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PubMed1976

Studies on the structural and functional organization of system II of photosynthesis. The use of trypsin as a structurally selective inhibitor at the outer surface of the thylakoid membrane.

The effect of trypsin on the photosynthetic electron transport has been investigated in the presence of various electron acceptors (benzyl viologen, p-benzo-quinone, K3[Fe(CN)6]) by measurements of flash-induced oxygen evolution and of the absorption changes at 334 nm, indicating the primary electron acceptor of System II, X 320, and at 515 nm, indicating via electrochromism the electrical potential gradient across the thylakoid membrane. It was found that the effect of trypsin is strongly dependent on the nature of the electron acceptor: (1) Oxygen evolution is completely inhibited in the presence of p-benzo-quinone, but remains nearly unaffected by K3[Fe(CN)]6. (2) The initial amplitude deltaAO of the 334 nm absorption change is insensitive to trypsin in the presence of K3[Fe(CN)6], but the absorption change is abolished if benzyl viologen is used as acceptor. (3) The initial amplitude deltaAO of the 515 nm absorption change decreases by trypsin down to 50% with K3[Fe(CN)6] and is completely suppressed with benzyl viologen. (4) In trypsinated chloroplasts, the above-mentioned activities appear to be rather insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, in contrast to normal chloroplasts. On the basis of these results it is inferred that the primary electron acceptor of System II, X 320, is covered by a proteinaceous component susceptible to tryptic digestion. In addition, it is postulated that this component acts as well as an allosteric protein responsible for the regulation of the electronic interaction between X 320 and the plastoquinone pool, as for the inhibitory effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Various other possible effects caused by the proteinaceous shield and its modification by trypsin are discussed. The present results are in complete agreement with asymmetric membrane models postulating a zig-zag arrangement of the electron transport chain with the reducing side located towards the outer phase and the oxidizing side near the inner phase of the thylakoids.

2,6-Dichloroindophenol↗
PubMed1981

Trypsin cleavage of the alpha-subunit of beef heart F1-ATPase abolishes ATP synthesis and ATP-driven energy-transduction capabilities.

Previous work has shown that mild trypsin treatment eliminates energy-transduction capability and tight (non-exchangeable)nucleotide binding in beef heart mitochondrial F1-ATPase (Leimgruber, R.M. and Senior, A.E. (1976) J. Biol. Chem. 251, 7103-7109). The structural change brought about by trypsin was, however, too subtle to be identified by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, and was not defined. In this work we have applied two-dimensional electrophoresis (isoelectric focussing then sodium dodecyl sulfate polyacrylamide gradient electrophoresis) to the problem, and have determined that the alpha-subunit of F1 is altered by the mild trypsin treatment, whereas no change was detected in beta-, gamma-, delta- or epsilon-subunits. Binding of ADP to the trypsin-treated F1 was compared to binding to control enzyme over a range of 0-40 muM ADP in a 30 min incubation period. There was no difference between the two enzymes, KADPd in Mg2+ -containing buffer was about 2 muM in each. Since the tight (nonexchangeable)sites are abolished in trypsin-treated F1, this shows that tight exchangeable ADP-binding sites are different from the tight nonexchangeable ADP-binding sites. There was no effect of trypsin cleavage of the alpha-subunit on beta-subunit conformation as judged by aurovertin fluorescence studies. The cleavage of the alpha-subunit which occurred was judged to occur very close to the C- or N-terminus of the subunit and constitutes therefore a small and specific chemical modification which abolishes overall function in F1 but leaves partial functions intact.

Adenosine Diphosphate↗
PubMed1976

Effects of incorporated trypsin on anion exchange and membrane proteins in human red blood cell ghosts.

Varying concentrations of trypsin were sealed into human red cell ghosts and the effects on membrane proteins and sulfate equilibrium exchange were studied. After incubation for 45 min at 37 degrees C, pH 7.2, the following observations were made: above 10 ng/ml the ghosts undergo fragmentation without lysis. Dodecyl sulfate gel electrophoresis shows that the digestion of spectrin and of the protein in band 2.1 (nomenclature of Steck (1974) J. Cell. Biol. 62, 1-19) is nearly complete at 50 ng/ml, that of the protein in band 3 at 25 mug/ml. After digestion at 25 mug/ml, about 60% of the total protein of the membrane is released and the original bands of conventional dodecyl sulfate gel electropherograms of the remaining protein are nearly completely abolished. In their place three new bands appear representing peptides with molecular weights of 58 000, 48 000 and 34 000, respectively. Sometimes a fourth peptide with a molecular weight of approx. 13 000 is also observed. Using a radioactive labeling technique it is shown that the two peptides with the highest molecular weights are derived from the protein in band 3. Labeling with diazo[35S]sulfanilic acid indicates that in addition to the peptides in the described four Coomassie blue-stainable bands, other peptides with molecular weights up to 100 000 are still present in the exhaustively trypsinized ghosts. External trypsin has no effect on the sulfate equilibrium exchange in ghosts while internal trypsin causes inhibition. Inhibition becomes apparent at trypsin concentration exceeding those required to produce a complete digestion of spectrin. It remains incomplete, even at the highest intracellular concentrations which cause maximal effects on all membrane proteins, including the protein in band 3. Under these conditions strong further inhibition can be produced by agents which are known to inhibit anion transport in untreated red cells and ghosts. These agents include the penetrating 1-fluoro-2,4-dinitrobenzene and the nonpenetrating phlorizin, 4-acetamido-4'-isothiocyanato stilbene-2,2'-disulfonic acid, 4,4'-diacetamido stilbene-2,2'-disulfonic acid, and 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonic acid (APMB). Unlike the other nonpenetrating inhibitors APMB is not only capable of inhibiting at the outer but also at the inner membrane surface. Treatment with internal trypsin does not significantly reduce the inhibition by incorporated APMB. The described observations suggest that after exhaustive tryptic digestion of the major membrane proteins, the receptor sites for typical inhibitors of anion transport continue to exert their function.

Biological Transport↗
PubMed1981

A sepharose derivative coupled with a leupeptin-like peptide aldehyde, glycylglycyl-L-argininal, and its use as an affinity adsorbent for trypsin.

A Sepharose derivative containing a peptide aldehyde, glycylglycyl-L-argininal, the structure of which resembles that of leupeptin was prepared. It was a strong affinity adsorbent for trypsin (EC 3.4.21.4). Bovine trypsin showed higher affinity for this adsorbent at the optimum pH of catalysis (8.2) than at lower pH (5.0). This observation was in good agreement with the pH dependence of the interaction of leupeptin and trypsin (Kuramochi, H., Nakata, H. and Ishii, S. (1979) J. Biochem. 86, 1403-1410). Streptomyces griseus trypsin was also adsorbed while trypsinogen, alpha-chymotrypsin and TLCK-trypsin were not adsorbed. Though anhydrotrypsin, in which Ser-183 is converted to dehydroalanine, was not adsorbed, carbamoylmethylated (His-46) trypsin was adsorbed. Ser-183 proved to be essential for the binding. This adsorbent can also be used as a good tool to study the mechanism of action of leupeptin.

Adsorption↗
PubMed1986

Reduced trypsin binding capacity of alpha 2-macroglobulin in diabetes.

The plasma proteinase inhibitor, alpha 2-macroglobulin, is usually elevated in diabetes. The trypsin binding capacity and the concentration of alpha 2-macroglobulin in 90 diabetics sera were compared with 30 age- and sex-matched normal sera. The mean alpha 2-macroglobulin concentration determined by radial immunodiffusion was 313 mg/dl for the diabetics as compared to 240 mg/dl for the healthy subjects (significantly higher, p less than 0.01). The mean of the ratio, mol trypsin bound/mol alpha 2-macroglobulin (molar binding ratio) for the Type I diabetics (n = 54), 0.82, was significantly lower than the mean of the healthy subjects, 0.87, or the mean of the Type II diabetics, 0.87. No relationship between the molar binding ratios and the levels of glycosylated hemoglobin was found. The alpha 2-macroglobulin was isolated from the plasma of 11 Type I diabetics and 7 normals. The maximum molar trypsin binding capacities of the diabetic alpha 2-macroglobulin were significantly lower. The mean for the diabetic alpha 2-macroglobulin was 1.72 vs. 1.97 for the normal alpha 2-macroglobulin. These results indicate that the trypsin binding function of alpha 2-macroglobulin is moderately impaired in diabetes. No differences were found in the extent of proteolytic cleavage of the 'bait region' of diabetic alpha 2-macroglobulin, autolytic cleavage or the methylamine reaction at the thiolester site between diabetic and normal alpha 2-macroglobulin. Nonenzymatic glucosylation of normal alpha 2-macroglobulin did not lower the trypsin binding capacity. The nature of the modification of alpha 2-macroglobulin leading to reduced trypsin binding capacity or the physiological significance is not yet known.

Adult↗
PubMed1986

Acid stable trypsin inhibitor in bile.

Acid stable trypsin inhibitor having the same antigenicity as urinary trypsin inhibitor was first identified in the bile of patients with malignant tumors (biliary tract carcinoma or pancreas head carcinoma) and gallstones. Bile trypsin inhibitor from malignant tumor patients was partially purified by DEAE cellulose ion exchange column chromatography. Two molecular forms of the inhibitor were identified. The main form had a molecular weight of about 86,000 and the minor one a molecular weight of 31,000 as determined by gel filtration. Using isoelectric focussing, the larger molecular form gave a pI value of 2.0 and the smaller form, a pI value of 5.1. The isolated larger form migrated on the slightly cationic side of human serum albumin by analytical polyacrylamide gel electrophoresis. The larger form reacted and fused with anti-urinary trypsin inhibitor serum and strongly inhibited trypsin, partially inhibited chymotrypsin and plasmin, but did not inhibit urokinase. The clinical significance of acid stable trypsin inhibitor is discussed.

Bile↗
PubMed1987

Development and application of an enzyme-linked immunosorbent assay for acid stable trypsin inhibitor (ASTI) in human plasma.

An enzyme-linked immunosorbent assay (ELISA) for acid-stable trypsin inhibitor was developed using goat antibodies raised against urinary trypsin inhibitor purified from normal human urine. The assay was highly sensitive and precise, and gave reproducible results in the range of 1-1,000 ng/ml. The specificity and sensitivity of this assay permitted measurement of a low content of acid stable trypsin inhibitor in different tissue extracts and body fluids. Using the new method, the mean level in plasma samples taken from healthy individuals was found to be 54.9 +/- 17.4 (mean +/- SD) micrograms/ml and that of acid-treated plasma was 11.5 +/- 3.0 micrograms/ml (n = 27). The calculated specific activity of acid stable trypsin inhibitor in acid treated plasma was 300 U/mg, while that of highly purified inhibitor is 2,100 U/mg. These results suggest the presence of both acid unstable protein having the same antigenicity as urinary trypsin inhibitor and the inactive form of acid stable trypsin inhibitor.

Enzyme-Linked Immunosorbent Assay↗
PubMed1996

Peptic-tryptic digests of gliadin: contaminating trypsin but not pepsin interferes with gastrointestinal protein binding characteristics.

For many years, peptic-tryptic digests of gliadin, known as Frazer's fraction III, have been used in investigations of gliadin effects. Potential contamination by the proteases pepsin and trypsin, however, was not considered. To investigate the influence of contaminating proteases on binding of gliadin peptides to rat small intestinal brush border membranes we compared binding characteristics of different gliadin digests. Binding of biotinylated probes was studied in dot blots and Western blots with an enhanced chemiluminescence system. In gliadin peptide preparations only contaminating trypsin, but not pepsin, was detectable by specific antisera. Digestion with insoluble proteases attached to cross-linked beaded agarose yielded gliadin peptides free of contaminating pepsin and trypsin. These peptides bound 30% less to brush border membranes. Using these peptides, there was no trypsin-typical binding pattern to low molecular mass membrane proteins in contrast to peptide preparations which contained contaminating trypsin. In conclusion contaminating trypsin might alter gliadin peptide binding characteristics by direct binding to brush border membranes and by interfering with interactions between gliadin peptides and brush border membranes.

Animals↗
PubMed1990

Leishmania donovani: cell-surface heparin receptors of promastigotes are recruited from an internal pool after trypsinization.

With the use of [3H]heparin, we recently demonstrated that Leishmania donovani promastigotes express a cell-surface receptor that is specific for the glycosaminoglycan heparin (Mukhopadhyay et al. 1989, The Biochemical Journal, 264, 517-525.). Treatment of the parasite with trypsin abolishes 75-90% of this [3H]heparin-binding activity. When trypsinized promastigotes were resuspended in fresh culture medium in the absence and presence of cycloheximide (10 micrograms/ml), approximately 25-30% of the original heparin-binding capacity was restored within 1 hr, indicating that recruitment of receptors from an internal pool occurred without de novo protein synthesis. Scatchard analysis of the regenerated receptor revealed that the number of regenerated binding sites per cell was 2.3 x 10(5); these sites have a binding affinity of 6.7 x 10(-7) M. Like the native heparin receptors on the surface of freshly isolated cells, the receptors recruited after trypsinization are also highly specific for heparin, as a 25-fold excess of four other glycosaminoglycans displaced less than 10% of bound [3H]heparin from the trypsinized cells. The structural requirements of the ligand heparin, namely the number of monosaccharide units and degree of sulfation, were compared for both the native and regenerated receptor: for both receptors, oversulfated polysaccharide heparin fragments of at least six to eight sugar residues were most efficient at displacing [3H]heparin. The concentrations of oligosaccharide fragments required to displace 50% of [3H]heparin were 0.32 and 0.035 microM for the hexa- and octasaccharides, respectively. Colloidal gold-labeled heparin was bound to promastigotes and visualized by electron microscopy. This analysis revealed that the heparin bound almost exclusively to the flagella of control cells (not subjected to trypsin) and those which had regenerated receptor after trypsinization. The physiological significance of this heparin-binding activity on the surface of promastigotes is discussed.

Animals↗
PubMed1990

Pancreatic secretory trypsin inhibitor stimulates the growth of rat pancreatic carcinoma cells.

Pancreatic secretory trypsin inhibitor was examined for growth-promoting activity on five cell lines using standard cell culture techniques. One cell line, AR4-2J, derived from a rat pancreatic acinar cell carcinoma, responded with significantly increased incorporation of [3H]thymidine and colony formation. Pancreatic secretory trypsin inhibitor stimulated the incorporation of [3H]thymidine in liquid culture; the maximal increase was 61 +/- 10% above control (P less than 0.001) and was seen at a concentration of 10(-9) mol/L. Using a soft agarose clonogenic assay, pancreatic secretory trypsin inhibitor also consistently stimulated (3 assays) colony formation: the peak activity occurred at a concentration of 10(-10) mol/L which caused a 150 +/- 55% (mean +/- SE, P less than 0.05) increase above control. Aprotinin had no effect on the growth of AR4-2J cells and pancreatic secretory trypsin inhibitor did not bind to the epidermal growth factor receptor. AR4-2J cells were shown to produce pancreatic secretory trypsin inhibitor. The study raises the possibility that pancreatic secretory trypsin inhibitor provides autocrine stimulation of tumor cell growth.

Animals↗
PubMed1985

Factors affecting the trypsin induced release of aldosterone in rat adrenal zona glomerulosa tissue.

The yields of aldosterone obtained during incubation of whole adrenal capsule tissue from the rat (consisting of the connective tissue capsule itself, all of the glomerulosa tissue, and some fasciculata) cannot apparently be accounted for by the gland's capacity for de novo synthesis of this steroid. Recent studies with proteolytic enzymes and inhibitors suggest that in part aldosterone output may result from the activation of proteolytic events which release aldosterone from a sequestered intraglandular pool. These proteolytic events are mimicked by the addition of trypsin to whole tissue incubations in vitro. Experiments were carried out to determine what factors may govern the size of such intraglandular steroid pools. The most remarkable effect was that prior sodium depletion greatly enhanced the yield (2-3-fold) of aldosterone on subsequent incubation of adrenal capsules with trypsin, to an extent far greater than the increase in basal (non trypsin induced) aldosterone output in this tissue. Although betamethasone (20 micrograms/ml in drinking water) and the converting enzyme inhibitor captopril (7.2 mg/day) eliminated trypsin releasable steroid in control animals, they had no effect on the enhanced levels of trypsin releasable steroid seen with sodium depletion. The data suggest that trypsin releasable steroid pools are variable in accordance with the physiological requirements of the animal, particularly in sodium depletion.

Adrenal Glands↗
PubMed1996

Role of urinary trypsin inhibitor in the maintenance of pregnancy in mice.

OBJECTIVE: To investigate the mechanisms whereby urinary trypsin inhibitor prevents lipopolysaccharide-induced preterm delivery in mice. METHODS: On day 15 of pregnancy, C3H/HeNCrg female mice impregnated by Crg:B6D2F1 male mice were treated intraperitoneally with lipopolysaccharide (50 micrograms/kg, twice at a 3-hour interval) to induce preterm delivery. Urinary trypsin inhibitor (2.5 x 10(4), 7.5 x 10(4), or 25 x 10(4) units/kg, ten times at 1-hour intervals) or saline solution was administered intraperitoneally to the animals. RESULTS: The incidence of preterm delivery was significantly decreased on a dose-related basis by urinary trypsin inhibitor treatment. Urinary trypsin inhibitor prevented the morphologic and functional changes in fetal membranes and cervical ripening preceding the onset of preterm delivery. Urinary trypsin inhibitor also suppressed the increase in plasma and amniotic fluid concentrations of interleukin-1 alpha, interleukin-6, and tumor necrosis factor-alpha after the lipopolysaccharide dosing in this animal model for preterm delivery. CONCLUSION: Urinary trypsin inhibitor prevents the pathogenicity of preterm delivery through the suppression of cytokine production.

Animals↗
PubMed1984

Trypsin-induced aggregation of bovine pulmonary artery endothelial cells cultured on microcarriers.

We studied adherence between 'luminal' surfaces of pulmonary artery endothelial cells by standard aggregometry techniques, widely used for measuring aggregation of platelets and granulocytes. Using suspensions of bovine pulmonary artery endothelial cells cultured on microcarrier beads, in an aggregometer, we found that trypsin caused endothelial aggregation. The aggregation response occurred at trypsin concentrations as low as 0.001%. The degree of trypsin-induced aggregation indicated by the magnitude of the change in light transmission through the endothelial suspensions was related to the trypsin concentration, reaching a maximum level at trypsin concentrations of 0.01%. We conclude that trypsin, even in very low concentrations, causes adherence between 'luminal' surfaces of pulmonary endothelial cells probably because the enzyme destroys cell surface proteins which are necessary to prevent intercellular adherence. The method we describe may be useful for studying cell-cell interactions of endothelium.

Animals↗
PubMed1986

Trypsin-like activity and thromboxane release in adult respiratory distress syndrome.

Plasmatic immunoreactive trypsin (IRT), thromboxane and trypsin-like enzymatic activity were measured in 117 patients at risk of developing adult respiratory distress syndrome (ARDS) (53 multiple injury, 30 abdominal surgery, 17 acute pancreatitis, 12 burnt and 5 disseminated intravascular coagulation patients). 69 of these patients developed ARDS. Immunoreactive trypsin and thromboxane were measured by radio-immuno-assay and trypsin-like enzymatic activity by spectrophotometry, using a specific chromogenic substrate. Mean IRT value was 675 ng/ml in ARDS and 265 ng/ml in non ARDS patients (p less than 0.05). Mean IRT value was 685 ng/ml in septic and 170 ng/ml in non septic patients (p less than 0.01). An abnormal trypsin-like enzymatic activity was measured in 26 ARDS patients. In 60 patients (37 ARDS and 23 non ARDS), thromboxane appeared in plasma simultaneously or about 24 hours after the beginning of IRT release. The importance of thromboxane release parallels the intensity of IRT. Originating from pancreas, trypsin can appear in plasma either by absorption from gastrointestinal tract or after pancreatic ischemia.

Clinical Enzyme Tests↗
PubMed1995

Feeding the mosquito Aedes aegypti with TMOF and its analogs; effect on trypsin biosynthesis and egg development.

Female Aedes aegypti that were given a blood meal by enema deposited yolk in their oocytes and synthesized trypsinlike enzymes in their midgut. When females were given an enema of Aea-TMOF (Trypsin Modulating Oostatic Factor) (NH2-YDPAPPPPPP-COOH) and blood both egg development and trypsin biosynthesis were inhibited. Similar results were observed if TMOF was mixed with the blood meal and fed to female mosquitoes through a membrane. Renin inhibitor (NH2-PHPFHFFVYK-COOH) or poly proline given by enema with the blood meal did not affect egg development or trypsin biosynthesis. Feeding of TMOF analogs P1 (NH2-YDPAP-COOH) or P4 (NH2-YDPAPPPP-COOH) inhibited trypsin biosynthesis in the midgut. Injecting or giving an enema of an amidated peptide (NH2-WRPGPPPPPP-CONH2) of HIV-2 X-ORF protein also inhibited egg development and trypsin biosynthesis in the mosquito gut. When [3H]TMOF was purified by high performance liquid chromatography (HPLC) and fed with the blood meal through a membrane to female mosquitoes, [3H]TMOF outside the gut increased linearly for the first 24 h and 28% of the hormone was found outside the gut at 72 h. These results suggest that TMOF and its active analogs traverse the gut epithelial cells into the hemolymph, bind TMOF gut receptor(s) and modulate trypsin biosynthesis.

Aedes↗
PubMed1994

Refined 1.8 A resolution crystal structure of the porcine epsilon-trypsin.

Porcine epsilon-trypsin is a three-chain inactivated trypsin from the limited autolysis of porcine beta-trypsin. It is cleaved at positions Lys60-Ser61 and Lys145-Ser146. The crystal structure has been determined by using the molecular replacement method, and refined at 1.8 A resolution. The R-value of final model is 0.184. Comparison with the electron density map of porcine beta-trypsin (PTRY) in complex (BBIT), and with that of native bovine beta-trypsin (HTNA), revealed no obvious changes except at the autolysis positions, and no changes at the active center were observed. The autolysis at positions Lys60-Ser61 and Lys145-Ser146 does not affect the conformation of His-57 in the active center and therefore cannot explain for a loss in porcine epsilon-trypsin activity.

Animals↗
PubMed1985

Uptake and degradation of insulin and alpha 2-macroglobulin-trypsin complex in rat adipocytes. Evidence for different pathways.

The cell association and degradation of insulin and alpha 2-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 microM) reduced both the uptake of alpha 2-macroglobulin X trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2-3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of alpha 2-macroglobulin X trypsin markedly without affecting that of insulin. Leupeptin (100 microM) increased uptake and reduced degradation of alpha 2-macroglobulin X trypsin without affecting insulin. Dansylcadaverine (500 microM) almost abolished uptake and degradation of alpha 2-macroglobulin X trypsin but had little effect on insulin. Moreover, uptake and degradation of alpha 2-macroglobulin X trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin.

Adipose Tissue↗
PubMed1995

Inhibition of bovine beta-trypsin, human alpha-thrombin and porcine pancreatic beta-kallikrein-B by 4',6-diamidino-2-phenylindole, 6-amidinoindole and benzamidine: a comparative thermodynamic and X-ray structural study.

The inhibitory effect of 4',6-diamidino-2-phenylindole (DAPI) and 6-amidinoindole on the catalytic properties of bovine beta-trypsin (trypsin), human alpha-thrombin (thrombin) and porcine pancreatic beta-kallikrein-B (kallikrein) was investigated (between pH 3.0 and 7.0, I = 0.1 M; T = 30.0 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Next, the X-ray crystal structure of the trypsin:DAPI complex was solved at 1.9 A resolution (R = 0.161). Over the whole pH range explored, values of the association inhibition constant (Ki) for DAPI and 6-amidinoindole binding to trypsin, thrombin and kallikrein are higher than those found for benzamidine association, suggesting a binding mode of DAPI to the enzyme primary specificity pocket-based on the indole moiety of the inhibitor. On lowering the pH from 5.5 to 3.0, the decrease in affinity for DAPI, 6-amidinoindole and benzamidine binding to trypsin, thrombin and kallikrein reflects the acidic pK shift of the Asp189 invariant residue, present at the bottom of the primary specificity subsite of the serine proteinases considered, from 4.5, in the free enzyme, to 3.7, in the proteinase:inhibitor complexes. Inspection of the refined crystal structure of the trypsin:DAPI complex, however, does not allow a unique interpretation of the inhibitor binding mode. The present data were analysed in parallel with those reported for related serine (pro)enzyme/inhibitor systems.

Animals↗