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Trypsin-like Enzyme (trypsin-like + enzyme)

Distribution by Scientific Domains

Chemistry	66%

Selected Abstracts

Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) Larvae

ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2010
Nikola Lon
Abstract Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5,9.5) and temperature optimum of 45°C with the KM ratio of 0.065,mM for benzoyl-arginine- p -nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (KI value of 0.012,mM, Ic50 value of 0.204,mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5,kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38,kDa, suggesting that the enzyme is a monomer. © 2010 Wiley Periodicals, Inc. [source]

cDNA sequence, mRNA expression and genomic DNA of trypsinogen from the Indianmeal moth, Plodia interpunctella

INSECT MOLECULAR BIOLOGY, Issue 1 2000
Y. C. Zhu
Abstract Trypsin-like enzymes are major insect gut enzymes that digest dietary proteins and proteolytically activate insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indianmeal moth, Plodia interpunctella, was linked to the absence of a major trypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like proteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt-susceptible and -resistant strains of the Indianmeal moth were compared. Proteinase activity blots of gut extracts indicated that the susceptible strain had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides from the susceptible strain and 848 nucleotides from the resistant strain contained an open reading frame of 783 nucleotides which encoded a 261-amino acid trypsinogen-like protein. There was a single silent nucleotide difference between the two cDNAs in the open reading frame and the predicted amino acid sequence from the cDNA clones was most similar to sequences of trypsin-like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Northern blotting analysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the strains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were different. Based on an enzyme size comparison, the cDNA isolated in this study corresponded to the gene for the smaller of two trypsin-like proteinases, which is found in both the Bt-susceptible and -resistant strains of the Indianmeal moth. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF064525 for the RC688 strain and AF064526 for HD198). [source]

PARTIAL PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE TRYPSIN FROM PYLORIC CAECA OF TAMBAQUI (COLOSSOMA MACROPOMUM)

JOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2001
RANILSON S. BEZERRA
ABSTRACT A 38.5 kDa alkaline protease from pyloric caeca of tambaqui (Colossoma macropomumj, a tropical freshwater fish, was partially purified in three steps: thermal treatment (45Cfor 30 min), salting-out (ammonium sulfate at 40,80% of saturation) and gel filtration (Sephadex G-75), The purification and yield were 51.2-fold and 40%, respectively. The effects of pH, temperature, inhibitors, and substrates on proteolytic activities of partially purified enzyme were investigated. The optimum pH was 9.5, while the optimum temperature was 60C. This alkaline proteolytic activity remained unaltered after 30 min incubation at 55C. Active site inhibition provided additional evidence that this activity is attributed to a trypsin-like enzyme. [source]

Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) Larvae

A heterozygous null mutation combined with the G1258A polymorphism of SPINK5 causes impaired LEKTI function and abnormal expression of skin barrier proteins

BRITISH JOURNAL OF DERMATOLOGY, Issue 2 2009
W-L. Di
Summary Background, Loss-of-function mutations in the Kazal-type serine protease inhibitor, LEKTI, encoded by the SPINK5 gene cause the rare autosomal recessive skin disease Netherton syndrome (NS). G1258A polymorphism in SPINK5 may be associated with atopic dermatitis, which shares several clinical features with NS. Objectives, To determine if the phenotype of NS can be caused by a single null mutation in SPINK5 combined with the homozygous G1258A polymorphism. Methods, We screened mutations in the gene SPINK5 by direct DNA sequencing and position cloning and examined the expressions of the SPINK5 -encoded protein LEKTI and other relevant proteins by immunostaining and immunoblot. Results, We describe here a patient who was clinically diagnosed with NS and carried a single null mutation in SPINK5 combined with the homozygous G1258A polymorphism. SPINK5 mRNA was present at normal levels and LEKTI was expressed in the epidermis. Nonetheless, the putative downstream LEKTI substrates stratum corneum trypsin-like enzyme (SCTE), desmoglein 1 and protein markers of keratinocyte differentiation were expressed abnormally, similar to that seen in NS if two null mutant alleles are present. Conclusion, This finding indicates that haploinsufficiency of SPINK5 can cause the NS phenotype in the presence of one null mutation with homozygous G1258A polymorphisms in SPINK5, and this could impair the function of LEKTI and therefore acts as a true mutation. [source]

Synthesis and biological activity of homoarginine-containing opioid peptides

JOURNAL OF PEPTIDE SCIENCE, Issue 1 2007
Jan Izdebski
Abstract Two tris-alkoxycarbonyl homoarginine derivatives, Boc-Har{,,,,-[Z(2Br)]2}-OH and Boc-Har{,,,,-[Z(2Cl)]2}-OH, were prepared by guanidinylation of Boc-Lys-OH, and used for the synthesis of neo-endorphins and dynorphins. The results were compared with that obtained in the synthesis in which Boc-Lys(Fmoc)-OH was incorporated into the peptide chain, and after removing Fmoc protection, the resulting peptide-resin was guanidinylated with N,N,-[Z(2Br)]2 - or N,N,-[Z(2Cl)]2 - S -methylisourea. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. The results indicated that replacement of Arg by Har may be a good avenue for the design of biologically active peptides with increased resistance to degradation by trypsin-like enzymes. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [source]