The digestive proteinase trypsin, alkaline A contributes to anti-BmNPV activity in silkworm (Bombyx mori).

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious pathogenic microorganism that causes tremendous loss to sericulture. Previous studies have found that some proteins of serine protease family in the digestive juice of B. mori larvae have anti-BmNPV activity. In our previous publication about proteome analysis of the digestive juice of B. mori larvae, the digestive enzyme trypsin, alkaline A (BmTA) was filtered as a differentially expressed protein possibly involved in BmNPV resistance. Here, the biological characteristics and anti-BmNPV functions of BmTA were comprehensively analysed. The cDNA sequence of BmTA had an ORF of 768 nucleotides encoding 255 amino acid residues. Domain architecture analysis showed that BmTA contained a signal peptide and a typical Tryp_SPc domain. Quantitative real-time PCR analysis showed that BmTA was highly expressed in the larval stages and specifically expressed in the midgut of B. mori larvae. The expression level of BmTA in BmNPV resistant strain A35 was higher than that in susceptible strain P50. After BmNPV infection, the expression of BmTA increased in both strains from 24 to 72 h. Virus amplification analysis showed that the relative levels of VP39 in B. mori larvae and BmN cells infected with the appropriate concentration of recombinant-BmTA-treated BmNPV were significantly lower than in the control groups. Moreover, overexpression of BmTA in BmN cells significantly inhibited the amplification of BmNPV. Taken together, the results of this study indicated that BmTA possessed anti-BmNPV activity in B. mori, which broadens the horizon for virus-resistant breeding of silkworms.

Maternally derived trypsin may have multiple functions in the early development of turbot (Scopthalmus maximus).

Trypsin is an important serine protease that is considered to be involved in digestion of protein in teleost fish. Nevertheless, studies on trypsin/trypsinogen in fish embryos are very limited. In this study, the trypsinogen of turbot (Scophthalmus maximus) (tTG) was identified and the expression patterns and activity of trypsinogen/trypsin were investigated. The results showed that the tTG mRNA was evenly distributed in the oocytes and was also expressed along the yolk periphery in early embryos. At later embryo stages and 1 days after hatching (dph), the tTG mRNA concentrated at the alimentary tract and head. Quantitative expression analysis showed that the tTG transcripts decreased after fertilization until the gastrula stage, then increased with the embryo and larvae development. This result was also confirmed by the specific activity analysis of trypsin and in-situ-hybridization (ISH). All of the results indicated that tTG in early embryo stages was maternally derived and expressed by itself after gastrula stages. Additionally, location of tTG mRNA in embryos and larvae was investigated; we considered that trypsin may have multiple functions during the embryo development process. Based on our results regarding trypsinogen in embryos and early development, we concluded that the trypsin/trypsinogen in turbot embryos was inherited from a maternal source and we suggested that trypsin in early development has multiple functions in the process of development.

Development of activity-based probes for trypsin-family serine proteases.

A series of diphenylphosphonate-based probes were developed for the trypsin-like serine proteases. These probes selectively target serine proteases rather than general serine hydrolases that are targets for fluorophosphonate-based probes. This increased selectivity allows detection of low abundance serine proteases in complex proteomes using simple SDS-PAGE methods. We present here the application of multiple probes in enzyme activity profiling of intact mast cells, a type of inflammatory cell implicated in allergy and autoimmune diseases.

Trypsins from yellowfin tuna (Thunnus albacores) spleen: purification and characterization.

Two anionic trypsins (A and B) were purified to homogeneity from yellowfin tuna (Thunnus albacores) spleen by a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. Purity was increased to 70.6- and 91.5-fold with approximately 2.8% and 15.6% yield for trypsin A and B, respectively. The apparent molecular weight of both trypsins was estimated to be 24 kDa by size exclusion chromatography and SDS-PAGE. Both trypsin A and B appeared as a single band on native-PAGE. Trypsin A and B exhibited the maximal activity at 55 and 65 degrees C, respectively, and had the same optimal pH at 8.5 using TAME as a substrate. Both trypsins were stable to heat treatment up to 50 degrees C and in the pH range of 6.0 to 11.0. Both trypsin A and B were stabilized by calcium ion. The activities were inhibited effectively by soybean trypsin inhibitor, TLCK and partially inhibited by EDTA, but were not inhibited by E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). Apparent Km and Kcat of trypsin A and B for TAME were 0.2-0.33 mM and 66.7-80 S(-1), respectively. The N-terminal amino acid sequences of trypsin A, IVGGYECQAHSQPHQVSLNA, and trypsin B, IVGGYECQAHSQPPQVSLNA, indicated the high homology between both enzymes.

The denaturation of alpha, beta and psi bovine trypsin at pH 3.0: evidence of intermediates.

The conformational stability and the folding process of alpha, beta and Psi bovine trypsin at pH 3.0 followed by circular dichroism (CD) and size exclusion in HPLC have been analyzed as a function of urea concentration. The thermodynamic stability for a and b are deltaG = 15.91 +/- 0.28 kcal/mol, deltaG = 15.54 +/- 2.39 kcal/mol. respectively, and y trypsin is deltaG = 16.10 +/- 2.51 kcal/mol. The transition curves for alpha, beta and Psi forms suggest a molten globule state.

Molecular cloning and partial characterization of a trypsin-like protein in salivary glands of Lygus hesperus (Hemiptera: Miridae).

Trypsin-like enzymes from the salivary gland complex (SGC) of Lygus hesperus Knight were partially purified by preparative isoelectric focusing (IEF). Enzyme active against Nalpha-benzoyl-L-arginine-p-nitroanilide (BApNA) focused at approximately pH 10 during IEF. This alkaline fraction gave a single activity band when analyzed with casein zymograms. The serine proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor, completely inhibited or suppressed the caseinolytic activity in the crude salivary gland extract as well as the IEF-purified sample. Chicken egg white trypsin inhibitor also inhibited the IEF-purified sample but was not effective against a major caseinolytic band in the crude salivary gland extract. These data indicated the presence of serine proteinases in the SGC of L. hesperus. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for serine proteinases in L. hesperus. The encoded trypsin-like protein included amino acid sequence motifs, which are conserved with five homologous serine proteinases from other insects. Typical features of the putative trypsin-like protein from L. hesperus included residues in the serine proteinase active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides.

A clade of trypsins found in cold-adapted fish.

A clade of trypsins, known as group III, is identified by phylogenetic analysis. These trypsins occur in fish that spend all or part of their lives at temperatures near 0 degrees C and may represent extreme psychrophilic enzymes. A principal component analysis of amino acid compositions distinguishes group III from mesophilic trypsins, as do molecular trees and multidimensional scaling of molecular sequence distances. The primary sequences of group III trypsins, in conjunction with the known structures of mesophilic trypsins, permit insight into function and mechanisms of cold adaption. The techniques employed are broadly applicable to phylogenies characterized by a markedly different, or "fast-evolving," clade. An updated lactate dehydrogenase molecular tree illustrates an additional fast-evolving clade.

Three-dimensional structure analysis of PROSITE patterns.

Pattern matches for each of the sequence patterns in PROSITE, a database of sequence patterns, were searched in all protein sequences in the Brookhaven Protein Data Bank (PDB). The three-dimensional structures of the pattern matches for the 20 patterns with the largest numbers of hits were analysed. We found that the true positives have a common three-dimensional structure for each pattern; the structures of false positives, found for six of the 20 patterns, were clearly different from those of the true positives. The results suggest that the true pattern matches each have a characteristic common three-dimensional structure, which could be used to create a template to define a three-dimensional functional pattern.

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

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

The peptidolytic capacity of the spirochete system.

Relatively scant chemical information has been available on the proteinases and peptidases of spirochetes in spite of the association of spirochetes with several serious infections known to plague humans and other animal species. This situation has partly resulted from difficulties in growing some spirochetes under laboratory conditions. The cells of Treponema denticola, a spirochete suggested to be associated with periodontal infections, have turned out to be a good source of new chemical information on those enzymes. Latest studies suggest that the outer cell envelope or the periplasmic space of T. denticola contains several novel proteinases and peptidases (hence called "ectoenzymes") which may contribute to the chronicity of periodontal infections. Some of the oligopeptidases discovered are specific for proline-containing host tissue peptides such as substance P, bradykinin, neurotensin, etc., and possibly small collagen fragments. The only spirochetal peptidases purified to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been obtained from T. denticola. One particular peptidase, suggested to be similar to the oligopeptidase B (EC 3.4.21.83) of Escherichia coli seems to be present in the cell envelope or in the periplasmic space at quite large concentrations. The presence of this and several other peptidases in the outer cell structures of the treponemes suggests that such enzymes are important for the nutrition of these highly motile and invasive organisms. The biological role of these enzymes can thus be envisaged in the peptidolytic processing of host tissue proteins and peptides to gradually smaller molecules to fulfill the nutritional requirements of these organisms. Although the genetic similarity between T. denticola and some other treponemes and spirochetes can be hotly debated, it is nevertheless now possible to use T. denticula enzymes as suitable objects for comparison when the chemistry of other spirochetes is studied.

Effects of dehulling and storage conditions on cooking requirements and chemical composition of soybeans / Efecto del descascarado y de las condiciones de almacenamiento en los requerimientos de cocción y composición química de la soya

Changes in cooking requirements and chemical composition of whole and dehulled soybeans, stored in 2 different environments [25ºC/75 per cent RH. (Environment 1) and 38ºC/90 per cent RH. (Environment 2)], were studied. Rate of water absorption and solid losses during cooking were higher for the dehulled soybeans at both storage conditions. However, cooking requirements to archieve the same degree of texture cotyledons were similar for whole and dehulled seeds. Cooking time increased with prolonged storage; the effect was more noticeable in samples stored under Environment 2. Samples kept for 6 months almost twice as much cooking than control samples. dehulled soybeans had a lower fiber content, relatively higher amounts of protein and fat, but similar amino acid compositions than whole soybeans. Cooking caused losses of carbohydrates and ash and, therefore, significantly increased levels of protein and fat reflected by losses of solids during soaking and cooking. Among the amino acids, only cysteine suffered subtantial decrease as a result of cooking. Cooking and storage inactivated 90 per cent and from 20-35 per cent of the trypsin inhibitors, respectively; the latter effect was more accentuated in samples stored under Environment 2

Effects of dehulling, cooking and storage conditions on protein quality and digestibility of soybeans / Efecto del descascarado, cocción y almacenamiento sobre la calidad de la proteína y digestibilidad de la soya

Soybeans were debulled, stored under two environmental conditions [25ºC/75 por ciento RH (Env. 1) and 38ºC/90 por ciento RH (Env. 2)], optimally cooked and assayed for trypsin inhibitor and protein quality with laborary rats. Dehulling not significantly effects protein quality (PER and NPR) and protein digestibility of raw and cooked soybeans. Raw soybeans diets were significantly poorer in protein quality and digestibility when compared with cooked counterparts. PER values of dehulled-cooked soybean diets decreased significantly (p<0.05) as seeds were stored for up to 3 months under either environment. These were no significant differences in PER values due to etorage suring the period from 3 a 6 months. PER values for whole-cooked soybean diets exhibited a significant decline only when stored for 6 months under Env. 2

Isolation, sequencing and characterization of two cDNA clones coding for trypsin-like enzymes from the midgut of Aedes aegypti.

In order to understand the regulation of trypsin genes by the blood meal, we constructed a cDNA library from mRNA isolated from midguts of blood-fed female Aedes aegypti. The library was screened with a Drosophila melanogaster trypsin-like gene; twelve cDNAs were isolated and sequenced. Two clones were 846 bp and 788 bp long with 762 bp and 716 bp open reading frames, respectively. The cDNAs were identified as coding for serine proteases by the presence of conserved serine, histidine and aspartic acid residues; the presence of an aspartate residue at position 176 suggests that the clones were derived from trypsin-like gene transcripts rather than chymotrypsin or other serine proteases. One of the clones contained a 5' untranslated region and coding regions for putative signal and activation peptides, suggesting that the product is secreted as an inactive zymogen and processed by autoactivation. Southern analysis of genomic DNA suggests that trypsin is encoded by a multigene family in A. aegypti.

Characterization of a human trypsin resistant to Kunitz soybean trypsin inhibitor. Studies of duodenal juices after tube instillation of raw soybean extract.

Human duodenal juices collected during tube instillation of raw soybean extract into the duodenum contained free trypsin and free Kuntiz soybean trypsin inhibitor (KTI) in the simultaneous presence of trypsin-KTI complexes. It has previously been suggested that this KTI-non-inhibitable trypsin has a general resistance to serine protease inhibitors. Four different trypsin forms have been found and partly characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing followed by Western immunoblotting or enzyme staining. In addition, crossed immunoelectrophoresis and affinity chromatography with antibody-coupled gels have been used for identification of free and inhibitor-complexed trypsin.

Protein degradation in health and disease. Introduction: the classification of proteinases.

Twenty years after B.S. Hartley's 1960 review, on which the present scheme for classification of the proteinases is based, most of the new information that has been obtained appears fully consistent with Hartley's views. A slight amendment is proposed of the name of the four groups of these enzymes to 'serine', 'cysteine', 'aspartic' and 'metallo'-proteinases.