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At least 19 recordsLinked to original sources
PubMedDate not supplied

Methods for dual, site-specific derivatization of bovine pancreatic trypsin inhibitor: trypsin protection of lysine-15 and attachment of fatty acids or hydrophobic peptides at the N-terminus.

To produce a series of model membrane proteins, bovine pancreatic trypsin inhibitor (BPTI) has been modified by specifically attaching reporter groups to Lys-15 and fatty acids or hydrophobic peptides at the N-terminus. Lys-15 of BPTI was protected by trypsin bound to BPTI, then O-methylisourea (OMIU) was used to guanidinate all unprotected lysines. The N-terminal amine was then reacted with several saturated fatty acid anhydrides from 8 to 18 carbons in length, or with an SMCC cross-linker. Cysteine-containing hydrophobic peptides, cleaved from resin in the presence of sodium dodecyl sulfate (SDS), were then attached to the protein via the N-terminal cross-linker. The methods described yield a unique, chemically modified protein which can carry site-specific modifications at two distinct residues. The resulting proteins are ideal for diffusional or partitioning studies on model and biological membranes.

Acylation↗
PubMedDate not supplied

[Determination and comparative analysis of the conformation of bovine pancreatic trypsin inhibitor and trypsin inhibitors E and K from the data of two-dimensional 1H-NMR spectroscopy].

On the basis of joint consideration of distance dependences between amide proton NH and protons C alpha H, NH, C beta H of the preceding in amino acid sequence residue from the torsion angles phi psi, chi 1, the correlation diagram of these proton-proton distances with the regions of sterically allowed conformational space (phi, psi) is presented and the method for the determination of the L-amino acid residues backbone conformations is proposed. The diagram was used for the determination of backbone conformations of bovine pancreatic trypsin inhibitor and trypsin inhibitors E and K from Dendroaspis polylepis using the data from two-dimensional 1H-NMR spectroscopy. The analysis of backbone conformations was carried out. The individual elements of these protein molecules secondary structure were characterized and their high conformational homology was shown. The inference about qualitative coincidence of three protein molecules conformation in solution, preservation of secondary structure basic elements and their similarity with bovine pancreatic trypsin inhibitor crystalline structure was made.

Amino Acid Sequence↗
PubMed2005

Discovery, structural determination, and putative processing of the precursor protein that produces the cyclic trypsin inhibitor sunflower trypsin inhibitor 1.

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.

Amino Acid Sequence↗
PubMed1977

Isolation and immunochemical determination of sow colostrum trypsin inhibitor.

Trypsin inhibitor from sow colostrum was isolated by ion exchange chromatography on DEAE-Sephadex A-50 followed by gel filtration chromatography on Sephadex G-100 and affinity chromatography. Antiserum against sow colostrum trypsin inhibitor was produced by immunization with the purified inhibitor, and made specific by absorption with normal porcine serum. The specific antiserum was used for immunoquantitation by single radial immunodiffusion (SRI). In sow colostrum whey, good agreement was found between the results obtained by SRI and the total trypsin-inhibiting activity as determined by radial diffusion in a casein-containing agarose gel (r = 0.97, n = 10). In sow's milk there was only a very low inhibiting activity, and no colostral inhibitor was demonstrable by SRI. Also in baby-pig urine agreement was found between the two methods (r = 0.97, n = 14). In baby-pig serum such an agreement was not seen, undoubtedly becuase of the presence of genuine serum trypsin inhibitors. By the SRI technique it is possible specifically to determine the colostral inhibitor even in the presence of other trypsin inhibitors.

Animals↗
PubMed1989

Analysis of inter-alpha-trypsin inhibitor and a novel trypsin inhibitor, pre-alpha-trypsin inhibitor, from human plasma. Polypeptide chain stoichiometry and assembly by glycan.

The polypeptide chain composition of protein material referred to in the literature as "inter-alpha-trypsin inhibitor" was investigated. The material was found to consist of distinct proteins of 125,000 and 225,000 Da, each of which contained more than one polypeptide chain. The links that assemble each protein were found to be stable to various strong denaturants, but susceptible to treatment with trifluoromethanesulfonic acid or hyaluronidase, indicating a glycan nature. The 225,000-Da protein migrated with inter-alpha mobility on agarose gel electrophoresis and is designated inter-alpha-trypsin inhibitor, whereas the 125,000-Da protein migrated with pre-alpha mobility, and we designate it pre-alpha-trypsin inhibitor. Analysis of the proteins, the separated chains, and proteolytic derivatives thereof revealed that each protein contained a single, identical, trypsin-inhibitory chain of 30,000 Da. Inter-alpha-trypsin inhibitor contains noninhibitory heavy chains of 65,000 and 70,000 Da, whereas pre-alpha-trypsin inhibitor contains a heavy chain of 90,000 Da. Our data allow identification of several recently reported cDNA clones and clarify the confusion surrounding the composition of plasma proteins referred to as inter-alpha-trypsin inhibitor.

Alpha-Globulins↗
PubMed1975

Hydrogen exchange kinetics changes upon formation of the soybean trypsin inhibitor-trypsin complex.

The hydrogen exchange kinetics of the complex of trypsin-soybean trypsin inhibitor (Kunitz) have been compared to the calculated sum of the exchange kinetics for the inhibitor and trypsin measured separately. The exchange rates observed for the complex are substantially less than the sum of the exchange rates in the two individual proteins. These results cannot be accounted for by changes in intermolecular or intramolecular hydrogen bonding. The decrease in exchange rates in the complex are ascribed to changes in solvent accessibility in the component proteins.

Benzamidines↗
PubMed2006

The role of trypsin, trypsin inhibitor, and trypsin receptor in the onset and aggravation of pancreatitis.

Trypsin activity is properly suppressed in the pancreatic acinar cells under normal conditions. A small amount of trypsinogen is converted to active trypsin and inactivated by pancreatic secretory trypsin inhibitor (PSTI), thereby preventing damage to pancreatic acinar cells as a first line of defense. However, if trypsin activation (due to excessive stimulation of pancreatic acinar cells) exceeds the capacity of PSTI, a subsequent cascade of events leads to the activation of various proteases that damage cells. This can be interpreted as the main causative event of pancreatitis onset. Trypsin produced in and secreted from the pancreatic acinar cells activates protease activated receptor-2 (PAR-2), which is present at high densities on the luminal surfaces of pancreatic acinar cells and duct cells. Results of PAR-2 activation are the production of cytokines and the regulation of exocrine function via a negative feedback loop. Thus, the actions of trypsin, trypsin inhibitor (PSTI), and trypsin receptor (PAR-2) in the pancreas are strongly interconnected.

Animals↗
PubMed2003

Enzymatic cyclization of a potent bowman-birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide.

The most potent known naturally occurring Bowman-Birk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine beta-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI-1[6,5] is approximately 9:1 regardless of whether trypsin is incubated with SFTI-1[6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[6,5] revealed high heterogeneity, and multiple species of SFTI-1[6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a beta-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an iso-aspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.

Amino Acids↗
PubMed1987

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor.

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.

Animals↗
PubMed2005

Impact of oilseed rape expressing the insecticidal serine protease inhibitor, mustard trypsin inhibitor-2 on the beneficial predator Pterostichus madidus.

Abstract Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators feeding on crop pests, through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the serine protease inhibitor, mustard trypsin inhibitor -2 (MTI-2), on the predatory ground beetle Pterostichus madidus were investigated, using diamondback moth, Plutella xylostella as the intermediary pest species. As expected, oilseed rape expressing MTI-2 had a deleterious effect on the development and survival of the pest. However, incomplete pest mortality resulted in survivors being available to predators at the next trophic level, and inhibition studies confirmed the presence of biologically active transgene product in pest larvae. Characterization of proteolytic digestive enzymes of P. madidus demonstrated that adults utilize serine proteases with trypsin-like and chymotrypsin-like specificities; the former activity was completely inhibited by MTI-2 in vitro. When P. madidus consumed prey reared on MTI-2 expressing plants over the reproductive period in their life cycle, no significant effects upon survival were observed as a result of exposure to the inhibitor. However, there was a short-term significant inhibition of weight gain in female beetles fed unlimited prey containing MTI-2, with a concomitant reduction of prey consumption. Biochemical analyses showed that the inhibitory effects of MTI-2 delivered via prey on gut proteolysis in the carabid decreased with time of exposure, possibly resulting from up-regulation of inhibitor-insensitive proteases. Of ecological significance, consumption of MTI-2 dosed prey had no detrimental effects on reproductive fitness of adult P. madidus.

Animals↗
PubMed1984

Photoreactive derivative of Kunitz's soybean trypsin inhibitor. Preparation by selective modification of a tryptophan residue and formation of a covalent complex of the modified inhibitor with trypsin.

The photoreactive arylsulfenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (SBTI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-SBTI indicated that only one of the two tryptophans (93 or 117) present in SBTI was modified. CNBr cleavage of 2,4-NAPS-SBTI resulted in two fragments 1-114 and 115-181. Amino acid analysis of the two separated fragments showed that only tryptophan 93 underwent modification. 2,4-NAPS-SBTI fully retained its inhibitory activity against trypsin. The photoaffinity labeling of trypsin with 2,4-NAPS-Cl was performed on tritiated trypsin prepared by reacting bovine trypsin with [3H]-succinimidyl propionate. The covalent attachment of 2,4-NAPS-SBTI to the tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride.

Affinity Labels↗
PubMed1977

Effect of dietary raw soybean and soybean trypsin inhibitor on trypsin and chymotrypsin activities in the pancreas and in small intestinal juice of growing swine.

Sixty-eight growing gilts with a 12 kg average initial weight were used in seven trials to study the effect of dietary raw soybean (Harosov) and SBTI (Kunitz soybean trypsin inhibitor) on pancreatic and small intestinal trypsin and chymotrypsin activities. A solvent-extracted, heated soybean meal (SBM) was used, cause reduced growth. Both a single-meal and continuous feeding of the raw soybean diet caused a decrease in the pancreatic trypsin and chymotrypsin activities. In contrast, to the rat and the chick, the pancreas of the pigs did not enlarge subsequent to consumption of the raw soybean or SBTI diets. Raw soybean feeding also resulted in an inhibition of the intestinal trypsin and chymotrypsin activities. This inhibiting effect was greater than that of the SBTI, especially the chymotrypsin-inhibiting effect. This suggested that soybean constituents other than the SBTI, such as the Bowman-Birk inhibitor, caused inhibition. In the pig the inhibition of the intestinal proteolysis may be a major cause of reduced growth when raw soybean is fed.

Animals↗
PubMed1993

Bovine pancreatic trypsin inhibitor-trypsin complex as a detection system for recombinant proteins.

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10(-14) and 1.1 x 10(-13) M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

Animals↗
PubMed1998

Human neutrophil elastase degrades inter-alpha-trypsin inhibitor to liberate urinary trypsin inhibitor related proteins.

Urinary trypsin inhibitor (UTI) is a physiological protease inhibitor and inter-alpha-trypsin inhibitor (ITI) is regarded as a precursor of UTI. The purpose of this study is to determine the mechanism of the UTI release from ITI. To examine this, ITI was digested by human neutrophil elastase at various concentrations, and UTI-related proteins which were of the same size as UTI were obtained. The amino acid sequence of the 15 amino acid residues at the N-terminal of UTI-related proteins, corresponded to that of UTI. The amino acid sequences of the small amount of peptides detected corresponded to those of peptides from the heavy chain1 (H1) and the heavy chain2 (H2) of ITI, suggesting that most UTI-related proteins do not combine with peptides from the H1 and H2 of ITI. It was also revealed that UTI-related proteins have several physiological activities similar to those of UTI, i.e., human trypsin inhibitory activity, human neutrophil elastase inhibitory activity, inhibition of tumor necrosis factor-alpha (TNF-alpha) production from rat macrophages and of superoxide production from rabbit leukocytes. These results demonstrated that ITI is a precursor of UTI which is digested by human neutrophil elastase to release UTI, and that its elastase inhibitory activity is derived from UTI.

Alpha-Globulins↗