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Chymotrypsin-like Activity (chymotrypsin-like + activity)
Selected AbstractsDigestive peptidases in Tenebrio molitor and possibility of use to treat celiac diseaseENTOMOLOGICAL RESEARCH, Issue 3 2007Elena N. ELPIDINA Abstract Digestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post-proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin-like and chymotrypsin-like, are compartmentalized to the PM. Trypsin-like activity is due to one cationic and three anionic proteinases. Chymotrypsin-like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N-like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM , cathepsin L and post-proline cleaving peptidase , in the treatment of celiac disease is discussed. [source] Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae)AQUACULTURE RESEARCH, Issue 7 2009Diego Souza Buarque Abstract Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 °C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2,9.0) and 55 °C. CaCl2 did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 °C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated (P<0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols. [source] Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)INSECT SCIENCE, Issue 5 2009Brian David Shaw Abstract, The protein digestive capability of the larvae of the longhorn beetle (Oemona hirta, Coleoptera: Cerambycidae, Fabricius, 1775) was investigated. This species feeds only on wood where there is a high proportion of vascular tissue. The pH of the midgut, the major digestive organ, was alkaline and protein hydrolysis was maximal at alkaline pH. Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases, trypsin and chymotrypsin-like activity, and the exopeptidase, leucine aminopeptidase and the pH curves corresponded to that with protein substrate. Studies using a range of serine protease inhibitors as well as specific inhibitors of metalloproteases, cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids. Control of these insect pests using protease inhibitors is discussed. [source] Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosisJOURNAL OF NEUROCHEMISTRY, Issue 5 2002Makoto Urushitani Abstract Accumulating evidence indicates that abnormal conformation of mutant superoxide dismutase 1 (SOD1) is an essential feature underlying the pathogenesis of mutant SOD1-linked familial amyotrophic lateral sclerosis (ALS). Here we investigated the role of ubiquitin-proteasome pathway in the mutant SOD1-related cell death and the effect of oxidative stress on the misfolding of mutant SOD1. Transient overexpression of ubiquitin with human SOD1 (wild-type, ala4val, gly85arg, gly93ala) in Neuro2A cells decreased the amount of mutant SOD1, but not of wild-type, while only mutants were co-immunoprecipitated with poly-ubiquitin. Proteasome inhibition by lactacystin augmented accumulation of mutant SOD1 in the non-ionic detergent-insoluble fraction. The spinal cord lysates from mutant SOD1 transgenic mice showed multiple carbonylated proteins, including mutant SOD1 with SDS-resistant dimer formation. Furthermore, the treatment of hSOD1-expressing cells with hydrogen peroxide promoted the oligomerization, and detergent-insolubility of mutant SOD1 alone, and the oxidized mutant SOD1 proteins were more heavily poly-ubiquitinated. In Neuro2A cells stably expressing human SOD1 protein, the proteasome function measured by chymotrypsin-like activity, was decreased over time without a quantitative alteration of the 20S proteasomal component. Finally, primary motor neurons from the mouse embryonic spinal cord were more vulnerable to lactacystin than non-motor neurons. These results indicate that the sustained expression of mutant SOD1 leads to proteasomal inhibition and motor neuronal death, which in part explains the pathogenesis of mutant SOD1-linked ALS. [source] Decreased Proteasome Activity Is Associated With Increased Severity of Liver Pathology and Oxidative Stress in Experimental Alcoholic Liver DiseaseALCOHOLISM, Issue 8 2004Terrence M. Donohue Jr Background: Because of its role in degrading the bulk of intracellular proteins and eliminating damaged proteins, the proteasome is important in maintaining cell viability. Previously, we showed a 35,40% decrease in proteasome peptidase activity when ethanol was administered to rats by intragastric infusion. We hypothesized that this reduction was caused by ethanol-elicited oxidative stress, the degree of which varies depending on the method of ethanol administration. This study examined the relationship of proteasome activity and content with ethanol-induced oxidative stress and the degree of liver injury. Methods: Rats were given ethanol or isocaloric dextrose-containing liquid diets by intragastric infusion for 1 month. The diets contained medium-chain triglycerides (MCT), palm oil (PO), corn oil (CO), or fish oil (FO) as the principal source of fat. Results: Rats given ethanol and MCT exhibited no significant liver pathology, whereas cumulative pathology scores in ethanol-fed rats given PO, CO, or FO were 2.5, 5.4 and 7.0, respectively, indicating that ethanol and FO caused the greatest liver damage. The severity of liver pathology in the last three groups of animals correlated with levels of lipid peroxides and serum 8-isoprostanes. Alpha smooth muscle actin, an indicator of stellate cell activation, was increased relative to controls in the livers of all ethanol-fed rats except FO-fed animals, in which both control and ethanol-fed rats had similar levels of this protein. In livers of CO and FO ethanol-fed rats, proteasome chymotrypsin-like activity was decreased by 55,60%, but there was no quantitative alteration in 20S proteasome subunit content. In contrast, ethanol affected neither proteasome activity nor its content in MCT- and PO-treated animals. Conclusions: Our findings indicate that the severity of liver injury and ethanol-induced oxidative stress is associated with a reduction in proteasome catalysis. [source] Purification and characterization of a subtilisin-like serine protease induced during the senescence of wheat leavesPHYSIOLOGIA PLANTARUM, Issue 4 2003Irma N. Roberts A senescence-specific protease accounting for almost 70% of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc-AAPF-pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent Km and Vmax for the peptide were 1.18 mm and 2.27 mmol pNA mg,1 h,1, respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60°C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity. Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65,75% identity) with a highly conserved region of several plant subtilisin-like serine proteases. The absence of this enzymatic activity in fractionated extracts from non-senescent tissues suggests that it might play a role in the senescing process. [source] | ||||||||||