Potent Trypsin Inhibitor Research Articles

Total synthesis of the macrocyclic cysteine knot microprotein MCoTI-II

The first total synthesis of MCoTI-II, a cysteine knot microprotein and potent trypsin inhibitor, is described; a synthetic strategy has been developed that combines efficient backbone construction via optimised solid phase peptide synthesis with one-pot 'thia-zip' native chemical ligation and refolding to yield the natural product.

Purification and characterization of an irreversible serine protease inhibitor from skin secretions of Bufo andrewsi

Amphibian skin secretions contain many bioactive compounds. In the present work, an irreversible serine protease inhibitor, termed baserpin, was purified for the first time from the skin secretions of toad Bufo andrewsi by successive ion-exchange and gel-filtration chromatography. Baserpin is a single chain glycoprotein, with an apparent molecular weight of about 60 kDa in SDS-PAGE. Baserpin is an irreversible inhibitor and effectively inhibits the catalytic activity of trypsin, chymotrypsin and elastase. SDS-stable baserpin–trypsin complex could be seen in SDS-PAGE indicates that it possibly belongs to the serpin superfamily. According to the association rates determined, baserpin is a potent inhibitor of bovine trypsin (4.6×10 6 M −1 s −1), bovine chymotrypsin (8.9×10 6 M −1 s −1) and porcine elastase (6.8×10 6 M −1 s −1), whereas it shows no inhibitory effect on thrombin. The N-terminal sequence of baserpin is HTQYPDILIAKPXDK, which shows no similarity with other known serine protease inhibitors.

Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B.maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1:1 molar ratio. The equilibrium dissociation constants (KD) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys53-Cys62), which comprises the scissile bond Arg58(P1)-His59(P1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

Single mutation at P1 of a chymotrypsin inhibitor changes it to a trypsin inhibitor: X-ray structural (2.15 Å) and biochemical basis

Change in specificity, caused by the mutations at P1 site, of the serine protease inhibitors of different families is reported in the literature, but Kunitz (STI) family inhibitors are almost unexplored in this regard. In this paper, we present the crystal structure of a P1 variant of winged bean chymotrypsin inhibitor (WCI) belonging to Kunitz (STI) family, supplemented by biochemical, phylogenetic and docking studies on the mutant. A single mutation (Leu → Arg) at P1 converted WCI to a strong inhibitor of trypsin with an association constant of 4.8 × 10 10 M −1 which is comparable to other potent trypsin inhibitors of the family. The crystal structure (2.15 Å) of this mutant (L65R) shows that its reactive site loop conformation deviates from that of WCI and adopts a structure similar to that of Erythrina caffra trypsin inhibitor (ETI) belonging to the same family. Mutation induced structural changes have also been propagated in a concerted manner to the neighboring conserved scaffolding residue Asn14, such that the side chain of this residue took an orientation similar to that of ETI and optimized the hydrogen bonds with the loop residues. While docking studies provide information about the accommodation of non-specific residues in the active site groove of trypsin, the basis of the directional alteration of the reactive site loop conformation has been understood through sequence analysis and related phylogenetic studies.

Proinflammatory Role of Trypsin and Protease-activated Receptor-2 in a Rat Model of Acute Pancreatitis

The pathophysiology of acute pancreatitis is strongly associated with autoactivation of trypsin. The biologic activity of trypsin on cells is attributed to the activation of protease-activated receptor-2 (PAR-2). We hypothesize that trypsin may activate acinar cells or inflammatory cells through PAR-2 signals in acute pancreatitis. We immunochemically analyzed the expression of PAR-2 in the rat acinar cell line, ARIP, and the rat pancreas, using anti-rat PAR-2 cleavage site (PCS) and anti-rat PAR-2 N-terminal fragment (PNF) antibodies. Plasma levels of PNF were determined. Furthermore, the effects of the anti-rat PCS antibody and nafamostat mesylate, a potent trypsin inhibitor, on PAR-2 activation during acute pancreatitis were also analyzed. ARIP cells expressed PAR-2, which was activated by exogenous trypsin activity. We also showed that PAR-2 is strongly expressed in pancreatic acinar and duct cells and that it is activated in rat cerulein-induced acute pancreatitis. The anti-rat PCS antibody and nafamostat mesylate reduced interleukin-6 and interferon gamma production and alleviated distant organ injury. These results suggest that trypsin and its specific receptor, PAR-2, play an important role in cytokine production and the resultant development of distant organ injury during rat acute pancreatitis.

Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II

MCoTI-I and MCoTI-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTI-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue #10 within the open-chain variant of MCoTI-II by the non-natural isosteric nucleo amino acid AlaG [beta-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTI-I and MCoTI-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity.

Sunflower Trypsin Inhibitor-1

SFTI-1 is a bicyclic 14 amino acid peptide that was originally isolated from the seeds of the sunflower Helianthus annuus. It is a potent inhibitor of trypsin, with a sub-nanomolar K(i) value and is homologous to the active site region of the well-known family of serine protease inhibitors known as the Bowman-Birk trypsin inhibitors. It has a cyclic backbone that is cross-braced by a single disulfide bridge and a network of hydrogen bonds that result in a well-defined structure. SFTI-1 is amenable to chemical synthesis, allowing for the creation of synthetic variants. Alterations to the structure such as linearising the backbone or removing the disulfide bridge do not reduce the potency of SFTI-1 significantly, and minimising the peptide to as few as nine residues results in only a small decrease in reactivity. The creation of linear variants of SFTI-1 also provides a tool for investigating putative linear precursor peptides. The mechanism of biosynthesis of SFTI-1 is not yet known but it seems likely that it is a gene-coded product that has arisen from a precursor protein that may be evolutionarily related to classic Bowman-Birk inhibitors.

Expression of LEKTI domains 6–9 ′ in the baculovirus expression system: recombinant LEKTI domains 6–9 ′ inhibit trypsin and subtilisin A

The precursor lympho-epithelial Kazal-type-related inhibitor (LEKTI), containing two Kazal-type and 13 nonKazal-type domains, is an efficient inhibitor of multiple serine proteinases, among them plasmin, subtilisin A, cathepsin G, elastase, and trypsin. To gain insight into the structure and function of some of these domains, a portion of the cDNA coding for LEKTI domains 6–9 ′ was cloned and expressed in Sf9 cells using the baculovirus expression vector system (BEVS). Through a single purification step using a Co 2+ column, 3–4 mg of purified recombinant LEKTI-domains 6–9 ′ (rLEKTI6–9 ′) with the predicted molecular mass of 34.6 kDa was obtained from the cell pellet of a 1-L culture. Unlike full-length LEKTI, rLEKTI6–9 ′ inhibited trypsin and subtilisin A but not plasmin, cathepsin G, or elastase. The inhibition of trypsin and subtilisin A by rLEKTI6–9 ′ occurred through a noncompetitive mechanism, with inhibitory constants ( K i) of 356 ± 12 and 193 ± 10 nM, respectively. On the basis of the K i values, rLEKTI6–9 ′ was determined to be a more potent trypsin inhibitor and a less potent subtilisin A inhibitor than the full-length LEKTI. In contrast to LEKTI domains 6–9 ′, recombinant LEKTI domain 6 does not inhibit subtilisin A but competitively inhibited trypsin with a K i of 200 ± 10 nM. Taking LEKTI6-9 ′ as an example, the BEVS should facilitate the structure–function analysis of naturally occurring processed LEKTI forms that have physiological relevance.

Action of Bauhinia bauhinioides synthetic peptides on serine proteinases

The kallikrein inhibitor found in Bauhinia bauhinioides seeds (BbKI) differs from classical Kunitz plant inhibitors in the lack of disulfide bridges in its structure [Biochim. Biophys. Acta 1477 (2000) 64–74]. In this study, we examined whether structural properties may be involved in inhibitory specificity and, if so, whether those properties might be useful tools in designing compounds that interfere with enzyme activity. Peptides structurally related to the BbKI (RPGLPVRFESPLRINIIKE-NH 2) reactive site were synthesized by solid-phase method and assayed for serine proteinase activity. The peptides RPGLPVRFESPLRINIIKE-NH 2, RPGLPVRFESPL-NH 2, and GLPVRFES-NH 2 were efficient tissue kallikrein inhibitors, with I 50 values of 0.54 μM, 0.87 μM, and 0.5 mM, respectively. The lasting inhibitory effect was observed in incubation periods of up to 120 min. None of the studied peptides interfere with the activity of thrombin, factor Xa or trypsin, although the native protein BbKI is a potent trypsin inhibitor.

Significance of trypsin inhibitor gene mutation in the predisposition to pancreatitis

Pancreatic secretory trypsin inhibitor (PSTI) is a potent natural inhibitor of trypsin. We proposed a hypothesis: if the function of PSTI is impaired by its genetic mutation, trypsin may easily promote autodigestion causing pancreatitis, and performed mutational analysis of PSTI gene in patients with pancreatitis. Two exonic mutations (N34S and R67C) were suggested to be associated with the predisposition to pancreatitis. N34S mutation was co-segregated with two intronic mutations, IVS1-37T>C and IVS3-69insTTTT. Although we analyzed the function of recombinant N34S protein, we could not demonstrate the loss of function of this protein. Intronic mutations rather than N34S itself (IVS1-37T>C+N34S+IVS3-69insTTTT complex) may be associated with the decreased function of PSTI. Alternatively, increased digestion of N34S in vivo may be applicable. As for R67C, the conformational alteration of the protein by forming intramolecular or intermolecular disulfide bonds with 67Cys was strongly suggested. These results, along with the brand new findings in PSTI knockout mice, suggest that genetic mutation of PSTI is one of the important mechanisms for predisposition to pancreatitis by lowering the trypsin inhibitory function.

Purification and identification of a binding protein for pancreatic secretory trypsin inhibitor: a novel role of the inhibitor as an anti-granzyme A.

Pancreatic secretory trypsin inhibitor (PSTI) is a potent trypsin inhibitor that is mainly found in pancreatic juice. PSTI has been shown to bind specifically to a protein, distinct from trypsin, on the surface of dispersed cells obtained from tissues such as small intestine. In the present study, we affinity-purified the binding protein from the 2% (w/v) Triton X-100-soluble fraction of dispersed rat small-intestinal cells using recombinant rat PSTI. Partial N-terminal sequencing of the purified protein gave a sequence that was identical with the sequence of mouse granzyme A (GzmA), a tryptase produced in cytotoxic lymphocytes. We confirmed the formation of an affinity-cross-linked complex between (125)I-labelled PSTI and recombinant rat GzmA (rGzmA). In situ hybridization and immunostaining revealed the existence of GzmA-expressing intraepithelial lymphocytes in the rat small intestine. We concluded that the PSTI-binding protein isolated from the dispersed cells is GzmA that is produced in the lymphocytes of the tissue. The rGzmA hydrolysed the N -alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT), and the BLT hydrolysis was inhibited by PSTI. Sulphated glycosaminoglycans, such as fucoidan or heparin, showed almost no effect on the inhibition of rGzmA by PSTI, whereas they decreased the inhibition by antithrombin III. In the present paper, we propose a novel role of PSTI as a GzmA inhibitor.

Selection by phage display of a variant mustard trypsin inhibitor toxic against aphids.

The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 x 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 microg ml-1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.

A Family of 16-kDa Pancreatic Secretory Stress Proteins Form Highly Organized Fibrillar Structures upon Tryptic Activation

A group of 16-kDa proteins, synthesized and secreted by rat pancreatic acinar cells and composed of pancreatic stone protein (PSP/reg) and isoforms of pancreatitis-associated protein (PAP), show structural homologies, including conserved amino acid sequences, cysteine residues, and highly sensitive N-terminal trypsin cleavage sites, as well as conserved functional responses in conditions of pancreatic stress. Trypsin activation of recombinant stress proteins or counterparts contained in rat pancreatic juice (PSP/reg, PAP I and PAP III) resulted in conversion of 16-kDa soluble proteins into 14-kDa soluble isoforms (pancreatic thread protein and pancreatitis-associated thread protein, respectively) that rapidly polymerize into insoluble sedimenting structures. Activated thread proteins show long lived resistance to a wide spectrum of proteases contained in pancreatic juice, including serine proteases and metalloproteinases. In contrast, PAP II, following activation with trypsin or pancreatic juice, does not form insoluble structures and is rapidly digested by pancreatic proteases. Scanning and transmission electron microscopy indicate that activated thread proteins polymerize into highly organized fibrillar structures with helical configurations. Through bundling, branching, and extension processes, these fibrillar structures form dense matrices that span large topological surfaces. These findings suggest that PSP/reg and PAP I and III isoforms consist of a family of highly regulated soluble secretory stress proteins, which, upon trypsin activation, convert into a family of insoluble helical thread proteins. Dense extracellular matrices, composed of helical thread proteins organized into higher ordered matrix structures, may serve physiological functions within luminal compartments in the exocrine pancreas.

The Kinetic and Structural Characterization of the Reaction of Nafamostat with Bovine Pancreatic Trypsin

Nafamostat mesilate (FUT-175), a synthetic serine protease inhibitor, is active against a number of the serine proteases involved in coagulation. This has been proposed as the basis of its anticoagulant activity. We investigated the reaction of Nafamostat with bovine pancreatic trypsin as a model system. It was shown to act as a time-dependent competitive inhibitor, and the inhibition constants for the binding of Nafamostat to trypsin (i.e., K i) and the overall inhibition constants (i.e., K i *) were calculated to be 11.5 μM and 0.4±0.14 nM, respectively. The second-order rate constant for the reaction was 4.5±0.19×10 5 M −1s −1, and the product released following the acylation step, 6-amidino-2-naphthol, showed mixed-type inhibition. The competitive ( K ic) and uncompetitive ( K iu) inhibition constants were 14.7 μM and 19.5 μM, respectively. Formation of the acyl-enzyme intermediate was dissected into at least two steps, with rates of 0.9 s −1 and 195 s −1. The deacylation step was relatively much slower (3.2±0.19×10 −5 s −1), enabling the mass spectroscopic analysis of the acyl-enzyme intermediate, which confirmed the covalent attachment of 4-guanidinobenzoic acid to trypsin. The product of the deacylation step, 4-guanidinobenzoic acid, showed no inhibition up to a concentration of 200 μM. These data strongly suggest that while Nafamostat is a potent inhibitor of trypsin, it is actually an extremely poor substrate, and that apparent inhibition is due to the competitive formation of a very stable acyl-enzyme intermediate, analogous to some other active site titrants.

The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides.

The Ecballium elaterium trypsin inhibitor II (EETI-II), a member of the squash family of protease inhibitors, is composed of 28 amino acid residues and is a potent inhibitor of trypsin. Its compact structure is defined by a triple-stranded antiparallel beta-sheet, which is held together by three intramolecular disulfide bonds forming a cystine knot. In order to explore the potential of the EETI-II peptide to serve as a structural scaffold for the presentation of randomized oligopeptides, we constructed two EETI-II derivatives, where the six-residue inhibitor loop was replaced by a 13-residue epitope of Sendai virus L-protein and by a 17-residue epitope from human bone Gla-protein. EETI-II and derived variants were produced via fusion to maltose binding protein MalE. By secretion of the fusion into the periplasmic space, fully oxidized and correctly folded EETI-II was obtained in high yield. EETI-II and derived variants could be presented on the Escherichia coli outer membrane by fusion to truncated Lpp'-OmpA', which comprises the first nine residues of mature lipoprotein plus the membrane spanning beta-strand from residues 46-66 of OmpA protein. Gene expression was under control of the strong and tightly regulated tetA promoter/operator. Cell viability was found to be drastically reduced by high level expression of Lpp'-OmpA'-EETI-II fusion protein. To restore cell viability, net accumulation of fusion protein in the outer membrane was reduced to a tolerable level by introduction of an amber codon at position 9 of the lpp' sequence and utilizing an amber suppressor strain as expression host. Cells expressing EETI-II variants containing an epitope were shown to be surface labeled with the respective monoclonal antibody by indirect immunofluorescence corroborating the cell surface exposure of the epitope sequences embedded in the EETI-II cystine knot scaffold. Cells displaying a particular epitope sequence could be enriched 10(7)-fold by combining magnetic cell sorting with fluorescence-activated cell sorting. These results demonstrate that E.coli cell surface display of conformationally constrained peptides tethered to the EETI-II cystine knot scaffold has the potential to become an effective technique for the rapid isolation of small peptide molecules from combinatorial libraries that bind with high affinity to acceptor molecules.

Proteinase inhibitors from desert locust, Schistocerca gregaria: engineering of both P 1 and P 1′ residues converts a potent chymotrypsin inhibitor to a potent trypsin inhibitor

Two peptides, SGCI and SGTI, that inhibited chymotrypsin and trypsin, respectively, were isolated from the haemolymph of Schistocerca gregaria. Their primary structures were found to be identical with SGP-2 and SGP-1, two of a series of peptides isolated from ovaries of the same species (A. Hamdaoui et al., FEBS Lett. 422 (1998) 74–78). All these peptides are composed of 35–36 amino acid residues and contain three homologous disulfide bridges. The residues imparting specificity to SGCI and SGTI were identified as Leu-30 and Arg-29, respectively. The peptides were synthesised by solid-phase peptide synthesis, and the synthetic ones displayed the same inhibition as the natural forms: SGCI is a strong inhibitor of chymotrypsin ( K i=6.2×10 −12 M), and SGTI is a rather weak inhibitor of trypsin ( K i=2.1×10 −7 M). The replacement of P 1 then P 1′ residues of SGCI with trypsin-specific residues increased affinity towards trypsin 3600- and 1100-fold, respectively, thus SGCI was converted to a strong trypsin inhibitor ( K i=5.0×10 −12 M) that retained some inhibitory affinity towards chymotrypsin ( K i=3.5×10 −8 M). The documented role of both P 1 and P 1′ highlights the importance of S 1′P 1′ interactions in enzyme–inhibitor complexes.

High-resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds

Proteinaceous serine proteinase inhibitors are widespread throughout the plant kingdom where they play an important role in protection against pests and pathogens. Here, we describe the isolation and characterisation of a novel 14 amino acid residue cyclic peptide from sunflower seeds, which is a potent inhibitor of trypsin ( K i = 100 pM). The crystal structure of this peptide in complex with bovine β-trypsin shows both sequence and conformational similarity with the trypsin-reactive loop of the Bowman-Birk family of serine proteinase inhibitors. This inhibitor, however, is unique in being monofunctional, cyclic and far shorter (14 amino acid residues) than inhibitors belonging to this family (typically 60–70 amino acid residues). The high potency of this peptide is likely to arise from the considerable structural rigidity achieved through its cyclic nature which is further stabilised by a single internal disulphide bond. This study helps delineate the minimal unit required for effective peptide inhibitors of serine proteinases, and will assist in the further design of inhibitors to this widespread class of enzymes.

Quantification and characterization of human endothelial cell-derived tissue factor pathway inhibitor-2.

Tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5, is a serine protease inhibitor consisting of three tandemly-arranged Kunitz-type protease inhibitor domains. While TFPI-2 is a potent inhibitor of trypsin, plasmin, kallikrein, and factor XIa in the test tube, the function of this inhibitor in vivo remains unclear. In the present study, we investigated the synthesis and secretion of TFPI-2 by cultured endothelial cells derived from human umbilical vein, aorta, saphenous vein, and dermal microvessels to gain insight into its biological function. While all endothelial cells examined synthesized and secreted TFPI-2, dermal microvascular endothelial cells synthesized threefold to sevenfold higher levels of TFPI-2. Approximately 60% to 90% of the TFPI-2 secreted by endothelial cells was directed to the subendothelial extracellular matrix (ECM). When cultured human umbilical vein endothelial cells were stimulated with inflammatory mediators such as phorbol 12-myristate,13-acetate; endotoxin; and tumor necrosis factor-alpha, TFPI-2 synthesis by these cells increased twofold to 14-fold. Recombinant TFPI-2 bound to dermal microvascular endothelial cell monolayers and its ECM in a specific, dose-dependent, and saturable manner with Kd values of 21 and 24 nmol/L, respectively. TFPI-2 interacted with 4.5 X 10(10) sites/cm2 (3 X 10[5] sites/cell) and 2.3 X 10(11) sites/cm2 on endothelial cells and ECM, respectively. In the presence of rabbit anti-TFPI-2 IgG, but not preimmune IgG, endothelial cells dissociated from the culture flask in a time- and IgG concentration-dependent manner. Our findings provide evidence that endothelial cell-derived TFPI-2 is primarily secreted into the abluminal space and presumably plays an important role in maintaining the integrity of the ECM essential for cell attachment.

Effect of a new CCK-A receptor antagonist, dexloxiglumide, on the exocrine pancreas in the rat

The effect of dexloxiglumide, a new potent cholecystokinin (CCK) antagonist, on pancreatic enzyme secretion and growth was studied in the rat. Pancreatic exocrine secretion was studied both in vitro (isolated and perfused pancreatic segments) and in vivo (anaesthetized animals with cannulation of the common bile duct) whereas the trophic effect was investigated after short-term (7 days) administration of the CCK-agonist, caerulein, or camostate (a potent trypsin inhibitor), with or without dexloxiglumide. CCK-8 stimulated amylase release from in vitro pancreatic segments in a concentration-dependent manner. Dexloxiglumide displaced the concentration response curves to CCK-8 to the right without affecting the maximum response, suggesting a competitive antagonism. The Schild plot analysis of data gave a straight line with a slope (0.90±0.36) not significantly different from unity. The calculated pA 2 for dexloxiglumide was 6.41 ± 0.38. In vivo experiments confirmed results from in vitro studies since intravenous dexloxiglumide reduced pancreatic exocrine secretion induced by submaximal CCK-8 stimulation (0.5 nmol/kg/h) in a dose-dependent manner, the ID 50 being 0.64 mg/kg. Both exogenous and endogenous (released by camostate) CCK increased the weight of the pancreas, the total pancreatic protein and DNA, trypsin and amylase content. Dexloxiglumide (25 mg/kg), administered together with caerulein (1 μg/kg), reduced the peptide-induced increase in pancreatic weight, protein and enzyme content. Similarly, when dexloxiglumide was given together with camostate (200 mg/kg), all the observed changes were reduced by concomitant administration of the antagonist. These results demonstrate the ability of dexloxiglumide to antagonize the effects of CCK on pancreatic secretion and growth, suggesting that this compound is a potent and selective antagonist of CCK-A-receptors in the pancreas.

Enhanced protein flexibility caused by a destabilizing amino acid replacement in BPTI

A genetically engineered variant of bovine pancreatic trypsin inhibitor (Y35G BPTI) has been shown previously by X-ray crystallography to have a three-dimensional structure dramatically different from that of the wild-type protein, particularly in the protease-binding region of the molecule. Yet, the Y35G variant is a potent trypsin inhibitor. Described here are 15N NMR relaxation studies to compare the backbone dynamics of Y35G BPTI to those of the wild-type protein. The Tyr35 → Gly substitution increased the transverse relaxation rates of more than one third of all backbone amide groups, but had little effect on the longitudinal relaxation rates, indicating that the substitution facilitates relatively slow backbone motions, estimated to be on the microsecond time-scale. The results indicate that the residues making up the trypsin-binding site undergo large and relatively slow conformational changes in solution, estimated to be on the 5 to 20 μs time-scale. It is thus likely that the crystal structure represents only one of multiple interconverting conformations in solution, only a fraction of which may be competent for binding trypsin. The large thermodynamic destabilization associated with this substitution may arise, in part, from a loss in cooperativity among the multiple stabilizing interactions that are normally favored by the highly ordered structure of the wild-type protein. These results suggest that fully understanding the effects of amino acid replacements on the functional and thermodynamic properties of proteins may often require analysis of the dynamic, as well as the structural, properties of altered proteins.