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PubMed1976

Oral trypsin-inhibitor-induced improvement of the exocrine and endocrine pancreatic functions in alloxan diabetic rats.

Studies are presented dealing with the effect on the exocrine and endocrine pancreas of long-term oral trypsin inhibitor administration to alloxan diabetic rats. Three categories of rats were used: a) normal rats, b) alloxan diabetic rats, and c) alloxan diabetic rats treated with the trypsin inhibitor. 1. The concentration of amylase in both pancreas and intestinal contents were markedly decreased in alloxan diabetic rats as compared with normal rats, whereas the concentrations of lipase and trypsin(ogen) were practically unaffected by the diabetic state. 2. Trypsin inhibitor treatment of diabetic rats enhanced the concentrations of both amylase, trypsin(ogen), and lipase in pancreas and intestinal contents when compared with untreated diabetic controls. 3. Alloxan diabetic rats treated with trypsin inhibitor displayed an increased weight gain, increased pancreatic weight, and increased pancreatic protein concentration as compared with the untreated diabetic controls. 4. Alloxan diabetic rats treated for 3 or 5 weeks with the trypsin inhibitor were found to have decreased basal blood glucose levels and an increased plasma insulin: blood glucose ratio as compared to the untreated diabetic controls. 5. There was no apparent difference in the glucose elimination rate after I.V. glucose loads in diabetic rats given trypsin inhibitor and their diabetic controls. However, the insulin secretory response to the glucose stimulus was slightly improved in the trypsin inhibitor group. 6. Alloxan diabetic rats treated with trypsin inhibitor had an increased total pancreatic insulin content compared with their diabetic controls. It is concluded that long-term daily treatment with oral trypsin inhibitor in alloxan diabetic rats increased enzyme production and secretion in the exocrine pancreas, and produced an improvement of the diabetic condition of the animals. This improvement might partly be due to an increased pancreatic content and secretion of insulin.

Amylases↗
PubMed2002

Characterization of the coupling activity for the binding of inter-alpha-trypsin inhibitor to hyaluronan in human and bovine follicular fluid.

The plasma proteinase inter-alpha-trypsin inhibitor is necessary for normal expansion of the cumulus-oocyte complex (COC) and lack of inter-alpha-trypsin inhibitor results in severe infertility. After diffusion from the circulation into the follicles, inter-alpha-trypsin inhibitor is incorporated into the extracellular hyaluronan network of the expanding COC. However, mixing isolated inter-alpha-trypsin inhibitor with hyaluronan in vitro does not result in coupling to hyaluronan. Other components must be present. A recently developed electrophoretic technique by which hyaluronan-bound inter-alpha-trypsin inhibitor is immobilized was used to demonstrate coupling activity in human and bovine follicular fluid that is necessary for the formation of a firm binding between inter-alpha-trypsin inhibitor heavy chains and hyaluronan, as observed in vivo. No coupling activity could be detected in human serum. Coupling occurred only in the presence of follicular fluid. The coupling activity of follicular fluid was irreversibly destroyed by heat treatment, lowering of pH or tryptic digestion, indicating that the coupling activity is associated with a protein. Calcium ions are essential for the coupling reaction. The binding reaction in vitro using intact inter-alpha-trypsin inhibitor is slow and occurs over 24 h. The early-formed complexes between inter-alpha-trypsin inhibitor and hyaluronan contain small amounts of bikunin, whereas the end product contains heavy chains and essentially no bikunin. The heavy chains released from inter-alpha-trypsin inhibitor by NaOH treatment bound immediately to hyaluronan, indicating that the dissociation of heavy chains from inter-alpha-trypsin inhibitor is the rate-limiting step. In conclusion, at least four components are essential for the covalent binding of heavy chains to hyaluronan: inter-alpha-trypsin inhibitor and calcium from plasma, hyaluronan and one or more proteins found in follicular fluid.

Alpha-Globulins↗
PubMed1995

Unique specificity of in vitro inhibition of mosquito midgut trypsin-like activity correlates with in vivo inhibition of malaria parasite infectivity.

Synchrony in the egress of Plasmodium ookinetes from the food bolus and enzymatic digestion of the blood meal in the mosquito midgut suggests that digestive enzymes play a role in the successful transmission of malaria parasites. Previously, we found that parasite-produced chitinase is essential for parasite transmission and can be activated by mosquito midgut protease. To determine the suitability of developing a transmission-blocking vaccine directed against mosquito trypsin-like enzyme(s), Aedes aegypti midgut trypsin-like proteases were characterized biochemically and compared to a mammalian trypsin. Mosquito trypsin is more sensitive to inhibition by aprotinin and less sensitive to egg white trypsin inhibitor than is bovine pancreatic trypsin. Soybean trypsin inhibitor and leupeptin inhibit both enzymes to similar extent. Membrane-feeding assays with aprotinin, leupeptin, and egg white trypsin inhibitor revealed a correlation between in vitro inhibition of mosquito trypsin-like activity and transmission-blocking activity. The results suggest a role for mosquito midgut trypsin(s) in malaria parasite development and indicate that the protease(s) is a potential target for blocking malaria transmission.

Aedes↗
PubMed1981

Soybean trypsin inhibitor (Kunitz) is doubleheaded. Kinetics of the interaction of alpha-chymotrypsin with each side.

Further evidence is presented for the formation of a ternary complex between alpha-chymotrypsin (EC 3.4.21.1) and soybean trypsin inhibitor as well as between alpha-chymotrypsin and a performed complex of soybean trypsin and inhibitor (EC 3.4.21.1). This is well in agreement with our earlier sedimentation equilibrium studies. We report on different elution patterns of the ternary forms as compared to the inhibitor trypsin complex and the individual components in gel filtration studies. We also demonstrate the decrease of a given chymotryptic activity on a substrate if the solution is mixed with another one containing the preformed stoichiometric inhibitor-trypsin complex. A fourth piece of evidence for the formation of a chymotrypsin-inhibitor-trypsin complex is the appearance of a difference spectrum in absorbance, when chymotrypsin is mixed with the inhibitor-trypsin complex. Inhibition studies with purified inhibitor show that one molecule of inhibitor binds two molecules of alpha-chymotrypsin, with dissociation constants K1 about 1 microM and K2 about 300 nM at pH 8. The site with weaker affinity for chymotrypsin is specifically blocked by stoichiometric amounts of trypsin. Purification of commercially available preparations of soybean trypsin inhibitor (Kunitz) ("inhibitor") to apparent homogeneity using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and first-order association kinetics with beta-trypsin, is achieved by a combination of gel filtration and ion-exchange chromatography. The kinetics of the interaction of chymotrypsin with inhibitor or with inhibitor-trypsin complex were measured in a stopped-flow photometer by following the displacement of proflavine from the active site of chymotrypsin. A complete reaction scheme is presented with all rates and equilibrium constants as well as their pH-dependence.

Binding Sites↗
PubMed1999

Expression of trypsin in human cancer cell lines and cancer tissues and its tight binding to soluble form of Alzheimer amyloid precursor protein in culture.

It was recently found that overexpression of the trypsin gene in tumor cells stimulates their growth in culture and in nude mice. In the present study, expression of trypsin in various human cancer cell lines and tissues was studied by gelatin zymography and immunoblotting before and after enterokinase treatment and by immunohistochemistry. The analyses showed that many stomach, colon, and breast cancer cell lines secreted trypsinogens-1 and/or -2, as well as an unidentified serine proteinase of about 70 kDa, into culture medium. Lung cancer cell lines secreted 18- and 19-kDa unidentified trypsin-like proteins. Stomach cancer cell lines frequently secreted active trypsin, suggesting that they produced an endogenous activator of trypsinogen, most likely enterokinase. Active trypsin formed a complex with a soluble form of Alzheimer amyloid precursor protein (sAPP), a Kunitz-type trypsin inhibitor, which was secreted by all cell lines tested. This indicated that sAPP is a primary inhibitor of secreted trypsin. Immunohistochemical analysis showed that trypsin(ogen) was frequently expressed at high levels in stomach and colon cancers, but scarcely in breast cancers. In the stomach cancers, the trypsin immunoreactivity was higher in the malignant, non-cohesive type than in the cohesive type. These results support the hypothesis that tumor-derived trypsin is involved in the malignant growth of tumor cells, especially stomach cancer cells.

Amyloid beta-Protein Precursor↗
PubMed2002

Trypsin activity is not involved in premature, intrapancreatic trypsinogen activation.

A premature and intracellular activation of digestive zymogens is thought to be responsible for the onset of pancreatitis. Because trypsin has a critical role in initiating the activation cascade of digestive enzymes in the gut, it has been assumed that trypsin also initiates intracellular zymogen activation in the pancreas. We have tested this hypothesis in isolated acini and lobules from rat pancreas. Intracellular trypsinogen activation was induced by supramaximal secretagogue stimulation and measured using either specific trypsin substrates or immunoreactivity of the trypsinogen activation peptide (TAP). To prevent a trypsin-induced trypsinogen activation, we used the cell-permeant, highly specific, and reversible inhibitor Nalpha-(2-naphthylsulfonyl)-3-amidinophenylalanine-carboxymethylpiperazide (S124), and to prevent cathepsin-induced trypsinogen activation, we used the cysteine protease inhibitor E-64d. Incubation of acini or lobules in the presence of S124 completely prevented the generation of trypsin activity in response to supramaximal caerulein but had no effect whatsoever on the generation of TAP. Conversely, when trypsin activity was recovered at the end of the experiment by either washout of S124 from acini or extensive dilution of lobule homogenates, it was up to 400% higher than after caerulein alone and corresponded, in molar terms, to the generation of TAP. Both trypsin activity and TAP release were inhibited in parallel by E-64d. We conclude that caerulein-induced trypsinogen activation in the pancreas is caused by an E-64d-inhibitable mechanism such as cathepsin-induced trypsinogen activation, and neither involves nor requires intracellular trypsin activity. Specific trypsin inhibition, on the other hand, prevents 80% of trypsin inactivation or autodegradation in the pancreas.

Acute Disease↗
PubMed2001

Association of trypsin-2 with activation of gelatinase B and collagenase-2 in human bronchoalveolar lavage fluid in vivo.

BACKGROUND: Tissue injury mediated by matrix metalloproteinases (MMPs) is a hallmark of inflammatory lung diseases. Latent secreted proMMPs must be activated to be catalytically competent. AIM: Our aim was to analyse an involvement of the trypsin-2, trypsin-2-alpha1-proteinase inhibitor (PI) complex and tumour-associated trypsin inhibitor (TATI) in the in vivo activation of proMMP-8, -9 and -2. METHODS: Concentrations of trypsin-2, trypsin-2-alpha1-PI complex and TATI in bronchoalveolar lavage fluid (BALF) were analysed by immunofluorometry. Molecular forms and expression of trypsin-2 and trypsin-2-alpha1-PI complex were identified by Western immunoblot and immunocytochemistry. Gelatinolytic and collagenolytic activities were measured by substrate-based activity assays. RESULTS: BALFs from 16 of 43 patients and BALFs from five of 15 healthy controls contained trypsin-2-alpha1-PI complex. TATI was found in all healthy control BALFs (median 0.12 microg/L, range 0.02-0.66 microg/L) whereas 8 of 43 BALFs from patients (median 0, range 0-0.64 microg/L, P = 0.0001) contained TATI. Patient BALFs showed significantly increased activation of MMP-9 and MMP-8 compared with healthy controls. The concentrations of trypsin-2-alpha1-PI complex correlated with the in vivo activation of MMP-9 and -8 (r = 0.68, P = 0.002 and r = 0.61, P = 0.008) but not with the activation of MMP-2 in BALFs. CONCLUSION: Results show a key role of trypsin-2 in the in vivo activation of proMMP-8 and -9 in inflammatory lung diseases.

Adult↗
PubMed1981

Trypsin activation of inactive renin in human plasma. An assessment of some methodological aspects.

The following methodological aspects of the use of trypsin as activator of inactive renin in human plasma have been studied: a) the effect of SBTI on renin activity and angiotensin; b) the reaction velocity of trypsin on inactive renin; c) the optimum trypsin concentration; d) the ability of human plasma to neutralize exogenous trypsin. Our results show that: 1) Some commercially available SBTI may exert an angiotensinase-like effect which can be abolished by PMSF. 2) At 4 degrees C activation of inactive renin reached a maximum within the first two minutes then no further activation could be demonstrated. 3) Trypsin 2 mg/ml yielded more inactive renin than trypsin 1 or 0.5 mg/ml. A higher concentration (3 mg/ml) gave substantially equivalent activation as (with) trypsin 2 mg/ml whereas when using a still higher concentration (4 mg/ml) a degradation of the renin system components could be noted. 4) Endogenous trypsin inhibitors can eventually inactivate exogenous trypsin up to 3 mg/ml. About 20% of renin is destroyed by trypsin 4 mg/ml within 2 min at 4 degrees C while an additional 40% is lost during the incubation at 37 degrees C if no SBTI is added.

Enzyme Activation↗
PubMed1998

Engineering bidentate macromolecular inhibitors for trypsin and urokinase-type plasminogen activator.

Ecotin, a dimeric serine protease inhibitor from Escherichia coli, is a novel platform for inhibitor design. An approach using the three-dimensional structure of the ecotin-trypsin complex to guide combinatiorial design efforts was taken to create potent bidentate ecotin inhibitors for trypsin and human urokinase-type plasminogen activator (uPA). The ecotin surface loop that was redesigned is composed of residues 67 to 70 (60 s loop), and binds to the target protease at a region 25 A from the enzyme active site. Two ecotin phage display libraries were constructed to exploit the binding interactions at the 60 s loop. The ecotin 60X4 library, in which residues 67 to 70 of ecotin were randomized, was panned against rat and bovine trypsin in parallel for four rounds. Panning against bovine trypsin resulted in enrichment of ecotin phage but did not yield a consensus sequence. Panning against rat trypsin resulted in enrichment as well as the ecotin consensus sequence WGFP at positions 67 to 70. The variant ecotin encoded by this sequence inhibited rat trypsin at 80 pM, a 12-fold improvement over ecotin wild-type (WT). A second generation library, ecotin M84R+60X4 including an additional methionine to arginine substitution at position 84 in the primary binding site of ecotin, was generated for panning against uPA and rat trypsin. Panning against rat trypsin resulted in enrichment but no consensus sequence. Panning against uPA resulted in enrichment as well as the different ecotin consensus sequence WGYR at positions 67 to 70. Ecotin M84R+D70R bound to uPA at 50 pM, a 56,000-fold increase in binding compared to ecotin WT. Furthermore, ecotin M84R+D70R achieved a 13,680-fold preference of specificity towards uPA versus rat trypsin. The fact that the 60 s loop of ecotin plays different roles in binding to trypsin and uPA suggests this site can be used to introduce specificity and potency for other members of the serine proteases with a chymotrypsin fold.

Animals↗
PubMed1990

Influences of trypsin and collagenase on acetylcholine responses of physically isolated single neurons of Aplysia californica.

1. The influences of enzyme treatments (trypsin and collagenase) on responses to perfused acetylcholine were examined on physically isolated single Aplysia neurons, using the voltage-clamp, internal perfusion, and rapid external perfusion technique. 2. During treatment with trypsin (0.025 to 0.1%) for 10 to 30 min at room temperature (22 to 25 degrees C), the peak amplitude of the Na current induced by acetylcholine increased in a time- and dose-dependent manner, and the decay in the continued presence of acetylcholine was slowed. This effect of trypsin treatment was irreversible after washing for 60 min without enzyme. 3. Edrophonium, a cholinesterase inhibitor, has previously been shown to augment the Na acetylcholine response in this preparation by inhibition of acetylcholinesterase. After treatment of the neuron with trypsin, the augmentation after edrophonium was abolished. Furthermore, in the presence of edrophonium, trypsin also failed to increase the response. The dose-response curve for acetylcholine after treatment of trypsin was similar to that in the presence of edrophonium. These results suggest that the modification of the current response by trypsin is a result of removal of cholinesterase activity from the membrane. 4. In contrast to the effects of trypsin, collagenase (0.03 to 0.1%) for 10 to 60 min did not change the current amplitude of the acetylcholine response. However, collagenase treatment did alter the kinetics of the acetylcholine response in a dose-dependent manner, in that the rate of decay was accelerated. A similar acceleration was seen in the acetylcholine responses on other neurons which were due to Cl or K currents, suggesting that the effect was independent on the type of channel. This effect of collagenase was reversible after 30 to 60 min of washing of the neuron. 5. In the presence of edrophonium or after the treatment with trypsin, collagenase still accelerated the current kinetics of the acetylcholine response, indicating that cholinesterase activity is not related to this effect. Furthermore, heated collagenase (presumably inactivated) had a similar action, suggesting that the enzymatic activity of collagenase is not related to the modification of the response. 6. These results suggest that Aplysia acetylcholinesterase is sensitive to trypsin but not to collagenase. However, the preparation of a collagenase used in these studies contains some factor which alters the response to acetylcholine, but this effect is reversible and unrelated to enzymatic activity.

Acetylcholine↗
PubMed1975

Membrane bound and soluble adenosine triphosphatase of Escherichia coli K 12. Kinetic properties of the basal and trypsin-stimulated activities.

Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.

Adenosine Diphosphate↗
PubMed1990

Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells.

When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.

Animals↗
PubMed1985

Interactions of derivatives of guanidinophenylalanine and guanidinophenylglycine with Streptomyces griseus trypsin.

The rates of hydrolysis of the ester, amide and anilide substrates of p-guanidino-L-phenylalanine (GPA) by Streptomyces griseus trypsin (S. griseus trypsin) were compared with those of arginine (Arg) substrates. The specificity constant (kcat/km) for the hydrolysis of GPA substrates by the enzyme was 2-3-times lower than that for arginine substrates. The kcat and Km values for the hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) by S. griseus trypsin are in the same order of magnitude as those of N alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), although both values for the former when hydrolyzed by bovine trypsin are higher by one order of magnitude than those for the latter. The specificity constant for the hydrolysis of Bz-GPA-OEt by S. griseus trypsin is much higher than that for N alpha-benzoyl-p-guanidino-L-phenylglycine ethyl ester (Bz-GPG-OEt). As with the kinetic behavior of bovine trypsin, low values in Km and kcat were observed for the hydrolysis of amide and anilide substrates of GPA by S. griseus trypsin compared with those of arginine substrates. The rates of hydrolysis of GPA and arginine substrates by S. griseus trypsin are about 2- to 62-times higher than those obtained by bovine trypsin. Substrate activation was observed with S. griseus trypsin in the hydrolysis of Bz-GPA-OEt as well as Bz-Arg-OEt, whereas substrate inhibition was observed in three kinds of N alpha-protected anilide substrates of GPA and arginine. In contrast, no activation by the amide substrate of GPA could be detected with this enzyme.

Guanidines↗
PubMed1983

Aqueous two-phase partition studies of glucocorticoid receptors exposed to limited trypsination.

In a previous investigation the properties of glucocorticoid receptors exposed to partial proteolysis by chymotrypsin were studied by aqueous two-phase partitioning (Andreasen, P.A. and Gehring, U. (1981) Eur. J. Biochem. 120, 443-449). This paper describes studies of the properties of cytosolic glucocorticoid receptors submitted to limited trypsination, employing phase partitioning of rat thymocyte cytosol labelled with tritiated triamcinolone acetonide. Trypsin treatment of labelled cytosol at 0 degrees C does not result in any dissociation of steroid from the receptor. The partition properties of the trypsin-treated receptors exposed to receptor-activating conditions are indistinguishable from those of the activated native and chymotrypsin-treated receptors, although the trypsin-treated receptors have lost the affinity for DNA and dextran sulphate. Trypsin treatment of cytosol not exposed to receptor-activating conditions results in a rapid change in the receptor partition coefficients identical to that following chymotrypsin treatment. However, during incubations under conditions at which activation of native and chymotrypsin-treated receptors is very slow, the trypsin-treated receptor is converted to a form with partition properties indistinguishable from those of the activated receptors. During exposure of the cytosol to activating conditions, the time-course of the partition coefficient of the trypsin-treated receptors is only slightly different from that of the native and chymotrypsin-treated receptors, but the trypsin-treated receptors are far less susceptible to the activation inhibitors ATP, Li+ and MoO42-. We conclude that the proteolytic cleavages induced by trypsin in the non-activated receptor do not lead to any immediate changes in the charge and surface properties of the receptor different from those following chymotrypsin treatment, but that the trypsin-treated receptor is not able to maintain a non-activated state and a normal susceptibility to activation inhibitors.

Adenosine Triphosphate↗
PubMed1985

A solid-phase immunosorbent assay to determine the proteinase binding capacity of alpha 2-macroglobulin using 125I-trypsin as indicator proteinase.

Hyperimmune sera against human alpha 2 macroglobulin were raised in rabbits following immunization with 's' alpha 2-macroglobulin over half a year. Immunoglobulins were prepared by DEAE-Sephacel anion exchange chromatography. The immunoglobulin preparations showed a remarkably high and equal titer for 's' and 'f' alpha 2-macroglobulin (plasma alpha 2-macroglobulin fully saturated with pig pancreas trypsin), which amounted to 6.4 X 10(-6) as revealed by passive hemagglutination. Immunoimmobilization experiments revealed that at equilibrium, 's' alpha 2-macroglobulin and both 'f' alpha 2-macroglobulins (27 and 82% saturation of 's' alpha 2-macroglobulin with trypsin) had been bound to the same degree from the fluid phase to the monospecific antibodies that had been adsorbed to polystyrene tubes. Comparison of quantitative gel scans for disappearance of the intact alpha 2-macroglobulin subunit (Mr 182000) with 125I-labeled trypsin binding capacity of immunoimmobilized alpha 2-macroglobulin-trypsin complexes showed conspicuous agreement. Rocket immunoelectrophoresis did not give significant differences between 's' alpha 2-macroglobulin and 'f' alpha 2-macroglobulin. In the fluid phase, a binding ratio of 2.4 mol trypsin/mol alpha 2-macroglobulin was observed. Saturation of solid phase immunoimmobilized 's' alpha 2-macroglobulin with trypsin could be accomplished by incubation with a 100-200-fold molar excess of enzyme for 10 min. The solid-phase experiments showed a binding ratio of 2.0 mol trypsin/mol alpha 2-macroglobulin. The high molar excess of trypsin needed to saturate solid-phase immunoimmobilized alpha 2-macroglobulin, which binds 20% less trypsin than in the liquid phase, is partially explained by an enhancement of the negative cooperativity of trypsin binding to alpha 2-macroglobulin found in the liquid-phase system. Assessment of the trypsin-binding capacity of alpha 2-macroglobulin immunoadsorbed from synovial fluids (n = 19) of patients with seropositive rheumatoid arthritis yielded an inactive alpha 2-macroglobulin of 0-53% when compared to the trypsin-binding capacity of normal plasma alpha 2-macroglobulin.

Antibodies↗
PubMed1994

Sequence of three cDNAs encoding an alkaline midgut trypsin from Manduca sexta.

We have purified trypsin from the midgut of Manduca sexta and shown it has an alkaline pH optimum of 10.5. In order to clone the midgut trypsin, a DNA probe was generated using the polymerase chain reaction (PCR) with template isolated from a midgut cDNA library phage stock, a mixture of degenerate primers synthesized to code for the highly conserved region around the active site serine found in trypsins, and the T7 sequencing primer. Three different trypsin cDNAs were isolated each of which encodes a preproenzyme of 256 amino acids with a putative signal sequence of 17 amino acids, an activation peptide of seven amino acids and a mature trypsin of 232 amino acids. The encoded midgut trypsins contain the highly conserved residues, Asp, His, Ser, involved in catalysis in serine proteases, along with the residues which define the trypsin specificity pocket. Sequence comparisons show that all sequences are similar to other invertebrate and vertebrate serine proteases, but they differ in that two of the three encoded trypsins have an odd number of cysteines. Northern analysis localizes the trypsin mRNA to the middle third of the midgut. A large number of arginines (19, 20 and 21) are encoded by the three cDNAs which may stabilize the trypsin, by remaining protonated, in the alkaline midgut of M. sexta.

Amino Acid Sequence↗
PubMed2006

Trypsin immobilization by direct adsorption on metal ion chelated macroporous chitosan-silica gel beads.

Silica gel bead coated with macroporous chitosan layer (CTS-SiO(2)) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu(2+), Zn(2+), Ni(2+) ions, respectively on CTS-SiO(2), and trypsin could be adsorbed on the IMAC adsorbent through metal-protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu(2+)-CTS-SiO(2) with the value of 4980+/-125 IUg(-1) of the adsorbent, while the maximal adsorption capacity for trypsin on Zn(2+) and Ni(2+) loaded adsorbent was 3762+/-68 IUg(-1) and 2636+/-53 IUg(-1), respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5-10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO(2) beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 degrees C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO(2) could effectively digest BSA revealed by HPLC peptide mapping.

Adsorption↗
PubMed1996

Separation by heparin-affinity chromatography of catalytically active and inactive forms of trypsin which retain the (Na-K)ATPase stimulating property.

The possibility that different structural determinants on trypsin, other than catalytic sites, are involved in the cell membrane (Na-K)ATPase stimulating property was investigated by submitting bovine trypsin to two purification procedures: gel filtration on Sephadex G-50 and heparin-Sepharose chromatography. The latter procedure was also chosen in consideration of the known affinity for heparin displayed by serine proteinases. Trypsin peaks eluted from both columns were analysed by measuring esterolytic and proteolytic activities, the beef heart (Na-K)ATPase stimulating property and amino acid content. Fluorescence emission spectra and both non-denaturing and SDS-gel electrophoresis were also performed to test structural modifications on trypsin peaks. Four peaks eluted from Sephadex G-50 with variable estero-proteolytic and (Na-K)ATPase stimulating activities; the latter was also present in two peaks which displayed the lowest estero-proteolytic activities. All peaks proved to be trypsin in amino acid composition. Two peaks eluted from the heparin-Sepharose column with distinct biological activities: a first minor peak, eluted with the void volume, was catalytically inactive but it retained the (Na-K)ATPase stimulating activity. The second, major peak eluted mostly with 0.5 mol/l NaCl, displayed only esteroproteolytic activities, but no (Na-K)ATPase-stimulating activity. It overlapped control trypsin in both electrophoretic patterns, fluorescence emission spectrum and amino acid composition. The first peak showed differences with the parent compound, as revealed by the amino acid composition and tryptophan fluorescence emission spectrum. Marked differences were also observed in the electrophoretic pattern which only showed bands of low molecular mass mostly confined to the anode. NH2-terminus analysis confirmed that the first peak contained trypsin fragments originated from the parent compound after passage through the heparin column. It is hypothesized that trypsin binding to heparin causes structural alteration of the proteinase and primes the catalytic cleavage of fragments which lose heparin affinity and elute in the void volume. The results also confirm that the proteolytic mechanism is not involved in trypsin-mediated (Na-K)ATPase stimulation and indicate that heparin-Sepharose chromatography is a useful tool to separate catalytically active and inactive forms of trypsin.

Amino Acids↗