Monoclonal antibodies to human pancreatic trypsin 1 inhibit the activation of human trypsinogens 1 and 2.

Two monoclonal antibodies (Mab) raised against human pancreatic trypsin 1, Mab G6 and A8, were previously isolated and characterized. The two Mab which recognize trypsinogen 1 are found to inhibit the activation of trypsinogen 1 by enterokinase. The inhibition of activation by the two Mab is concentration-dependent, rapid and virtually complete with Mab G6. Activation of trypsinogen 2 is totally inhibited by Mab G6, while Mab A8 has no effect on the activation of trypsinogen 2. The two monoclonal antibodies have opposite effects on the proteolytic activity of trypsin 1; Mab G6 increases proteolytic activity while Mab A8 inhibits trypsin activity by as much as 40%. This inhibition is concentration dependent but cannot account for the complete inhibition of activation of trypsinogen 1. Neither monoclonal antibody significantly inhibits the esterolytic activity of either form of human trypsin. Western-blot analysis of the reactivity of the two monoclonal antibodies with trypsinogens of various species shows that only Mab G6 cross-reacts with dog trypsinogen.     

On the complex formation between basic pancreatic trypsin inhibitor and TLCK-, TPCK-derivatives of beta-trypsin

The study of the interaction of the pancreatic inhibitor with different alkylated derivatives of alpha- and beta-trypsin shows that: 1) TLCK-beta-trypsin forms a complex with pancreatic inhibitor in tris buffer and tris-ethanol 40% system. 2) TLCK-alpha-trypsin and TLCK-TPCK-beta-trypsin have lost their ability to complex formation with pancreatic inhibitor. TLCK-alpha-trypsin and TLCK-TPCK-beta-trypsin are in derivatives in which the "chymotryptic" active site is destroyed. The results presented in this paper prove the participation of the "chymotrypic" active site in the interaction between trypsin and pancreatic inhibitor. This is the second interaction beside that of the electrostatic bond between Asp-117 of trypsin and Lys-15 of the inhibitor which we proved earlier.     

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.     

The two alpha 2-macroglobulin-bound trypsin molecules have different affinities for the basic pancreatic trypsin inhibitor

We have investigated the enzymatic properties of alpha 2-macroglobulin-bound porcine trypsin using a substrate: Z-Gly-Gly-Arg-p-nitroanilide and two inhibitors: p-aminobenzamidine and basic pancreatic trypsin inhibitor. The ternary alpha 2-macroglobulin-(trypsin)2 complex behaves like a mixture of two enzymes which bind basic pancreatic trypsin inhibitor with widely different affinities (Ki = 0.11 microM and 23 microM). About one-half of the trypsin molecules of the ternary complex are covalently bound to alpha 2-macroglobulin. Preparation of the complex in the presence of hydroxylamine prevents covalent bond formation, but the two trypsins of this artificial complex still exhibit large differences in affinity for basic pancreatic trypsin inhibitor. The trypsin molecules of the ternary complex also exhibit small differences in their affinity for Z-Gly-Gly-Arg-p-nitroanilide and p-aminobenzamidine.     

Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens

Two cDNA clones encoding two major human trypsinogen isozymes were isolated from a human pancreatic cDNA library. The deduced amino acid (aa) sequences of the two trypsinogen precursors are found to have 89% sequence homology, and have the same number of aa (247), including 15 aa for a signal peptide and 8 aa for an activation peptide. Southern blot analysis of human genomic DNA using the cloned cDNA as a probe, revealed that the human trypsinogen genes constitute a multigene family of more than ten genes.     

Eukaryotic initiation complex formation. Evidence for two distinct pathways.

Two distinct pathways have been elucidated which lead to the formation of an AUG-dependent initiation complex. One pathway involves the use of initiation factor M1 (IF-M1) to promote AUG-dependent binding of the initiator tRNA to the 40 S subunit, followed by joining of the 60 S subunit in the presence of IF-M2A, IF-M2B, and GTP. The second pathway involves the IF-MP-directed binding of initiator tRNA to the 40 S subunit via a ternary complex of IF-MP-GTP-Met-tRNAf. This reaction does not require AUG codon. However, subsequent formation of an 80 S initiation complex (as determined by methionyl-puromycin synthesis) required AUG as well as IF-M2A, IF-M2B, and GTP. Since both pathways require the same complementary initiation factors (at the same level), it would appear that the only difference is the manner in which the initiator tRNA is bound to the 40 S subunit, either by IF-M1 or IF-MP. Examination of the requirements for endogenous mRNA-directed methionyl-puromycin synthesis indicates a greater difference between IF-MP and IF-M1 in that only IF-MP was capable of forming an 80 S initiation complex which was sensitive to puromycin.     

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.     

Identification of two major proteins of bovine pancreatic stones as immunoreactive forms of trypsinogens.

Two major proteins have been identified in sodium citrate extracts of bovine pancreatic stones from 15 glands with lithiasis. They were found to have a molecular weight of about 24 000 and were further characterized by a variety of methods, including polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, isoelectric focusing, two-dimensional electrophoresis, immunodiffusion, immunoelectrophoresis and determination of N-terminal residues. These two immunologically and electrophoretically different proteins were definitely shown to be immunoreactive forms of anionic and cationic trypsinogens, which are normal components of pancreatic juice. However, in contrast with both secretory trypsinogens, the stone proteins displayed an important charge heterogeneity under isoelectric-focusing conditions. A possible role for both secretory trypsinogens in pancreatic lithogenesis is suggested by the reproducibility of the data. Finally, two minor proteins with a lower molecular weight (about 11 000--13 000) have also been found to be present in all extracts, but have not yet been identified.     

The multiplicity of human pancreatic secretory trypsin inhibitor

Four forms of pancreatic secretory trypsin inhibitor (PSTI; A1, A2, B, and C) were purified from human pancreatic juice. According to sequence results, the primary structure of B was different from that reported earlier (Greene, L.J., et al. (1976) Method Enzymol. 45, 813-825) at two positions, i.e. Asn21----Asp21, Asp29----Asn29. A1 and A2 were deamidated forms of B judging from peptide mappings with Staphylococcus aureus V8 protease. Gln45 in B was replaced by Glu in A1 and Gln51 in B was replaced by Glu in A2. C was an inhibitor lacking five amino acid residues from the amino terminal of B. B and C inhibited human cationic trypsin activity stoichiometrically with similar dissociation constants, but A1 and A2 showed poorer trypsin inhibitory activity than B and C.     

Characterization of trypsinogens 1 and 2 in two human pancreatic adenocarcinoma cell lines; CFPAC-1 and CAPAN-1.

Proteins with trypsin-like immunoreactivity (first detected by a specific immunoenzymatic assay) were isolated from CAPAN-1 and CFPAC-1 cell culture-conditioned media by chromatography on an immunoadsorbent prepared with a polyclonal antibody directed against trypsin 1. The adsorbed proteins were devoid of free trypsin activity but trypsin activity was present after enterokinase activation demonstrating that the immunoreactive trypsin present in cell supernatants corresponds to trypsinogens. When characterised by Western blotting using a monoclonal antibody directed against human trypsin 1 two protein bands corresponding to trypsinogen 1 (23 kDa) and trypsinogen 2 (25 kDa) gave a positive reaction. These results demonstrate the presence of trypsinogens 1 and 2 in CAPAN-1 and CFPAC-1 cells and in their culture-conditioned media.     

Raman studies of the conformation of the basic pancreatic trypsin inhibitor.

The vibrational Raman spectra of the basic pancreatic trypsin inhibitor in aqueous solution, as lyophilized powder and in a single crystal and presented. The thermal stability of this protein is demonstrated by the fact that minor alterations in the spectrum, mainly in the amide III band near 1260 cm-1, occur in the solution spectrum only at temperatures above 75 degrees C. No significant spectral changes appear when the pH value of the solution is varied in the range from 1.5 to 8.7. The distinct differences of the powder spectrum compared to that of the solution, show that lyophilization causes appreciable conformational changes both in the main-chain and in the side-chains. A difference in main chain conformation of the basic pancreatic trypsin inhibitor in single crystal and in solution is suggested by different amide III frequencies.     

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.     

Refolding of S-methylmethionyl basic pancreatic trypsin inhibitor

The normal CH₂CH₂SCH₃ side-chain of Met52 of bovine pancreatic trypsin inhibitor was converted to CH₂CH₂S⁺(CH₃)₂ with methyl iodide. After unfolding and breakage of the three disulphide bonds, the reduced protein refolded some three to six times more slowly than unmodified bovine pancreatic trypsin inhibitor. This was shown to be due to the decreased occurrence of the normal one- and two-disulphide initial intermediates. Modifications of the Met52 side-chain suggest that it normally has an important conformational role in the initial stages of folding, participating in extensive hydrophobic interactions with other parts of the protein. This role is different from that in the final folded state, where modification produced no detectable change in stability.     

Characterization of a high-molecular-weight form of human acrosin. Comparison with human pancreatic trypsin.

A high-molecular-weight form of acrosin (alpha-acrosin, EC 3.4.21.10) was extracted from spermatozoa obtained from frozen semen and purified over 300-fold. Purification was effected by sequential use of Sephadex G-150, CM-cellulose and DEAE-cellulose chromatography. Properties of human acrosin were compared with those of human pancreatic trypsin. The molecular weight (Mr) of acrosin (70000) was greater than that of trypsin (Mr 21000). Isoelectric points for acrosin (pI = 9.0) and trypsin (pI = 8.2) were also different. alpha-N-Benzoyl-L-arginine ethyl ester was hydrolysed 50% more rapidly by acrosin than by trypsin. Acrosin had similar kcat. values for the hydrolysis of esters with different acylating groups (i.e. benzoyl-L-arginine and p-tosyl-L-arginine esters). In contrast, trypsin had dissimilar kcat. values for the hydrolysis of esters with different acylating groups. Kinetic data argue against deacylation as the rate-limiting step in ester hydrolysis by acrosin. Acrosin was less sensitive than trypsin to inhibition by 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK'), di-isopropyl fluorophosphate and soya-bean trypsin inhibitor. D-Fructose and D-arabinose inhibited acrosin, but had no effect on trypsin. The data indicate that definite differences exist between human acrosin and trypsin.