the calculated ΔG° values allow to calculate all the equilibrium constants, including the pK_C2 for the thiazolium C2 atom whose ionization is believed to initiate the catalytic cycle.     

The kinetics of trypsinogen activation studied by analytical affinity chromatography

The product distribution in autoactivated bovine trypsinogen has been studied by analytical affinity chromatography. In the presence of Ca²⁺ more β- than α-trypsin is formed. In the absence of Ca²⁺ mainly α-trypsin is formed. These results and data on the autolysis of α- and β-trypsin show that α-trypsin is a hydrolysis product of an inactive intermediate, neo-trypsinogen, formed after hydrolysis of a peptide bond in the middle of the trypsinogen polypeptide chain.     

Effect of manganese(II) ion on trypsinogen activation

The effect of Mn²⁺ and Ca²⁺ 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 Mn²⁺ increases the conversion rate of trypsinogen into trypsin in a 25–40% larger extent than Ca²⁺. 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.