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PubMed2006

Comparative evaluation of bioactivity change of crystalline trypsin during compression by chemoinformatics and 2-D Fourier-transform infrared spectroscopy.

The purpose of this study is to develop a method of evaluating the enzymatic activity of trypsin in a solid-state based on Fourier transform infrared (FT-IR) spectra using chemoinformatics and two-dimensional (2-D) correlation spectroscopy. Crystalline trypsin powders are compressed at 0-4000 kg cm-2 by a compression/tension tester. The enzymatic activity of trypsin is assayed by the kinetic degradation method. Spectra of 10 calibration sample sets are recorded 3 times with a FT-IR spectrometer. The maximum intensity of FT-IR spectra and enzymatic activity of trypsin decrease as the compression pressure increases. The FT-IR spectra of trypsin samples are subjected to a principal component regression (PCR). A plot of the calibration data obtained is made between the actual and predicted trypsin activity based on a two-component model with gamma2=0.909 (n=30). The regression vector is almost the same as the loading vector for PC1. On the other hand, a generalized two-dimensional (2-D) correlation spectroscopic method is applied to FT-IR spectra of compressed trypsin. The result is consistent with that of the chemoinformatics method. The FT-IR chemoinformatics method allows for solid-state quantitative analysis of the bioactivity of the bulk powder of a polypeptide drug.

Animals↗
PubMed2003

Thermal stabilization of trypsin by enzymic modification with beta-cyclodextrin derivatives.

Streptoverticillum sp. transglutaminase was used as catalyst for the attachment of several beta-cyclodextrin derivatives to the glutamine residues in bovine pancreatic trypsin. The modifying agents used were mono-6-ethylenediamino-6-deoxy-beta-cyclodextrin, mono-6-propylenediamino-6-deoxy-beta-cyclodextrin, mono-6-butylenediamino-6-deoxy-beta-cyclodextrin and mono-6-hexylenediamino-6-deoxy-beta-cyclodextrin. The transformed trypsin preparations contained about 3 mol of oligosaccharides/mol of protein. The specific esterolytic activity of trypsin was increased by about 4-21% after conjugation. The K (m) values for cyclodextrin-trypsin complexes represented about 58-87% of that corresponding to the native enzyme. The optimum temperature for esterolytic activity of trypsin was increased by about 5-10 degrees C after enzymic modification with the cyclodextrin derivatives. The thermostability was increased by 16 degrees C for the modified trypsin. Thermal inactivation at different temperatures ranging from 45 to 60 degrees C was markedly increased for the oligosaccharide-trypsin complexes. This modification also protected the enzyme against autolysis at alkaline pH.

Animals↗
PubMed1991

Changes in insulin-receptor structure associated with trypsin-induced activation of the receptor tyrosine kinase.

The tyrosine kinase of the insulin receptor can be activated by trypsin treatment. The concomitant abolition of insulin binding has been postulated to result from proteolytic destruction of the receptor. A discrepancy between the decrease in insulin binding and receptor immunoreactivity after trypsin treatment led us to investigate more closely the structure of the trypsin-treated receptor. After trypsin treatment of the CHOT cell line, which over-expresses transfected human insulin receptors, insulin binding was significantly decreased, but reactivity with five alpha-subunit monoclonal antibodies was either unaffected or only moderately decreased, indicating that the alpha-subunit was substantially intact. Examination of receptor structure after trypsin treatment, receptor autophosphorylation and gel electrophoresis revealed a single band at 110 kDa in non-reduced gels, comprising a small fragment (21 kDa) of the alpha-subunit linked to the beta-subunit by class II disulphides. When the receptor was radio-labelled with 125I, two additional alpha-subunit bands of 142 kDa and 81 kDa (composed of identical reduced bands) were observed on non-reduced gels, which contained disulphide-linked (class I) fragments. All fragments could be precipitated by antibodies to both alpha- and beta-subunits. However, only antibodies directed towards the N-terminus of the receptor could immunoblot trypsin-treated fragments. Thus activation of the receptor tyrosine kinase by trypsin occurs after cleavage, but not loss of the alpha-subunit. This finding has implications for the mechanism of transmembrane activation of the receptor kinase by insulin.

Animals↗
PubMed1997

Juvenile hormone controls early trypsin gene transcription in the midgut of Aedes aegypti.

Early trypsin is a female-specific protease present in the Aedes aegypti midgut during the first few hours after ingestion of a blood meal. The enzymatic activity of early trypsin plays an essential role in the transcriptional activation of the late trypsin gene, which encodes the major midgut endoprotease involved in blood meal protein digestion. Transcription of the early trypsin gene is part of the normal post-emergence maturation of the midgut in the adult female. Abdominal ligation within 1 h of emergence completely prevented the transcription of the early trypsin gene. Topically applied JH III or methoprene induced transcription of the early trypsin gene in ligated abdomens to levels similar to those observed in non-ligated females. The induction of early trypsin transcription by JH is dose-dependent and 'head-independent', suggesting that factors coming from the neuro-secretory axis are not required.

Aedes↗
PubMed1999

The three-dimensional structure of Asp189Ser trypsin provides evidence for an inherent structural plasticity of the protease.

Trypsin mutant Asp189Ser, first described by Gráf et al. [Gráf, L., Jancsó, A., Szilágyi, L., Hegyi, G., Pintér, K., Náray-Szabó, G., Hepp, J., Medzihradszky, K. & Rutter, W.J. (1988) Proc. Natl Acad. Sci. USA 85, 4961-4965] has played an important role in recent studies on the structural basis of substrate-specific catalysis by serine proteases. The present work reports the three-dimensional structure of this mutant crystallized in unliganded form: the first unliganded rat trypsin structure reported. The X-ray structure of the Asp189Ser trypsin mutant in complex with bovine pancreatic trypsin inhibitor is already known. The X-ray structure of free Asp189Ser rat trypsin revealed that the single amino acid mutation at the bottom of the substrate binding pocket of trypsin resulted in extensive structural changes around the mutated site and in dimerization of the mutant, in contrast with the complexed enzyme the structure of which is practically the same as that of wild-type trypsin. The structural rearrangement in the mutant was shown to be restricted to the activation domain region providing further evidence for the allosteric property of this structural-functional unit of the enzyme. This study supports our view that the plasticity of the activation domain may play an important role in the mechanism of substrate-specific serine protease action.

Allosteric Site↗
PubMed1999

Fluorescence and FTIR study of the pressure-induced denaturation of bovine pancreas trypsin.

The pressure denaturation of trypsin from bovine pancreas was investigated by fluorescence spectroscopy in the pressure range 0. 1-700 MPa and by FTIR spectroscopy up to 1000 MPa. The tryptophan fluorescence measurements indicated that at pH 3.0 and 0 degrees C the pressure denaturation of trypsin is reversible but with a large hysteresis in the renaturation profile. The standard volume changes upon denaturation and renaturation are -78 mL.mol-1 and +73 mL.mol-1, respectively. However, the free energy calculated from the data in the compression and decompression directions are quite different in absolute values with + 36.6 kJ.mol-1 for the denaturation and -5 kJ. mol-1 for the renaturation. For the pressure denaturation at pH 7.3 the tryptophan fluorescence measurement and enzymatic activity assays indicated that the pressure denaturation of trypsin is irreversible. Interestingly, the study on 8-anilinonaphthalene-1-sulfonate (ANS) binding to trypsin under pressure leads to the opposite conclusion that the denaturation is reversible. FTIR spectroscopy was used to follow the changes in secondary structure. The pressure stability data found by fluorescence measurements are confirmed but the denaturation was irreversible at low and high pH in the FTIR investigation. These findings confirm that the trypsin molecule has two domains: one is related to the enzyme active site and the tryptophan residues; the other is related to the ANS binding. This is in agreement with the study on urea unfolding of trypsin and the knowledge of the molecular structure of trypsin.

Anilino Naphthalenesulfonates↗
PubMed1974

Dissection of chromosome structure with trypsin and nucleases.

Exhaustive digestion of chromatin with trypsin leads to the cleavage of only 20-30 amino acids from each of histones III (f3), IV (f2a1), IIb2 (f2b), and IIb1 (f2a1), the remainder of these chains being resistant. This resistance is not altered by removing the histones from the DNA with 2 M NaCl, but is dramatically reduced in 6 M urea. Histones III, IV, IIb2, and possibly IIb1 are cleaved at their N-termini. Histones I and V and the nonhistone proteins are the first to be attacked by trypsin and have no detectable trypsin-resistant fragments. The arginine rich histones, III and IV, are then cleaved as a pair, followed by most of IIb2 and IIb1, also as a pair. This data is consistent with a model in which basic N-terminal "arms" extend from a trypsin-resistant histone complex. The structural arrangement of these arms relative to the trypsin-resistant histone complex may define the spatial coordinates of DNA binding sites and, consequently, the folding of the DNA fiber in the chromosome. Accompanying the tryptic digestion of the N-terminals of histones III, IV, IIb2, and possibly IIb1, is an increased sensitivity of chromatin to staphylococcal nuclease. As analyzed by electrophoresis, untrypsinized chromatin is digested into eight discrete limit-digest fragments by nuclease. Trypsinization results in the nuclease digestion of some, but not all, of these DNA bands. Together with the information on the way trypsin cleaves histones in chromatin, the analysis of the resistant DNA suggests that histone N-terminals are associated with some DNA bands and histone C-terminals with other DNA bands. We propose that histones fold the chromosome by crosslinking the DNA corresponding to these bands.

Animals↗
PubMed1993

Design of peptide enzymes (pepzymes): surface-simulation synthetic peptides that mimic the chymotrypsin and trypsin active sites exhibit the activity and specificity of the respective enzyme.

Two 29-residue peptides were prepared, one of which (ChPepz) was designed by surface-simulation synthesis to mimic the active site of alpha-chymotrypsin, and the other (TrPepz), which contained four substitutions relative to ChPepz, was fashioned after the active site of trypsin. Each peptide was cyclized by a disulfide bond. The ChPepz monomer effected hydrolysis of the ester group in N-benzoyl-L-tyrosine ethyl ester, an alpha-chymotrypsin substrate, with Km and kcat values that were comparable to those of alpha-chymotrypsin. ChPepz was completely inactivated by diisopropyl fluorophosphate (DIFP), L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK), or reduction of the disulfide bond. It had no catalytic activity on N-tosyl-L-arginine methyl ester, a trypsin substrate. On the other hand, TrPepz, which had no effect on N-benzoyl-L-tyrosine ethyl ester, hydrolyzed N-tosyl-L-arginine methyl ester with a Km value that was essentially identical to that of trypsin, but its kcat value was almost half that of trypsin. TrPepz was fully inactivated by reduction of the disulfide bond, by DIFP, or by phenylmethylsulfonyl fluoride but not by TPCK. It was also completely inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, and human alpha 1-antitrypsin. ChPepz and TrPepz hydrolyzed proteins (myoglobin and casein) to give panels of peptides that were similar to those of the same protein obtained with the respective enzyme. However, TrPepz was more efficient than trypsin at hydrolyzing the C bonds of two or more consecutive lysine and/or arginine residues. Like its esterase activity, the proteolytic activity of ChPepz was inhibited by either DIFP or TPCK whereas that of TrPepz was inhibited by either DIFP or phenylmethylsulfonyl fluoride but not by TPCK. Finally, ChPepz and TrPepz were each more active at low temperature than the respective enzyme. This ability to construct fully functional peptide enzymes (pepzymes) of chosen specificities should find many practical applications.

Amino Acid Sequence↗
PubMed2001

The serine protease trypsin cleaves C termini of beta- and gamma-subunits of epithelial Na+ channels.

Both extracellular and intracellular proteases can activate epithelial Na(+) channels (ENaC). The mechanism by which serine proteases activate ENaC is unknown. We investigated the effect of the serine protease trypsin on in vitro translated and immunopurified alpha-, beta-, and gamma-rENaC subunits. Immunopurified subunit proteins were exposed to increasing concentrations of trypsin ranging from 0.002 to 2 microg/ml in Tris-buffered saline buffer for 2 h. The proteolytic mixture was subjected to SDS-PAGE and analyzed by autoradiography. Our results demonstrate that the beta- and gamma-subunits of ENaC were most susceptible to trypsin proteolysis, and exposure to as little as 0.002 microg/ml trypsin resulted in a reduction in the size of the beta- and gamma-transcripts by 7-8 kDa. By using N- and C-terminally truncated beta- and gamma-subunits, we determined that trypsin cleaved the C termini of both subunits, resulting in a channel structure resembling that seen in Liddle's disease. Exposure to 2 microg/ml trypsin completely digested all three subunits. Our results suggest different susceptibilities of proteolytic sites of ENaC subunits to trypsin. Thus, we propose that limited intracellular proteolysis may be one of the potential physiological mechanisms of sodium channel regulation.

3T3 Cells↗
PubMed1991

Trypsin-induced changes in cell shape and chromatin structure result in radiosensitization of monolayer Chinese hamster V79 cells.

Trypsin is the enzyme commonly used to prepare single cell suspensions from monolayer and spheroid cultures, both to determine survival and to assay DNA damage. Trypsin induces rounding, dissociation and radiosensitization of anchorage-dependent cells. Radiosensitivity and chromatin structure were compared between trypsin-treated (0.05%) round V79 cells from monolayers and spheroids vs. untreated spread monolayer cells in situ. The fluorescent halo technique was used to measure the changes in DNA supercoiling in nucleoids isolated from control and irradiated round and spread cells. Maximal halo diameters, the amount of initial and residual radiation-induced DNA damage (estimated from nucleoid halo diameter changes), and the radiosensitivity were higher in round cells than in spread monolayer V79 cells. The effects on cellular radiosensitivity and maximal halo diameter of other agents which also round and dissociate cells, e.g. 0.25% trypsin, pronase E and a non-enzymatic cell-dissociation solution, were similar to those of 0.05% trypsin. In LY-S cells, which are anchorage-independent, DNA loop size, the initial amount of DNA damage and radiosensitivity were not affected by trypsin. We suggest that the higher radiosensitivity of anchorage-dependent cells under immediate trypsinization and plating conditions, compared to cells with postirradiation in situ repair incubation, is due to correlated changes in cell shape and chromatin structure.

Animals↗
PubMed1980

Macrophage component gp160, a major trypsin-sensitive surface glycoprotein.

Macrophages secrete a large number of proteases, implying in vivo exposure of the cell surface to proteolytic conditions. Mild trypsin treatment of 125I-labeled guinea pig peritoneal macrophages preferentially cleaves one surface component of apparent 160,000 mol wt. Similar trypsin treatment of macrophages with 3H-labeled carbohydrate surface moieties also cleaves a single 3H-labeled 160,000 mol wt glycoprotein, referred to as gp160. Nonreducing sodium dodecyl sulfate (SDS)-electrophoresis established that gp160 of trypsinized cells remains assembled in the membrane as a multichain disulfide-bonded molecule. gp160 was purified by detergent extraction, L. culinaris lectin affinity chromatography and DEAE-cellulose chromatography. The corresponding molecule from trypsinized cells was purified by the same procedure. Reducing SDS-electrophoresis of purified trypsinized 125I-labeled gp160 revealed two proteolytic fragments with apparent molecular weights of 85,000 and 71,000. Thus, mild trypsin treatment of macrophages preferentially cleaves a single surface protein, possibly at a single site. Because the two fragments of gp160 are accessible to lactoperoxidase and trypsin, both must be exposed on the membrane surface. The reactive carbohydrate site was found on the 85,000 mol wt fragment, which alone contains the 3H-label introduced into intact cells by neuraminidase, galactose, oxidase, and [3H]KBH4.

Animals↗
PubMed1991

Trypsin inhibitor from the seeds of Acacia confusa.

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.

Acacia↗
PubMed1995

Amino acid sequences of trypsin inhibitors from the melon Cucumis melo.

Two squash family trypsin inhibitors, CMeTI-A and CMeTI-B, were isolated from the melon (Cucumis melo) seeds, by ion exchange chromatography, gel filtration, affinity chromatography, and high-performance liquid chromatography, and their amino acid sequences were determined. All inhibitors contain 29 amino acid residues including 6 half-cystine residues. They differ by twelve amino acid residues. These polypeptides are strong inhibitors of bovine trypsin, with Ki values of 1.6 x 10(-10) M (CMeTI-A) and 4.7 x 10(-10) M (CMeTI-B). The products of CMeTI-A and CMeTI-B cleaved at their reactive sites by tryptic digestion during the purification by trypsin-Sepharose 4B affinity column chromatography are active against trypsin activity, but a molar ratio of inhibitor to trypsin of 2:1 for trypsin-treated CMeTI-B or 1:1 for trypsin-treated CMeTI-A is required.

Amino Acid Sequence↗
PubMed1975

Affinity chromatography of trypsin and related enzymes. I. Preparation and characteristics of an affinity adsorbent containing tryptic peptides from protamine as ligands.

An absorbent for the affinity chromatography of trypsin [EC 3.4.21.4] (AP Sepharose) was prepared. The ligand was a mixture of oligopeptides (mainly di- and tripeptides) containing L-arginine as carboxyl termini, and was obtained from a tryptic digest of protamine. Trypsin was absorbed at relatively low pH (7-4), but was not absorbed at the optimum pH of catalysis (8.2). This was clearly explained on the basis of the pH dependence of the interaction of trypsin with its products. Inactivated trypsin, trypsinogen, and chymotrypsin were not absorbed. The absorption of active trypsin was interferred with by either benzamidine or urea. From these observations, it is evident that AP Sepharose is an affinity adsorbent. AP Sepharose was useful for purification of commercial bovine trypsin. A preliminary application for the purification of Streptomyces griseus trypsin was also successful.

Amino Acids↗
PubMed1976

Affinity chromatography of trypsin and related enzymes. II. An affinity adsorbent containing glycylglycyl-L-arginine.

An affinity adsorbent for trypsin [EC 3.4.21.4] (GGA Sepharose) was prepared. Glycylglycyl-L-arginine (GGA) was synthesized by a simple procedure and was immobilized on agarose gel. This adsorbent proved to have essentially the same characteristics as AP Sepharose, which is an affinity adsorbent containing tryptic peptides of protamine (1). GGS Sepharose was specific for native trypsin and had a stronger affinity at lower pH's (6-5) than at the optimum pH of trypsin action (8.2). It also proved to be suitable for analytical experiments because of its relatively weak affinity. By comparison of the elution profiles of trypsin from GGA Sepharose under various conditions, the nature of the interaction of trypsin with the adsorbent could be studied. It was found that alpha- and beta-trypsin could be distinguished. In the presence of arginine and N-substitute arginines, the elution of trypsin was accelerated. From the extents of the accelerating effects, the affinities of these compouunds could be compared.

Animals↗
PubMed2000

Structural and interactional homology of clinically potential trypsin inhibitors: molecular modelling of cucurbitaceae family peptides using the X-ray structure of MCTI-II.

Several trypsin inhibitor peptides (with 28-32 amino acid residues) belonging to the Cucurbitaceae (LA-1, LA-2, MCTI-I, CMTI-I, CMTI-III, CMTI-IV), characterized by a distinctive tertiary fold with three conserved disulphide bonds and with mostly arginine at their active centre, were modelled using the high-resolution X-ray structure of a homologous inhibitor, MCTI-II, isolated from bitter gourd. All the inhibitors were modelled in both their native and complexed state with the trypsin molecule, keeping the active site the same as was observed in the trypsin-MCTI-II complex, by homology modelling using the InsightII program. The minimized energy profile supported the binding constants (binding behaviour) of the inhibitor-trypsin complexes in the solution state. A difference accessible surface area (DASA) study of the trypsin with and without inhibitors revealed the subsites of trypsin where the inhibitors bind. It revealed that the role of mutation of these peptides through evolution is to modulate their inhibitory function depending on the biological need rather than changing the overall structural folding characteristics which are highly conserved. The minor changes of amino acids in the non-conserved regions do not influence significantly the basic conformational and interactional sequences at the trypsin binding subsites during complex formation.

Amino Acid Sequence↗
PubMed1985

Serial alterations in the forms of immunoreactive pancreatic cationic trypsin in plasma from patients with acute pancreatitis.

We employed a radioimmunoassay for human cationic trypsin to define the time course for alterations in the molecular forms of this enzyme in plasma from patients with pancreatitis. Six patients developed acute pancreatitis as a complication of a known disorder [three, Reye's syndrome; two, hemolytic uremic syndrome (HUS); one, choledochal cyst]. The immunoreactive forms of cationic trypsin were determined by gel filtration of each plasma sample followed by radioimmunoassay of the column fractions. Early in the course of the disease, predominantly free trypsinogen was released into the circulation in five patients. In the three patients with Reye's syndrome, subsequent plasma samples showed, in addition to free trypsinogen, increasing amounts of immunoreactive trypsin complexed to alpha 2-macroglobulin and alpha 1-protease inhibitor. In contrast, subsequent samples from the two patients with HUS contained little or no inhibitor-bound trypsin. The remaining patient had intermediate concentrations of cationic trypsin complexed to these two circulating protease inhibitors. Five patients died and postmortem studies showed a striking correlation between the histological severity of acute pancreatic inflammation and the amount of immunoreactive trypsin complexed to alpha 2-macroglobulin and alpha 1-protease inhibitor. This preliminary study suggests that measurement of alpha 2-macroglobulin or alpha 1-protease inhibitor-bound trypsin may be a useful method of monitoring the progression and severity of disease in patients with acute pancreatitis. Characterization of serial changes in the forms of circulating pancreatic proteases may enhance our understanding of time-dependent pathophysiologic events, possibly leading to improved forms of specific therapy.

Acute Disease↗
PubMed1990

Immunoreactive trypsin(ogen) in the sera of children with recent-onset insulin-dependent diabetes and matched controls. The Swedish Childhood Diabetes Group.

To evaluate the exocrine pancreatic function at the time of diagnosis of insulin-dependent diabetes mellitus, we determined immunoreactive anodal and cathodal trypsin(ogen) levels in sera from almost all children (n = 375) 0-14 years of age in Sweden in whom diabetes developed during 1 year, and in sex-, age-, and geographically matched control subjects (n = 312). The median level of anodal trypsin(ogen) was 5 (quartile range, 3-7) micrograms/L in children with newly diagnosed diabetes, compared with a median level of 7 (quartile range, 4-8) micrograms/L in control subjects (p less than 0.0001). Similarly, the median level of cathodal trypsin(ogen) was 8 (quartile range, 4-10) micrograms/L in children with diabetes, compared with a median level of 11 (quartile range, 7-15) micrograms/L in control subjects (p less than 0.0001). The median of the individual ratios between cathodal and anodal trypsin(ogen) was 1.4 in the diabetic patients and 1.7 in the control children (p less than 0.001). In a multivariate test, however, only the decrease in cathodal trypsin(ogen) concentration was associated with diabetes. The levels of trypsin(ogen)s did not correlate with levels of islet cell antibodies, present in 81% of the diabetic children. Several mechanisms may explain our findings, for example, similar pathogenetic factors may affect both the endocrine and exocrine pancreas simultaneously, a failing local trophic stimulation by insulin on the exocrine cells may decrease the trypsinogen production, and there may be an increased elimination of trypsin(ogen) because of higher filtration through the kidneys in the hyperglycemic state.

Adolescent↗