Turkey serum trypsin inhibitors: description and characterization

1. Turkey serum trypsin inhibitors were studied on whole and chromatographically fractionated normal turkey serum using both quantitative (trypsin inhibitory capacity measurement) and qualitative (antitryptic activity detection methods) determinations, coupled to electrophoretic and isoelectrophoretic studies. 2. Five proteins with trypsin inhibitory activity were described, the most important ones being alpha 2 and beta-globulins with a multibanded pattern revealed by isoelectric focusing. 3. Trypsin inhibitory capacity assays, performed on individual sera, as well as isoelectric focusing studies, failed to find any quantitative and/or qualitative deficiency of these antiproteases. 4. Evidence is given that round heart disease in turkeys is not related to serum trypsin inhibitor deficiency.     

Assay for enzyme inhibition: detection of natural inhibitors of trypsin and chymotrypsin

A technique for quickly detecting nanogram quantities of low- and high-molecular-weight inhibitors of some serine proteases is described. The inhibitor solutions are spotted onto agar films which contain either L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK)-trypsin or tosyl lysine chloromethyl ketone (TLCK)-chymotrypsin. Enzyme inhibition is visualized as colorless zones on a pink background after the films were stained with the chromogenic substrate N-acetyl-DL-phenylalanine-beta-naphthyl ester. The method is used for rapidly testing both high-performance liquid chromatography fractions and thin-layer chromatograms to identify the inhibitors of trypsin and chymotrypsin in complex microbial extracts. The assay is quantitative so that it is possible to compare the specificity of the inhibitory fractions for trypsin and chymotrypsin. Results with standard inhibitors demonstrate the high sensitivity of the method, e.g., inhibition is detected with 1 ng of soybean trypsin inhibitor and 0.3 ng of antipain or chymostatin.     

Radioimmunoassay of human plasma trypsin.

A radioimmunoassay has been developed for the determination of human trypsin (3.4.21.4) in plasma. It allows the measurement of trypsin concentration in spite of the presence of plasma or pancreatic inhibitors. The human trypsin used as a standard and for labelling was isolated from pancreatic tissue and purified by affinity chromatography. The antiserum was obtained from guinea-pigs immunized with partially purified human trypsin. In the radioimmunoassay, the values of trypsin in serial dilutions of plasma were parallel to those of the standard curves. The assay was shown to be reproducible, sensitive and specific. However, the two antisera used did not distinguish between the enzyme and its proenzyme. In normal subjects, plasma values were found to be around 400 ng/ml. They were 10-40 times higher in patients with acute pancreatitis. The method appears to be much more specific for the diagnosis of acute pancreatitis than the current determinations of amylase and lipase activity.     

Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases.     

Trypsin inhibitor paradoxically stabilizes trypsin activity in sodium dodecyl sulfate, facilitating proteolytic fingerprinting

Normally trypsin has negligible activity after being dissolved in sodium dodecyl sulfate (SDS), and so it has had little utility for proteolytic fingerprinting during gel electrophoresis. Here it is demonstrated that trypsin retained activity in SDS if it was first complexed to either of two soybean-derived protease inhibitors: trypsin inhibitor (Kunitz) or trypsin-chymotrypsin inhibitor (Bowman-Birk). The inhibitors alone did not cause proteolysis. Heating or acidification in SDS inactivated the inhibitor-dependent tryptic activity, as did prior treatment with tosyl lysine chloromethyl ketone, a covalent affinity reagent for trypsin. Quenching of samples with acid at intervals prior to gel electrophoresis revealed that proteolysis did not occur in sample buffer (pH 6.8), but only at higher pH and during gel electrophoresis. Exposure of trypsin to SDS prior to addition of trypsin inhibitor resulted in an irreversible loss of activity with a half-life of about 10 s. It is proposed that the trypsin inhibitors stabilize trypsin by retarding its denaturation in SDS. The substrate for these experiments was the alpha subunit of the Na,K-ATPase. The same pattern of Na,K-ATPase fragments was obtained with bovine and porcine trypsin and with rat and porcine Na,K-ATPases. Different fragments resulted when chymotrypsin or elastase were substituted for trypsin; these proteases were active in the absence of an inhibitor, and were not markedly stabilized by interaction with soybean trypsin-chymotrypsin inhibitor (Bowman-Birk).     

Role of the NADP/thioredoxin system in the reduction of alpha-amylase and trypsin inhibitor proteins

Thioredoxin, reduced either enzymatically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol, reduced alpha-amylase and trypsin inhibitor proteins from several sources. Included were cystine-rich seed representatives from wheat (alpha-amylase inhibitors), soybean (Bowman-Birk trypsin inhibitor), and corn (kernel trypsin inhibitor). This system also reduced other trypsin inhibitors: the soybean Kunitz inhibitor, bovine lung aprotinin, and egg white ovoinhibitor and ovomucoid proteins. The ability of these proteins to undergo reduction by thioredoxin was determined by 1) a coupled enzyme activation assay with chloroplast NADP-malate dehydrogenase or fructose-1,6-bisphosphatase, 2) a dye reduction assay with 5',5'-dithiobis(2-nitrobenzoic acid), and 3) a direct reduction method based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduction experiments with the seed proteins were carried out with thioredoxin from wheat germ (h-type) or Escherichia coli; the corresponding experiments with the animal trypsin inhibitors were carried out with thioredoxin from calf thymus or E. coli. In all cases, thioredoxin appeared to act catalytically; the reduced form of glutathione was without effect. When considered in conjunction with earlier results on purothionin (confirmed and extended in the current study), the new findings suggest that the NADP/thioredoxin system functions in the reduction of protein inhibitors of seeds and animal tissues. These results also raise the question of the occurrence of glutaredoxin in plants, as E. coli glutaredoxin was found to promote the reduction of some but not all of the proteins tested.     

Induced resistance of trypsin to sodium dodecylsulfate upon complex formation with trypsin inhibitor

The stabilities of trypsin and soybean trypsin inhibitor in sodium dodecylsulfate (SDS) were examined by SDS-polyacrylamide gel electrophoresis (PAGE). Both samples contained several bands, all of which migrated to positions corresponding to the appropriate molecular weight or less, even when the samples were unheated, suggesting that both the trypsin and trypsin inhibitor are susceptible to SDS-induced denaturation. When they were mixed together prior to addition of SDS-PAGE sample buffer (1% SDS), a new smearing band appeared which corresponded to a molecular weight of around 46,000, suggesting that these proteins form a stable complex in SDS. This was confirmed by electroblotting and sequence analysis, which indicated that this band contains both the trypsin and inhibitor sequences. At a fixed concentration of the inhibitor, increasing concentrations of the trypsin resulted in an increase in the intensity of the complex band. When the mixture was heated for 10 min in 1% SDS, the complex band disappeared in a temperature-dependent manner. The melting temperature determined under the experimental conditions used was about 35|MoC. Similar results were obtained with Bowman-Birk trypsin inhibitor, except that the complex with the above inhibitor had a higher melting temperature, around 41|MoC, suggesting that the Bowman-Birk inhibitor/trypsin complex is more stable than the soybean inhibitor/trypsin complex.     

A possible evolutionary relationship between plant trypsin inhibitor, alpha-amylase inhibitor, and mammalian pancreatic secretory trypsin inhibitor (Kazal)

The amino acid sequence of the carboxyl-terminal half of barley trypsin inhibitor was found to be significantly similar to the whole sequence of bovine pancreatic secretory trypsin inhibitor (Kazal). Kazal type inhibitors and related proteins are known for the extraordinary mode of divergence among animals, and the present observation extends this to a plant for the first time. The present observation together with our previous finding of sequence homology between barley trypsin inhibitor and wheat alpha-amylase inhibitor (Odani, S., Koide, T., & Ono, T. (1982) FEBS Lett. 141, 279-282) suggest an unusual evolutionary relationship between cereal enzyme inhibitors and animal proteinase inhibitors of the Kazal type.     

Human mesotrypsin defies natural trypsin inhibitors: from passive resistance to active destruction

More than twenty years ago Rinderknecht et al. identified a minor trypsin isoform resistant to natural trypsin inhibitors in the human pancreatic juice. At the same time, Estell and Laskowski found that an inhibitor-resistant trypsin from the pyloric caeca of the starfish, Dermasterias imbricata rapidly hydrolyzed the reactive-site peptide bonds of trypsin inhibitors. A connection between these two seminal discoveries was made recently, when human mesotrypsin was shown to cleave the reactive-site peptide bond of the Kunitz-type soybean trypsin inhibitor, and degrade the Kazal-type pancreatic secretory trypsin inhibitor. These observations indicate that proteases specialized for the degradation of protease inhibitors are ubiquitous in metazoa, and prompt new investigations into their biological significance. Here we review the history and properties of human mesotrypsin, and discuss its function in the digestive degradation of dietary trypsin inhibitors and possible pathophysiological role in pancreatitis.     

Immunochemical studies of human urinary trypsin inhibitor

Antisera against purified urinary trypsin inhibitor (UTI-I, molecular weight 67,000) and UTI-III (molecular weight 23,000) were first produced in rabbits. Both anti-UTI-I and anti-UTI-III sera formed a single immunoprecipitin line with human plasma inter-alpha-trypsin inhibitor (I alpha TI), whereas two immunoprecipitin lines were formed with crude urine. It was speculated that both UTI-I and UTI-II might be present in normal human urine. In the present study, the inhibitory effects of anti-UTI sera on UTI activity were examined by three different assay methods. The results indicated that the inhibitory effect was almost immediate. Although the inhibitory effect of anti-UTI-III serum on UTI-III was almost of the same degree of completeness for the three assay methods. UTI-I was partially inhibited by the anti-UTI-I serum when residual trypsin activity was measured by the caseinolytic or fibrinolytic assay method. This discrepancy was considered to be due to the difference in conformational change between UTI-I and UTI-III by antigen-antibody reaction.     

The two alpha 2-macroglobulin-bound trypsin molecules have different affinities for the basic pancreatic trypsin inhibitor

We have investigated the enzymatic properties of alpha 2-macroglobulin-bound porcine trypsin using a substrate: Z-Gly-Gly-Arg-p-nitroanilide and two inhibitors: p-aminobenzamidine and basic pancreatic trypsin inhibitor. The ternary alpha 2-macroglobulin-(trypsin)2 complex behaves like a mixture of two enzymes which bind basic pancreatic trypsin inhibitor with widely different affinities (Ki = 0.11 microM and 23 microM). About one-half of the trypsin molecules of the ternary complex are covalently bound to alpha 2-macroglobulin. Preparation of the complex in the presence of hydroxylamine prevents covalent bond formation, but the two trypsins of this artificial complex still exhibit large differences in affinity for basic pancreatic trypsin inhibitor. The trypsin molecules of the ternary complex also exhibit small differences in their affinity for Z-Gly-Gly-Arg-p-nitroanilide and p-aminobenzamidine.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

Wheat germ trypsin inhibitors. Isolation and structural characterization of single-headed and double-headed inhibitors of the Bowman-Birk type

A number of trypsin inhibitors were isolated from wheat germs by affinity chromatography on immobilized trypsin, gel-filtration, and ion-exchange and reverse-phase chromatography. These inhibitors were classified into two groups, inhibitors I (Mr = 14,500) and II (Mr = 7,000), based on their molecular sizes. Inhibitors I and II inhibited bovine trypsin stoichiometorically at an enzyme to inhibitor ratio of 2 and 1, respectively. Sequence analysis of these inhibitors indicated a high degree of homology and that inhibitors I had a duplicated structure of inhibitors II. They are highly homologous to double-headed proteinase inhibitors (Bowman-Birk inhibitors) of Leguminosae plants. Inhibitors II are the first example of single-headed inhibitor corresponding to one inhibitory domain of the Bowman-Birk type double-headed inhibitors, which suggests that inhibitors II are relic of an ancestral single-headed inhibitor before the gene-duplication that led to the formation of present-day Bowman-Birk type inhibitors.     

Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe

Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests. We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L.) by biolistic bombardment of cultured immature embryos. Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe. BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 1.1% of total extracted protein. No expression was detected in untransformed wheat seeds. Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay. The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance. However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects.     

Human mesotrypsin is a unique digestive protease specialized for the degradation of trypsin inhibitors

Mesotrypsin is an enigmatic minor human trypsin isoform, which has been recognized for its peculiar resistance to natural trypsin inhibitors such as soybean trypsin inhibitor (SBTI) or human pancreatic secretory trypsin inhibitor (SPINK1). In search of a biological function, two conflicting theories proposed that due to its inhibitor-resistant activity mesotrypsin could prematurely activate or degrade pancreatic zymogens and thus play a pathogenic or protective role in human pancreatitis. In the present study we ruled out both theories by demonstrating that mesotrypsin was grossly defective not only in inhibitor binding, but also in the activation or degradation of pancreatic zymogens. We found that the restricted ability of mesotrypsin to bind inhibitors or to hydrolyze protein substrates was solely due to a single evolutionary mutation, which changed the serine-protease signature glycine 198 residue to arginine. Remarkably, the same mutation endowed mesotrypsin with a novel and unique function: mesotrypsin rapidly hydrolyzed the reactive-site peptide bond of the Kunitz-type trypsin inhibitor SBTI, and irreversibly degraded the Kazal-type temporary inhibitor SPINK1. The observations suggest that the biological function of human mesotrypsin is digestive degradation of trypsin inhibitors. This mechanism can facilitate the digestion of foods rich in natural trypsin inhibitors. Furthermore, the findings raise the possibility that inappropriate activation of mesotrypsinogen in the pancreas might lower protective SPINK1 levels and contribute to the development of human pancreatitis. In this regard, it is noteworthy that the well known pathological trypsinogen activator cathepsin B exhibited a preference for the activation of mesotrypsinogen of all three human trypsinogen isoforms, suggesting a biochemical mechanism for mesotrypsinogen activation in pancreatic acinar cells.     

Purification and characterization of two trypsin inhibitors from the hemolymph of Manduca sexta larvae

Trypsin inhibitory activity from the hemolymph of the tobacco hornworm (Manduca sexta) was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 14,000 (M. sexta hemolymph trypsin inhibitor (HLTI) A) and 8,000 (HLTI B) by molecular sieve chromatography on Sephadex G-75. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8,300 for HLTI A and 9,100 for HLTI B, suggesting that HLTI A is a dimer and HLTI B is a monomer. Isoelectrofocusing on polyacrylamide gels focused HLTI A as a single band with pI 5.7, whereas HLTI B was resolved into two components with pI values of 5.3 and 7.1. Both inhibitors were stable at 100 degrees C and pH 1.0 for at least 30 min. HLTIs A and B inhibited serine proteases such as trypsin, chymotrypsin, and plasmin, but did not inhibit elastase, papain, pepsin, subtilisin BPN', and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors. Both inhibitors formed low-dissociation complexes with trypsin in a 1:1 molar ratio. The inhibition constant for trypsin inhibition by HLTI A was estimated to be 1.45 x 10(-8) M. The HLTI A-chymotrypsin complex did not inhibit trypsin; similarly, the HLTI A-trypsin complex did not inhibit chymotrypsin, indicating that HLTI A has a common binding site for both trypsin and chymotrypsin. The amino-terminal amino acid sequences of HLTIs A and B revealed that both these inhibitors are homologous to bovine pancreatic trypsin inhibitor (Kunitz).     

The complete amino acid sequence of barley trypsin inhibitor

The amino acid sequence of barley trypsin inhibitor has been determined. The protein is a single polypeptide consisting of 121 amino acid residues and has Mr = 13,305. No free sulfhydryl groups were detected by Ellman's reagent, which indicates the presence of five disulfide bridges in the molecule. The primary site of interaction with trypsin was tentatively assigned to the arginyl-leucyl residues at positions 33 and 34. On comparison of the sequence of this inhibitor with those of other proteinase inhibitors, we found that the barley trypsin inhibitor could not be classified into any of the established families of proteinase inhibitors (Laskowski, M., Jr., and Kato, I. (1980) Annu. Rev. Biochem. 49, 593-626) and that this inhibitor should represent a new inhibitor family. On the other hand, this trypsin inhibitor showed a considerable similarity to wheat alpha-amylase inhibitor (Kashlan, N., and Richardson, M. (1981) Phytochemistry (Oxf.) 20, 1781-1784) throughout the whole sequence, suggesting a common ancestry for both proteins. This is the first case of a possible evolutionary relationship between two inhibitors directed to totally different enzymes, a proteinase and a glycosidase.     

Radioimmunological quantitation of urinary trypsin inhibitor

Using monospecific antibody to human urinary trypsin inhibitor, we developed a highly specific and sensitive radioimmunoassay (RIA) for measuring human urinary trypsin inhibitor. No cross-reactivity of the antibody with protein standard serum, which contained albumin, alpha 1-antitrypsin, haptoglobin, alpha 2-macroglobulin, transferrin, IgG and IgA, was observed. The sensitivity of the system was 10 ng of trypsin inhibitor per assay tube, and 5-10 microliters of urine was sufficient to determine the concentration of trypsin inhibitor in urine. The amounts excreted in the urine of 10 healthy men and 10 healthy women were 4.83 +/- 2.46 (mean +/- SD) and 3.86 +/- 1.35 mg/day, respectively. The correlation between estimates by RIA and those by enzymic assay was r = 0.96 (p less than 0.005). The method proposed here can be used to determine the concentration of urinary trypsin inhibitor in a small amount of biological fluids and cells.     

Three high molecular weight protease inhibitors of rat plasma. Reactions with trypsin

We have compared the reactions of trypsin with human alpha 2-macroglobulin (alpha 2M), and three rat plasma protease inhibitors, alpha 1-macroglobulin (alpha 1M), alpha 1-inhibitor III (alpha 1I3), and alpha 2M. All four of these proteins appear to contain reactive thiol esters. The electrophoretic mobility in agarose gels of human and rat alpha 2M is increased by 1 mol of trypsin, while the mobility of alpha 1M and alpha 1I3 is decreased. Treatment with methylamine causes similar mobility changes, except in the case of rat alpha 2M. Titration of human and rat macroglobulins by repeated small additions of trypsin and by assay of liberated SH groups or enhanced ligand fluorescence revealed a stoichiometry of about 1 mol of trypsin/mol of inhibitor. In contrast, addition of macroglobulin to a fixed amount of trypsin and detection of residual amidase or protease activity revealed a stoichiometry of about 2 mol of trypsin for 1 mol of human alpha 2M, about 1.4 mol for rat alpha 1M, and about 1 mol for rat alpha 2M. One mol of trypsin reacted with 2 or more mol of alpha 1I3 by the criteria of SH groups liberated or protease inhibition. Methylamine-treated rat alpha 2M binds a significant amount of trypsin releasing about 2 mol of SH. Radioactive beta-trypsin was covalently bound to subunits of the purified plasma inhibitors. The Mr of the labeled products with rat and human alpha 2M had molecular weights which suggested trypsin was bound to intact as well as cleaved subunit chains and also to multiple chains via cross-linking. Rat alpha 1M also produced a product which may be an intact subunit alpha chain plus trypsin. Greater than 80% of the trypsin was bound covalently to these inhibitors at low molar ratios.     

The interaction of a range of serine proteinase inhibitors with bovine trypsin and Costelytra zealandica trypsin

Kinetic and other properties of the interaction between two serine proteinases, bovine trypsin and Costelytra zealandica (grass grub) larval trypsin and a range of proteinaceous serine proteinase inhibitors were investigated. Twenty-six inhibitors or isoinhibitors from 10 different inhibitor families were analysed. A 1700-fold range in equilibrium dissociation constant (K_d) values was obtained for bovine trypsin and a 10⁵-fold range for grass grub trypsin. The ratios of K_d (grass grub)/K_d (bovine) also spanned a range of 10⁵-fold. Qualitative observations indicated that the second order association rate constants were high for all except two inhibitors. Two classes of first order dissociation rate constant were determined from the dissociation of trypsin-inhibitor complexes induced by substrate. While most inhibitors were cleaved by grass grub trypsin, they still inhibited larval midgut crude extracts during long incubations. We suggest using the K_d value to assess the potential for any inhibitor to act as a grass grub larval resistance factor in plants, in preference to other parameters.