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PubMed1992

Ascaris suum: are trypsin inhibitors involved in species specificity of Ascarid nematodes?

Inhibitors of porcine trypsin were prepared from aqueous extracts of the parasitic nematodes Ascaris suum (hogs) and Ascaris lumbricoides (human). In this study three experiments were performed. (1) Polyclonal antibodies were prepared against one isoform of trypsin inhibitor from each parasitic nematode. Each antibody reacted with all isoforms from itself as well as all isoforms from the other parasite. (2) Association equilibrium constants were measured by titrating host trypsins (porcine or human) with the isoforms of trypsin inhibitors from A. suum and A. lumbricoides. While three of the combinations formed tight complexes that can be precipitated, the fourth complex, A. suum trypsin inhibitor-human trypsin has a Ka that is a 300 to 1000 times weaker interaction than the three other titration pairs. (3) Live A. suum worms were incubated in isosmotic media that contained either porcine trypsin or human trypsin. A suum worms survived in porcine trypsin and in the controls but were killed and digested after exposure for 5 days in human trypsin. The first experiment suggests that the trypsin inhibitors from A. suum and A. lumbricoides have similar epitopes, while the second experiment suggests that there are differences near the reactive site of the inhibitors. The consequences of these differences are dramatically demonstrated by the third experiment in which live A. suum worms in the presence of human trypsin die and are digested but those in porcine trypsin survive. These experiments suggest that in order to parasitize a host, a nematode requires a complement of protease inhibitors that interact strongly with those host proteases that are in their environment.

Animals↗
PubMed1986

Correlation of trypsin-plasma inhibitor complexes with mortality in experimental pancreatitis in rats.

Activation of trypsinogen in acute pancreatitis results in subsequent increases in plasma levels of trypsin bound to the inhibitors alpha 1-protease inhibitor (alpha 1-PI) and alpha-macroglobulin (alpha-M). It seems logical to speculate that plasma levels of these inhibitor-bound forms of trypsin may reflect the degree of intrapancreatic zymogen activation and that determination of such parameters may be of diagnostic and prognostic value. In order to test this hypothesis, the concentrations of trypsinogen and of trypsin bound to alpha 1-PI have been determined in serial plasma samples from rats who died (N = 7) and survived (N = 5) following induction of pancreatitis with taurocholate. Since the other major reaction product of active trypsin in plasma, alpha-macroglobulin-bound trypsin, cannot be measured directly, the plasma levels of trypsin-like amidase activity were determined to estimate the concentration of trypsin-alpha-M complex. Shortly after induction of pancreatitis, elevated levels of trypsinogen were present in plasma, but no alpha 1-PI-bound trypsin could be detected. Trypsin-alpha 1-PI complex continuously increased over the time course of pancreatitis in animals that died. In contrast, the plasma levels of trypsin-alpha 1-PI complex were lower in animals that survived, peaked around 15 hr postinduction at levels (182 +/- 53 ng/ml) significantly lower than those in dying animals (543 +/- 346 ng/ml), and fell during the following 48 hr. There was a significant correlation between plasma trypsin-like amidase activity and plasma alpha 1-PI-bound trypsin. Our data demonstrate that the concentration of activated forms of plasma trypsin in the bloodstream are correlated with mortality in experimental pancreatitis.

Animals↗
PubMed1986

In vivo metabolism of inter-alpha-trypsin inhibitor and its proteinase complexes: evidence for proteinase transfer to alpha 2-macroglobulin and alpha 1-proteinase inhibitor.

Inter-alpha-trypsin inhibitor was purified by a modification of published procedures which involved fewer steps and resulted in higher yields. The preparation was used to study the clearance of the inhibitor and its complex with trypsin from the plasma of mice and to examine degradation of the inhibitor in vivo. Unlike other plasma proteinase inhibitor-proteinase complexes, inter-alpha-trypsin inhibitor reacted with trypsin did not clear faster than the unreacted inhibitor. Studies using 125I-trypsin provided evidence for the dissociation of complexes of proteinase and inter-alpha-trypsin inhibitor in vivo, followed by rapid removal of proteinase by other plasma proteinase inhibitors, particularly alpha 2-macroglobulin and alpha 1-proteinase inhibitor. Studies in vitro also demonstrated the transfer of trypsin from inter-alpha-trypsin inhibitor to alpha 2-macroglobulin and alpha 1-proteinase inhibitor but at a much slower rate. The clearance of unreacted 125I-inter-alpha-trypsin inhibitor was characterized by a half-life ranging from 30 min to more than 1 h. Murine and human inhibitors exhibited identical behavior. Multiphasic clearance of the inhibitor was not due to degradation, aggregation, or carbohydrate heterogeneity, as shown by competition studies with asialoorosomucoid and macroalbumin, but was probably a result of extravascular distribution or endothelial binding. 125I-inter-alpha-trypsin inhibitor cleared primarily in the liver. Analysis of liver and kidney tissue by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis showed internalization and limited degradation of 125I-inter-alpha-trypsin inhibitor in these tissues. No evidence for the production of smaller proteinase inhibitors from 125I-inter-alpha-trypsin inhibitor injected intravenously or intraperitoneally was detected, even in casein-induced peritoneal inflammation. No species of molecular weight similar to that of urinary proteinase inhibitors, 19,000-70,000, appeared in plasma, liver, kidney, or urine following injection of inter-alpha-trypsin inhibitor.

Alpha-Globulins↗
PubMed1987

Insulin-mimetic effect of trypsin on the insulin receptor tyrosine kinase in intact adipocytes.

It has previously been demonstrated that the insulin-mimetic agent trypsin stimulates autophosphorylation of purified insulin receptors and activates the insulin receptor tyrosine kinase in vitro. We now report the effects of trypsin on whole cell tyrosine kinase activation and insulin receptor autophosphorylation. Trypsin treatment of intact adipocytes produces a time-dependent stimulation of tyrosine kinase activity as measured in lectin extracts containing the insulin receptor, or specifically immunoprecipitated insulin receptor samples. Trypsin treatment of adipocytes also results in a loss of insulin binding capacity, and a linear correlation exists between loss of binding and stimulation of tyrosine kinase activity. Exposure of adipocytes to trypsin is known to result in a time- and dose-dependent activation of intracellular glycogen synthase. Examination of the time courses of stimulation of tyrosine kinase and glycogen synthase activation in our system indicates that the stimulation of tyrosine kinase activity by trypsin occurs with sufficient rapidity and magnitude to be consistent with a role of phosphorylation in the activation of glycogen synthase. Trypsin has further been demonstrated to stimulate autophosphorylation of the beta-subunit of the insulin receptor in intact adipocytes. Cells prelabeled with [32P]PO4 for 2 h were exposed to trypsin, and receptors were partially purified over wheat germ agglutinin-agarose columns. Receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the beta-subunit was identified by autoradiography. The protein was extracted and hydrolyzed, and the phosphoamino acids were separated by electrophoresis and quantitated. Two- and five-fold increases in phosphotyrosine were observed with 3 and 10 min of trypsin treatment, respectively. We conclude that trypsin-induced cleavage of the insulin receptor alpha-subunit is relevant to the ability of trypsin to activate the insulin receptor tyrosine kinase in intact adipocytes. We further conclude that autophosphorylation of the insulin receptor and activation of its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic effects of trypsin.

Adipose Tissue↗
PubMed1993

Modification of the adenosine 5'-triphosphate-sensitive K+ channel by trypsin in guinea-pig ventricular myocytes.

1. The adenosine 5'-triphosphate (ATP)-sensitive K+ channel current was recorded in guinea-pig ventricular myocytes using the patch clamp technique with inside-out patch configuration. Modification of the channel activity by intracellular application of an endoprotease trypsin was studied, and was related to a possible model of regulation of this channel. 2. Maximal ATP-sensitive K+ channel activity was observed immediately upon formation of inside-out patches in the ATP-free internal solution, thereafter activity declined both spontaneously and gradually with time; a phenomenon known as rundown. When trypsin (1 mg/ml) was applied to the intracellular side of the membrane upon formation of inside-out patches, spontaneous run-down did not occur, and this trypsin action was irreversible. Neither trypsin (1 mg/ml) applied with trypsin inhibitor (0.25 mg/ml) nor heat-denatured trypsin (1 mg/ml) could mimic this effect. When trypsin was applied to the patches after run-down, channels were reactivated at approximately 13 min. 3. Treatment with trypsin did not affect unitary current amplitude, channel gating kinetics, or sensitivity to intracellular ATP. 4. Intracellularly applied Ca2+ induced run-down of channel activity in a dose-dependent manner. In membrane patches that were treated with trypsin (1 mg/ml) for 20 min, intracellularly applied Ca2+ up to 1 mM did not induce run-down of channel activity. 5. Intracellular application of an exopeptidase, carboxypeptidase A (1 mg/ml), but not Leu-aminopeptidase (0.5 mg/ml), prevented spontaneous or Ca(2+)-induced run-down of channel activity. 6. As postulated for several other channels, such as Na+ and Ca2+ channels, there may be a possible 'chemical gate' that is responsible for run-down of this channel activity. Application of trypsin might somehow modify this 'chemical gate', resulting in prevention of spontaneous or Ca(2+)-induced run-down. This target site for trypsin may be situated on the carboxy-terminus of the channel proteins, or of associated regulatory units. Because ATP sensitivity remained intact after trypsin treatment, the trypsin-selective site for channel inhibition is not related physically to the ATP binding site.

Adenosine Triphosphate↗
PubMed1996

Comparison of the structures of the cyclotheonamide A complexes of human alpha-thrombin and bovine beta-trypsin.

Thrombin, a trypsin-like serine protease present in blood, plays a central role in the regulation of thrombosis and hemostasis. A cyclic pentapeptide, cyclotheonamide A (CtA), isolated from sponges of the genus Theonella, inhibits thrombin, trypsin, and certain other serine proteases. Enzyme inhibition data for CtA indicate that it is a moderate inhibitor of alpha-thrombin (K(i) = 1.0 nM), but substantially more potent toward trypsin (K(i) = 0.2 nM). The comparative study of the crystal structures of the CtA complexes of alpha-thrombin and beta-trypsin reported here focuses on structure-function relationships in general and the enhanced specificity of trypsin, in particular. The crystal structures of the CtA complexes of thrombin and trypsin were solved and refined at 1.7 and 2.0 A resolution, respectively. The structures show that CtA occupies the active site with the Pro-Arg motif positioned in the S2 and S1 binding sites. The alpha-keto group of CtA is involved in a tetrahedral intermediate hemiketal structure with Ser 195 OG of the catalytic triad and is positioned within bonding distance from, and orthogonal to, the re-face of the carbonyl of the arginine of CtA. As in other productive binding modes of serine proteases, the Ser 214-Gly 216 segment runs in a twisted antiparallel beta-strand manner with respect to the diaminopropionic acid (Dpr)-Arg segment of CtA. The Tyr 60A-Thr 60I insertion loop of thrombin makes a weak aromatic stacking interaction with the v-Tyr of CtA through Trp 60D. The Glu 39 Tyr and Leu 41 Phe substitutions in trypsin produce an enhanced aromatic interaction with D-Phe of CtA, which also leads to different orientations of the side chains of D-Phe and the v-Tyr. The comparison of the CtA complexes of thrombin and trypsin shows that the gross structural features of both in the active site region are the same, whereas the differences observed are mainly due to minor insertions and substitutions. In trypsin, the substitution of Ile 174-Arg 175 by Gly 174-Gln 175 makes the S3 aryl site more polar because the Arg 175 side chain is directed away from thrombin and into the solvent, whereas Gln 175 is not. Because the site is occupied by the Dpr group of CtA, the occupancy of the S3 site is better in trypsin than in thrombin. In trypsin, the D-Phe side chain of CtA fits between Tyr 39 and Phe 41 in a favorable manner, whereas in thrombin, these residues are Glu 39 and Leu 41. The higher degree of specificity for trypsin is most likely the result of these substitutions and the absence of the fairly rigid Tyr 60A-Thr 60I insertion loop of thrombin, which narrows access to the active site and forces less favorable orientations for the D-Phe and v-Tyr residues.

Animals↗
PubMed2006

Trypsins from yellowfin tuna (Thunnus albacores) spleen: purification and characterization.

Two anionic trypsins (A and B) were purified to homogeneity from yellowfin tuna (Thunnus albacores) spleen by a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. Purity was increased to 70.6- and 91.5-fold with approximately 2.8% and 15.6% yield for trypsin A and B, respectively. The apparent molecular weight of both trypsins was estimated to be 24 kDa by size exclusion chromatography and SDS-PAGE. Both trypsin A and B appeared as a single band on native-PAGE. Trypsin A and B exhibited the maximal activity at 55 and 65 degrees C, respectively, and had the same optimal pH at 8.5 using TAME as a substrate. Both trypsins were stable to heat treatment up to 50 degrees C and in the pH range of 6.0 to 11.0. Both trypsin A and B were stabilized by calcium ion. The activities were inhibited effectively by soybean trypsin inhibitor, TLCK and partially inhibited by EDTA, but were not inhibited by E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). Apparent Km and Kcat of trypsin A and B for TAME were 0.2-0.33 mM and 66.7-80 S(-1), respectively. The N-terminal amino acid sequences of trypsin A, IVGGYECQAHSQPHQVSLNA, and trypsin B, IVGGYECQAHSQPPQVSLNA, indicated the high homology between both enzymes.

Amino Acids↗
PubMed2005

Trypsin-2 degrades human type II collagen and is expressed and activated in mesenchymally transformed rheumatoid arthritis synovitis tissue.

It has traditionally been believed that only the human collagenases (matrix metalloproteinase-1, -8, and -13) are capable of initiating the degradation of collagens. Here, we show that human trypsin-2 is also capable of cleaving the triple helix of human cartilage collagen type II. We purified human trypsin-2 and tumor-associated trypsin inhibitor by affinity chromatography whereas collagen type II was purified from cartilage extracts using pepsin digestion and salt precipitation. Degradation of type II collagen and gelatin by trypsin-2 was demonstrated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymography, and mass spectrometry, and tumor-associated trypsin inhibitor specifically inhibited this degradation. Although human trypsin-2 efficiently digested type II collagen, bovine trypsin did not. Furthermore, immunohistochemical staining detected trypsin-2 in the fibroblast-like synovial lining and in stromal cells of human rheumatoid arthritis synovial membrane. These findings were confirmed by reverse transcriptase-polymerase chain reaction and nucleotide sequencing. Trypsin-2 alone and complexed with alpha(1)-proteinase inhibitor were also detected in the synovial fluid of affected joints by time-resolved immunofluorometric assay, suggesting that trypsin-2 is activated locally. These results are the first to assess the ability of human trypsin to cleave human type II collagen. Thus, trypsin-2 and its regulators should be further studied for use as markers of prognosis and disease activity in rheumatoid arthritis.

Adult↗
PubMed2004

Ala226 to Gly and Ser189 to Asp mutations convert rat chymotrypsin B to a trypsin-like protease.

In a previous successful attempt to convert trypsin to a chymotrypsin-like protease, 15 residues of trypsin were replaced with the corresponding ones in chymotrypsin. This suggests a complex mechanism of substrate recognition instead of a relatively simple one that only involves three sites, residues 189, 216 and 226. However, both trypsin-->elastase and chymotrypsin-->trypsin conversion experiments carried out according to the complex model resulted in non-specific proteases with low catalytic activity. Chymotrypsin used in the latter studies was of B-type, containing an Ala residue at position 226. Trypsins, however, contain a conserved Gly at this site. The substantially decreased trypsin-like activity of the G226A trypsin mutant also suggests a specific role for this site in substrate binding. Here we investigate the role of site 226 by introducing the A226G substitution into chymotrypsin-->trypsin mutants which were constructed according to both the simple (S189D mutant) and the complex model (S(1) mutant) of specificity determination. The kinetic parameters show that the A226G substitution in the S(1) mutant increased the chymotrypsin-like activity, while the trypsin-like activity did not change. In contrast, this substitution in the S189D chymotrypsin mutant resulted in a 100-fold increase in trypsin-like activity and a trypsin-like specificity profile as tested on a competing oligopeptide substrate library. Additionally, the S189D+A226G mutant is the first trypsin-like chymotrypsin that undergoes autoactivation, an exclusive property of trypsinogen among pancreatic serine proteases.

Alanine↗
PubMed1989

Purification and characterization of trypsins from the digestive tract of Locusta migratoria.

Two trypsin-like enzymes were isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides. Primary purification was carried out on a DEAE-cellulose column, from which the two trypsins emerged in the anionic fraction. Further purification was achieved by affinity chromatography on a p-aminobenzamidine (PABA)-Sepharose column, which also separated the two trypsins (TLEAff.1. and TLEAff.2.), or by HPLC on an anion exchange column. The purity and homogeneity of the trypsins were demonstrated by electrophoresis of cellulose acetate strips and in polyacrylamide gels, with and without SDS. The molecular weights of TLEAff.1 and TLEAff.2, as determined by SDS-PAGE, were 17,000 and 24,000 respectively. The amino acid compositions of the locust trypsins were similar to those of trypsins from the digestive systems of other insects, which are characterized by the lack or low content of half cystines. The isoelectric points were 3.2 for TLEAff.1 and 3.5 fold for TLEAff.2. Since most of the locust trypsin comprised TLEAff.2, the latter served as the main object of this study. TLEAff.2 was unstable at low pH, differing in this respect from mammalian trypsins. The optimum activity was at pH 8.5-9.0. The Km and kcat, values were similar to those for bovine trypsin. Activation by substrate, a phenomenon in bovine trypsin, was also observed for TLEAff.2. The locust trypsin was full inhibited by the proteinaceous trypsin inhibitors Bowman-Birk (BBI) and Kunitz from soybeans, CI from chickpeas, chicken ovomucoid (COM), and turkey ovomucoid (TOM). It was inactivated by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone (TLCK), indicating the involvement of serine and histidine in the active site.

Amino Acids↗
PubMed1978

Studies on the influence of Trasylol on the partition of trypsin between the human plasma protease inhibitors in vitro.

The distribution of trypsin between the protease inhibitors of human serum with and without Trasylol was studied in vitro. 1) Trypsin was preferentially bound by alpha2-macroglobulin on addition of small amounts of the enzyme to normal serum in both the presence and absence of Trasylol in a molar concentration equal to that of alpha2-macroglobulin. 2) On saturation of alpha2-macroglobulin, a considerable amount of trypsin was bound by Trasylol even when most of the serum alpha1-antitrypsin was in a free form. 3) In reaction mixtures containing small amounts of trypsin, Trasylol was identified in a free form as well as in complex with trypsin-alpha2-macroglobulin complex and to a limited extent with trypsin. 4) With larger amounts of trypsin, sufficient to saturate alpha2-macroglobulin, increasing amounts of Trasylol were bound to trypsin. The relative amount of Trasylol bound to trypsin-alpha2-macroglobulin complexes was now smaller. This was explained by a higher affinity (or binding rate) of Trasylol for trypsin than for trypsin-alpha2-macroglobulin complexes. 5) Trypsin-Trasylol complexes showed no signs of dissociation after 5 h incubation at 37 degrees C in serum.

Aprotinin↗
PubMed1976

Enzymatic modification of lymphocyte receptors for antigen. III. Resistance of receptors to trypsin at the peak of the immune response.

Using sheep erythrocytes (SRBC) as the antigen, two subpopulations of spleen antigen-binding lymphocytes could be distinguished by a marked difference in the susceptibility of their receptors to trypsin. In unimmunized animals, 30% of the antigen-binding cells were trypsin-resistant, whereas at 5 days after immunization, 80-90% were trypsin-resistant, indicating an increase of about 50-fold in trypsin-resistant antigen-binding cells per spleen. In contrast, trypsin-sensitive cells per spleen were only 4-fold higher on day 5 than before immunization. The rise in % trypsin sensitivity preceded the increase in rosettes per spleen, implying that immunization produced a preferential increase in trypsin-resistant antigen binding cells partly by converting sensitive cells to resistant cells. After the 5th day, the trypsin sensitivity of antigen-binding cells slowly returned toward the unimmunized level, but a booster injection of SRBC restored trypsin resistance. Trypsin resistance was not lost in the presence of sodium azide or protein synthesis inhibitors. But a slightly increased trypsin susceptibility was conferred by 2-deoxyglucose, implying that glycolysis or the glycosylation of protein may be involved in maintaining trypsin resistance.

Animals↗
PubMed1985

Correlation between trypsin binding to a specific receptor and prostacyclin production in cultured vascular endothelial cells.

The correlation between the binding and processing of trypsin and its effect on prostacyclin (PGI2) production in cultured adult bovine aortic endothelial (ABAE) cells was studied. ABAE cells demonstrated an ability to produce PGI2 in a dose-response manner to trypsin at the range of 0.1-2.0 micrograms/ml with a saturation at a concentration of 1 microgram/ml. Likewise, 125I-trypsin binding to the cultured cells increased in a dose-response way and reached saturation at a concentration of about 1 microgram/ml; 125I-trypsin was bound to a specific high-affinity cell-surface receptor with a dissociation constant (Kd) of 1.5 X 10(-8) M and each of the confluent ABAE cells has about 1.2 X 10(5) such receptors sites. The cell-surface receptor for trypsin displayed specific characteristics and an excess amount of unlabeled trypsin successfully abolished 125I-trypsin binding while thrombin in excess failed to compete for 125I-trypsin binding. Only a small fraction of the cell-surface-bound 125I-trypsin was internalized and subsequently degraded by ABAE cells as compared to the process of 125I-trypsin internalization by human skin fibroblasts (HSF). This study demonstrated that the stimulatory effect of trypsin on prostacyclin production and release by ABAE cells might be mediated by a specific cell-surface receptor for trypsin on these cells distinct from the thrombin receptor.

6-Ketoprostaglandin F1 alpha↗
PubMed1977

Surface antigens of the embryonic chick myoblast: expression on freshly trypsinized cells.

Using an antiserum raised in rabbits against embryonic chick skeletal myoblasts (Anti-M-24), we have examined the trypsin and neuraminidase sensitivity and physiological expression of myoenic cell surface antigens. It was found that trypsin-released muscle cells more effectively inhibited, on a cell to cell basis, the cytotoxicity of Anti-M-24 for 24-h-old myoblast monolayers than did identical cells that had received a 3-4 h suspension culture recovery period from trypsinization. There was no such difference in absorptive capacities observed for any other embryonic chick tissue tested (e.g. brain, retina, liver, heart, and red blood cells) when freshly trypsinized cells were compared to ones which were given a 3-4 h culture period. If freshly trypsinized muscle cells were treated with high concentrations (30,000 international units (IU)/0.1 ml packed cells) of trypsin or with neuraminidase (30,000 IU/ml packed cells), there was a selective loss of myoblast-specific surface antigens. When single cells that had been in suspension culture for 3.5 h were reexposed to low concentrations (10,000 IU/0.1 ml packed cells) of trypsin, more antigenic sites were revealed on their surfaces as detected by an increased absorptive capacity in removing myoblast-binding antibodies from Anti-M-24. This increase in antigenic expression was time-dependent and inversely related to the length of culture time after trypsinization. Immunofluorescence studies revealed that tissue specific myoblast cell surface antigens are present on both muscle cells that were freshly dissociated and those that had been in suspension culture for 3-4 h. Furthermore, freshly trypsinized myoblasts possessed cell surface components that were highly antigenic; antiserum to such cells reacted extensively with both trypsinized and recovered muscle cells as detected by complement-dependent 51Cr release cytotoxicity assays and immunofluorescence. We conclude that embryonic chick myoblasts possess surface antigens that may be selectively removed by neuraminidase or high concentrations of trypsin. These antigens may be progressively masked, with increasing time of culture after protease-dissociation, by molecules that are sensitive to low concentrations of trypsin. Such masking of tissue-specific cell surface antigens could result in the display of molecular mosaics which may play a role in facilitating intercellular recognition and subsequent differentiation and histogenesis.

Animals↗
PubMed2005

Receptor rigidity and ligand mobility in trypsin-ligand complexes.

The trypsin-like serine proteases comprise a structurally similar family of proteins with a wide diversity of biological functions. Members of this family play roles in digestion, hemostasis, immune responses, and cancer metastasis. Bovine trypsin is an archetypical member of this family that has been extensively characterized both functionally and structurally, and that preferentially hydrolyzes Arg/Lys-Xaa peptide bonds. We have used molecular dynamics (MD) simulations to study bovine trypsin complexed with the two noncovalent small-molecule ligands, benzamidine and tranylcypromine, that have the same hydrogen-bond donating moieties as Arg and Lys side-chains, respectively. Multiple (10) simulations ranging from 1 ns to 2.2 ns, with explicit water molecules and periodic boundary conditions, were performed. The simulations reveal that the trypsin binding pocket residues are relatively rigid regardless of whether there is no ligand, a high-affinity ligand (benzamidine), or a low-affinity ligand (tranylcypromine). The thermal average of the conformations sampled by benzamidine bound to trypsin is planar and consistent with the planar internal geometry of the benzamidine crystallographic model coordinates. However, the most probable bound benzamidine conformations are +/-25 degrees out of plane, implying that the observed X-ray electron density represents an average of densities from two mirror symmetric, nonplanar conformations. Solvated benzamidine has free energy minima at +/-45 degrees , and the induction of a more planar geometry upon binding is associated with approximately 1 kcal/mol of intramolecular strain. Tranylcypromine's hydrogen-bonding pattern in the MD differs substantially from that inferred from the X-ray electron density. Early in simulations of this system, tranylcypromine adopts an alternative binding conformation, changing from the crystallographic conformation, with a direct hydrogen bond between its amino moiety and the backbone oxygen of Gly219, to one having a bridging water molecule. This result is consistently seen with the CHARMM22, Amber, or OPLS-AA force fields. The trypsin-tranylcypromine hydrogen-bonding pattern observed in the simulations also occurs as the crystallographic binding mode of the Lys15 side-chain of bovine pancreatic trypsin inhibitor bound to trypsin. In this latter cocrystal, a bridging crystallographic water does reside between the side-chain's amino group and the trypsin Gly219 backbone oxygen. Furthermore, the trypsin-tranylcypromine simulations sample two different stable noncrystallographic binding poses. These data suggest that some of the electron density ascribed to tranylcypromine in the X-ray model is rather due to a bound water molecule, and that multiple tranylcypromine binding conformations (crystallographic disorder) may be the cause of ambiguous electron density. The combined trypsin-benzamidine and trypsin- tranylcypromine results highlight the ability of simulations to augment protein-ligand complex structural data by deconvoluting the effects of thermal and structural averaging, and by finding energetically optimal ligand and bound water positions for weakly bound ligands.

Animals↗
PubMed2005

Atlantic cod trypsins: from basic research to practical applications.

Atlantic cod trypsin I is an appropriate representative of the traditionally classified cold-adapted group I trypsins, and the recombinant form of cod trypsin Y is the only biochemically characterized member of the novel group III trypsins. Trypsin Y is adapted to lower temperatures than all other presently known trypsins. This review describes the basic characteristics of and practical uses for trypsins of Atlantic cod, as well as those of other organisms. Overexpression of the recombinant forms of cod trypsins I and Y in microorganisms is explained as well as the advantages of using site-directed mutagenesis to increase their stability toward autolysis and thermal inactivation. Trypsins appear to play a key role in the nutrition and development of marine fish. We discuss the potential use of cod trypsins as biomarkers to evaluate the nutritional status of cod larvae and describe the industrial applications of cod trypsin I and other trypsins.

Adaptation, Physiological↗
PubMed2006

Development of a qRT-PCR assay to determine the relative mRNA expression of two different trypsins in Atlantic cod (Gadus morhua).

A fluorescence resonance energy transfer (FRET) based quantitative RT-PCR method (qRT PCR) was developed in this study for measuring the mRNA expression of trypsins Y and I in the Atlantic cod. Atlantic cod beta-actin was used as the reference gene and standard curves were created for quantification of the mRNA expression levels. For yet unknown reasons, the Atlantic cod (Gadus morhua) produces several trypsins with different characteristics. Trypsin I is the most common and best characterized of these but trypsin Y is a recently discovered enzyme. The recombinant form of trypsin Y was found to have unique characteristics relative to trypsin I. The native form of trypsin Y has proven difficult to isolate from the cod and activity assays do not distinguish between the activities of trypsin I and trypsin Y. The results show that trypsin Y mRNA is expressed in a very low copy number relative to that of trypsin I (ratio of 1:1340), which may explain the difficulty of isolating the native form of trypsin Y.

Animals↗
PubMed2006

Structural characterization of Na,K-ATPase from shark rectal glands by extensive trypsinization.

Extensive trypsinization of Na,K-ATPase from the salt gland of Squalus acanthias removes about half of the extramembranous protein mass of the alpha-subunit, while leaving the beta-subunit intact. Sequence analysis and epitope recognition of the remaining alpha-peptides show that transmembrane segments M1/M2 and M3/M4 are present when trypsinization is performed in either NaCl or RbCl. The M5/M6 segment and the intact 19-kDa peptide (M7-M10) are detected in Rb-trypsinized membranes but not in Na-trypsinized membranes. The L7/L8 loop is associated with Na-trypsinized membranes, indicating the presence of an M7/M8 or M8/M9 fragment. Freeze-fracture electron microscopy of both Rb- and Na-trypsinized membranes reveals intramembranous particles that indicate a retained cluster of peptides, even in the absence of an intact 19-kDa fragment. The rotational diffusion of covalently spin-labeled trypsinized complexes is studied in the presence of poly(ethylene glycol) or glycerol by using saturation transfer electron spin resonance. Rotational correlation times in aqueous poly(ethylene glycol) are longer than in glycerol solutions of the same viscosity and increase nonlinearly with the viscosity of the suspending medium, indicating that poly(ethylene glycol) induces aggregation of the tryptic peptides (and beta-subunit) within the membrane. The aggregates of enzyme trypsinized in the presence of NaCl are larger than those for enzyme trypsinized in RbCl, at both low and high aqueous viscosities. Similarities in mobility for native and Rb-trypsinized enzymes suggest either a change in average orientation of the spin-label upon trypsinization or that trypsinization leads to a reorganized protein structure that is more prone to aggregation.

Amino Acid Sequence↗