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PubMed1986

Serum elastase 1 and immunoreactive trypsin in chronic pancreatic disease: is there any relationship with trypsin inhibitors?

In order to investigate modifications of serum levels of elastase 1, immunoreactive trypsin, alpha 1-antitrypsin and alpha 2-macroglobulin in chronic pancreatic disease, and to speculate on the possible relationships among these parameters, the enzymes and inhibitors were assayed in the sera of 33 control subjects, 34 pancreatic cancer, 28 chronic pancreatitis and 36 extra-pancreatic diseases. An increase of elastase 1, alpha 1-antitrypsin and alpha 2-macroglobulin was detected in pancreatic cancer, chronic pancreatitis and extra-pancreatic diseases; no changes were found for serum immunoreactive trypsin. Multiple regression analyses showed that only 7% of elastase 1 was explained by inhibitors with alpha 1-antitrypsin playing a major role. Inhibitors did not influence immunoreactive trypsin. Our data indicate that the variations of the serum levels of proteases and antiproteases in chronic pancreatic disease are probably independent of each other.

Adult↗
PubMed1998

Human neutrophil elastase degrades inter-alpha-trypsin inhibitor to liberate urinary trypsin inhibitor related proteins.

Urinary trypsin inhibitor (UTI) is a physiological protease inhibitor and inter-alpha-trypsin inhibitor (ITI) is regarded as a precursor of UTI. The purpose of this study is to determine the mechanism of the UTI release from ITI. To examine this, ITI was digested by human neutrophil elastase at various concentrations, and UTI-related proteins which were of the same size as UTI were obtained. The amino acid sequence of the 15 amino acid residues at the N-terminal of UTI-related proteins, corresponded to that of UTI. The amino acid sequences of the small amount of peptides detected corresponded to those of peptides from the heavy chain1 (H1) and the heavy chain2 (H2) of ITI, suggesting that most UTI-related proteins do not combine with peptides from the H1 and H2 of ITI. It was also revealed that UTI-related proteins have several physiological activities similar to those of UTI, i.e., human trypsin inhibitory activity, human neutrophil elastase inhibitory activity, inhibition of tumor necrosis factor-alpha (TNF-alpha) production from rat macrophages and of superoxide production from rabbit leukocytes. These results demonstrated that ITI is a precursor of UTI which is digested by human neutrophil elastase to release UTI, and that its elastase inhibitory activity is derived from UTI.

Alpha-Globulins↗
PubMed1993

Effects of heparin on the inhibitory activities of human urinary trypsin inhibitor (ulinastatin) on trypsin, chymotrypsin and leukocyte elastase.

Effects of heparin on the inhibitory activities of human urinary trypsin inhibitor (ulinastatin) on trypsin, chymotrypsin and leukocyte elastase were studied. Heparin per se neither influenced the enzymatic activities nor changed the mode of inhibition of ulinastatin on the enzymes. In the presence of heparin, inhibitory effects of ulinastatin on trypsin were enhanced, whereas its effects on chymotrypsin and elastase were attenuated. These results suggest that the two functional domains in ulinastatin are differently affected by heparin.

Amino Acid Sequence↗
PubMed1978

[The immunhistochemical investigation of trypsin and trypsin-like-activity in the small intestine, pancreas and isolated Langerhans islets. A light-, fluorescent- and electron microscopic study (author's transl)].

By means of the indirect immunofluorescence and immunenzyme technique trypsin and trypsin-like activity in fixed cryostate sections of pancreas and gut and pellets of isolated pancreatic islets respectively were investigated using specific antiserum against trypsin. The enzyme was demonstrated in the cytoplasma of the acinus cells, the epithel cell of the intercalated ducts and the excretory (interlobular) ducts of the exocrine pancreas. By light and fluorescence microscopy we could also localize a high activity in pancreatic islets. Results taken from electron microscope show the enzyme in the region of the peripheral endoplasmatic reticulum, the surface of the mitochondria, the zymogen granules of the acinus cells and in the region of the membranes of the endoplasmatic reticulum and in the granules of the B-cells of the pancreatic islets. These results are discussed in correlation with biochemical data concerning the importance of trypsin/trypsin-like activity in the specific conversion of prohormones into hormones.

Animals↗
PubMed1988

Preparation of heteroenzyme conjugates: trypsin-chymotrypsin and trypsin-alkaline phosphatase.

Hybrid enzymes which have two different enzyme activities linked together covalently may be useful reagents for various applications, such as the determination of complex biological structures. The present paper describes the preparation and purification of two such enzyme-enzyme conjugates, namely, trypsin-chymotrypsin and trypsin-alkaline phosphatase. Whereas the former has been prepared by using the well-known bifunctional reagent glutaraldehyde, the latter exploited the Schiff base formation between the oxidized carbohydrate moiety of alkaline phosphatase and the free amino groups of trypsin.

Alkaline Phosphatase↗
PubMed1983

Mammalian tissue trypsin-like enzymes. Comparative reactivities of human skin tryptase, human lung tryptase, and bovine trypsin with peptide 4-nitroanilide and thioester substrates.

The subsite specificity of human lung and skin tryptase (trypsin-like enzyme) has been studied at pH 7.5 using 17 amino acid and dipeptide thioester substrates and 14 tripeptide 4-nitroanilide substrates. The reactivity and specificity of the human tryptases were compared with bovine trypsin and other trypsin-like enzymes. Neither tryptase was similar to either kallikrein or factor XIIa (Hageman factor). The skin enzyme was the most reactive as measured by the specificity constant kcat/KM. The best substrate was benzyloxycarbonyl(Z)-Lys-Arg-S-CH2CH(CH3)2 which had a kcat/KM value of 59,000,000 M-1 S-1. Only a single substrate, Z-Glu-Phe-Arg-4-nitroanilide, was slightly more reactive with the lung tryptase. Both enzymes have extended substrate-binding sites and proline residues at P3 substantially decrease kcat/KM. Both enzymes preferred the tripeptide 4-nitroanilides with a P2 Gly residue over Phe, and both favored the substrate Z-Lys-Gly-Arg-4-nitroanilide over similar substrates containing six other representative amino acid residues at P3. The lung enzyme was inhibited over three times faster by p-amidinophenylmethanesulfonyl fluoride than the skin enzyme. The preference of the skin tryptase for substrates with two terminal basic residues indicates that this enzyme could process prohormones and proproteins which contain this structural feature at the cleavage site. The substrates reported in this paper should be useful for the further characterization of the physiologic function of tryptases.

Anilides↗
PubMed1982

Specificity of trypsin. Cleavage of aspartic acid 101 derivatives of lysozyme by trypsin.

The beta-carboxyl group of Asp 101 was modified with several amine nucleophiles by the amine-carbodiimide procedure and the trypsin susceptibilities of the derivatives were analyzed by peptide mapping. The (aminoethyl)asparaginyl peptide bond was completely cleaved by trypsin but the (3-aminopropyl)asparaginyl bond was not. When the positive charge was in the proper position, large residues such as (2-[imidazol-4(5)-ylmethylamino]ethyl) asparagine or (11-amino-3,-6,9-triazaundecyl)asparagine were susceptible to trypsin.

Amines↗
PubMed1983

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, VII. Characterization of the bovine inhibitor as double-headed trypsin-elastase inhibitor.

The acid-resistant 14-kDa inhibitor BI-14, released from bovine inter-alpha-trypsin inhibitor, consists of two tandem Kunitz-type domains, and is of a double-headed nature. The Arg-Thr bond connecting both domains was cleaved and the two inhibitory domains were separated. The N-terminal domain is an inhibitor of bovine chymotrypsin and elastases from porcine pancreases and human polymorphonuclear granulocytes, whereas the C-terminal domain interacts with trypsin, plasmin, and chymotrypsin. In the intact inhibitor BI-14 both domains interact independently with the proteinases.

Alpha-Globulins↗
PubMed1993

Crystallization and preliminary X-ray studies on the trypsin inhibitor I-2 from wheat germ and its complex with trypsin.

A Bowman-Birk type trypsin inhibitor I-2, M(r) = 14 000, 123 amino-acid residues, isolated from wheat germ, and its complex with trypsin have been crystallized. For I-2 two morphologically different crystal forms were obtained. Crystal form 1 is tetragonal, P4(1)22 or P4(3)22, with a = 55.45 (2), c = 129.1 (2) A and V = 3.97 (2) x 10(5) A(3). The crystals diffract X-rays very anisotropically, to less than 6 A resolution normal to the c* direction, but up to 3 A resolution in the other directions. Crystal form 2 is monoclinic, space group C2. The cell parameters show significant variation even for crystals in the same batch. The median parameters are: a = 83.9, b = 41.5, c = 45.7 A, beta = 95.9 degrees and V = 1.58 x 10(5) A(3). The diffraction pattern is isotropic and reflections up to 2.2 A resolution were observed. The crystals of the complex between bovine trypsin and I-2 (2:1) belong to the orthorhombic space group P2(1)2(1)2(1) with a = 73.49 (2), b = 120.56 (3), c = 70.04 (2) A and V = 6.206 (5) x 10(5) A(3). The crystals diffract up to 2.3 A resolution, and contain one complex of 60 100 Da in an asymmetric unit.

Journal Article↗
PubMed1992

Amino acid sequencing of a trypsin inhibitor by refined 1.6 A X-ray crystal structure of its complex with porcine beta-trypsin.

The stoichiometric complex formed between porcine beta-trypsin and the Momordica charantia, Linn. Cucurbitaceae trypsin inhibitor-A (MCTI-A) was crystallized and its X-ray crystal structure determined using molecular replacement method. The primary sequence and topology of the inhibitor was determined by recognizing the electron density and refined to a final R value of 0.167 (7.0-1.6 A) with RMS deviation of bond lengths from standard values 0.012 A. The sequence was compared with those obtained by other groups and was found to be similar to the squash proteinase inhibitor. Its spatial structure and the conformation of its primary binding segment from Cys-3I (P3) to Glu-7I (P3') which contains the reactive scissile bond Arg-5I C-Ile-6I N were also very similar with other squash family proteinase inhibitors.

Amino Acid Sequence↗
PubMed1998

Modifications outside the proteinase binding loop in Cucurbita maxima trypsin inhibitor III (CMTI-III) analogues change the binding energy with bovine beta-trypsin.

Five 26-peptide analogues of the trypsin inhibitor [Pro18]CMTI-III containing Leu or Tyr in position 7 and Val or Tyr in position 27: 1 (Leu7, Tyr27), 2 (Tyr7, Val27), 3 (Tyr7, Tyr27), 4 (Leu7, Val27) and 5 (Leu7, Ala18, Tyr27) were synthesized by the solid-phase method. Analogues 1-4 displayed Ka with bovine beta-trypsin of the same order of magnitude as the wild CMTI-III inhibitor, whereas for analogue 5, this value was lower by about 3 orders of magnitude. This indicated that for the analogues with Pro (but not with Ala) in position 18, the side-chain interactions between positions 7 and 27 did not play a critical role for the stabilization of the active structure. In addition, these results also suggest that Tyr7 is involved in an additional aromatic interaction with position 41 of the enzyme.

Amino Acid Sequence↗
PubMed1979

[Interaction between alpha-2 macroglobulin and trypsin with synthetic trypsin inhibitors].

The complex formed between trypsin (Tn) and alpha 2 Macroglobulin (alpha 2 M) retains the whole hydrolytic activity of the enzyme for synthetic substrates. Moreover synthetic inhibitors of low molecular weight stiel inhibit this activity. A comparative study of three inhibitors (Benzylamine, Butylamine, Benzamidine) has been carried out and shows that their behavior is similar. These inhibitors bind trypsin when it is bound to alpha 2 M and reciprocally alpha 2 M can bind Tn-inhibitor complex. Nevertheless the dissociation constant of the enzyme-inhibitor complex (Ki) is increased by alpha 2 M. In the case of Benzamidine the value of Ki is 2.22.10(-5) M for native enzyme and 13.4.10(-5) M for Tn-alpha 2 M and in the case of Butylamine this value increases from 0.5.10(-3) M to 2.95.10(-3) M. These variations of the Ki values are due to the modification of the accessibility of the inhibitor to the active site. Unpublished results show that the alpha 2 M molecule undergoes a deep structural modification in the course of the complex formation, which must lead to an increase of the value of Ki. This structural modification is probably irreversible so that the alpha 2 M complex has never been dissociated without altering the alpha 2 M molecule. The increase of the values of Ki cannot therefore result in an effective decrease of the association constant of the Tn-alpha 2 M complex.

Binding Sites↗
PubMed1999

Crystallization and preliminary X-ray analysis of a complex between the Bowman-Birk trypsin inhibitor from barley and porcine pancreatic trypsin.

A 1:2 complex between the Bowman-Birk trypsin inhibitor from barley seeds and porcine pancreatic trypsin has been crystallized at 291 K using polyethylene glycol as precipitant. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67. 10, b = 88.38 and c = 203.65 A. The asymmetric unit contains two monomers of the complex, with a corresponding Vm of 2.41 A3 Da-1 and a solvent content of 49%. Native data to 2.2 A resolution have been collected at 100 K using synchrotron X-rays.

Crystallization↗
PubMed1990

Tumour-associated trypsin inhibitor and tumour-associated trypsin.

Tumour-associated trypsin inhibitor (TATI) is a 6 kDa peptide, which is synthesized at low concentrations by several tumours and cell lines. Very high concentrations of TATI occur in mucinous ovarian tumours. Elevated levels of TATI occur in serum and urine in connection with most types of cancer at advanced stages. In mucinous ovarian cancer up to 85% of all cases have elevated serum levels. Because high levels also occur in early mucinous ovarian cancer TATI appears to be the marker of choice for this tumour. Elevated levels may also occur in nonmalignant disease, especially in patients with severe infections, tissue destruction and pancreatitis. Production of TATI in tumours is associated with expression of two new tumour-associated trypsin(ogen) (TAT) isoenzymes, TAT-1 and -2, TAT-2 being the major form. These enzymes are immunologically similar to trypsinogen-1 and -2, respectively. They activate prourokinase and may therefore trigger the tumour-associated protease cascade contributing to the invasiveness of malignant tumours.

Biomarkers, Tumor↗
PubMed1985

The crystallographic study of the complex of the Lys active fragment of the mung bean trypsin inhibitor with bovine trypsin.

The Lys fragment of mung bean trypsin inhibitor can combine with bovine trypsin to form a complex at an equal molar ratio. The single crystals of the complex were obtained by using the micro-still-setting method and the X-ray diffraction extended to 1.8A resolution. Its space group is P212121 with cell dimensions a = 62.9(1)A, b = 63.4(1)A and c = 69.7 (2)A. There is one complex molecule in a crystallographic asymmetric unit.

Animals↗
PubMed1980

Hydrogen-deuterium exchange in the black-eyed pea trypsin and chymotrypsin inhibitor and its complex with beta-trypsin.

The H-D exchange of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) in D2O was studied by an ultraviolet spectroscopic method recently proposed (J. J. Englander, D. B. Calhoun, and S. W. Englander, (1979) Analytical Biochemistry, 92, 517-524). Isotopic exchange data are presented as plots of X (the fraction of unexchanged peptide hydrogen atoms at time t) versus log(kot), where ko is the pH dependent rate constant for peptide groups exposed to the solvent. In the range of pD 2.25-6.9, at 20 degrees C, BTCI shows a continuous exchange curve which indicates that the exchange mechanism is of the EX2 type and no detectable conformational changes occur in the protein. Deviations from this exchange curve are found at pD 7.3 and 8.0. About 60% of the peptide hydrogens of BTCI are exchanged for delta Go less than or equal to 2 kcal/mol, and 90% for delta Go less than 6 kcal/mole. For reduced and carboxymethylated BTCI, exchange data suggest a much more open conformation in comparison with the unmodified protein. However, some residual structure appears to be maintained, after scission of the disulfide bonds. The exchange data indicate that, as a consequence of the formation of the beta-trypsin-BTCI complex, part of the peptide groups of the enzyme and/or inhibitor become less accessible to the isotopic exchange.

Deuterium↗
PubMed1980

[Soluble high molecular weight derivatives of pancreatic trypsin inhibitor. Modification of pancreatic trypsin inhibitor by soluble polysaccharides activated by titanium tetrachloride].

The activation of soluble polysaccharide matrices by titan tetrachloride was studied. A method for obtaining soluble preparations of pancreatic trypsin inhibitor bound to the activated polysaccharide matrix completely retaining its inhibiting activity was developed. It was shown that the equilibrium and kinetic constants for association of modified preparations of pancreatic inhibitor with commercial trypsin did not practically differ from those for native inhibitor. It was found that modification by titan tetrachloride-activated polysaccharides stabilized the protein molecule.

Kinetics↗
PubMed1994

[Monoclonal antibodies to the trypsin-binding domain of trypsin inhibitor from human urine].

Three MAb M2, B6, P1 (IgG1 type) against human urinary trypsin inhibitor (UTI), a glycoprotein with antiinflammatory properties, have been produced by hybridization of mouse myeloma cells P3O1 with spleen cells of immunized mice BALB/c. Competitive ELISA-examination of the peroxidase conjugates of M2, B6, and P1 MAb in the presence of the trypsin binding domain shows the M2 antibody to possess the highest affinity for this domain. On the basis of the MAb M2, a competitive ELISA of UTI concentration in urine is proposed. ELISA-detectable changes in the UTI content of urine from patients with nephritis without renal failure can be considered as an early index of renal parenchyma damage.

Animals↗