Trypsin

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E N Elpidina - One of the best experts on this subject based on the ideXlab platform.

  • digestive peptidases in tenebrio molitor and possibility of use to treat celiac disease
    Entomological Research, 2007
    Co-Authors: E N Elpidina, Irina A Goptar
    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.

  • fractionation of digestive proteinases from tenebrio molitor coleoptera tenebrionidae larvae and role in protein digestion
    Comparative Biochemistry and Physiology B, 2006
    Co-Authors: K S Vinokurov, Brenda Oppert, Y E Dunaevsky, D P Zhuzhikov, E N Elpidina, S Prabhakar, M A Belozersky
    Abstract:

    Abstract Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of “heavy” Trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy Trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.

  • digestive proteinases of yellow mealworm tenebrio molitor larvae purification and characterization of a Trypsin like proteinase
    Biochemistry, 2005
    Co-Authors: T A Tsybina, Brenda Oppert, Y E Dunaevsky, M A Belozersky, D P Zhuzhikov, E N Elpidina
    Abstract:

    A new Trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI7.4. The enzyme was also characterized by temperature optimum at 55°C, pH optimum at 8.5, and K m value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a Trypsin-like serine proteinase stable within the pH range of 5.0–9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N- terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50–72% identity with other insect Trypsin-like proteinases, and 44–50% identity to mammalian Trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.

Brenda Oppert - One of the best experts on this subject based on the ideXlab platform.

  • characterization of cdnas encoding serine proteases and their transcriptional responses to cry1ab protoxin in the gut of ostrinia nubilalis larvae
    PLOS ONE, 2012
    Co-Authors: Lawrent L Buschman, Brenda Oppert, Chitvan Khajuria
    Abstract:

    Serine proteases, such as Trypsin and chymoTrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding Trypsins, chymoTrypsins and their homologs from the European corn borer (Ostrinia nubilalis) larval gut. Our analyses of the cDNA-deduced amino acid sequences indicated that 12 were putative Trypsins, 12 were putative chymoTrypsins, and the remaining 10 were Trypsin and chymoTrypsin homologs that lack one or more conserved residues of typical Trypsins and chymoTrypsins. Reverse transcription PCR analysis indicated that all genes were highly expressed in gut tissues, but one group of phylogenetically-related Trypsin genes, OnTry-G2, was highly expressed in larval foregut and midgut, whereas another group, OnTry-G3, was highly expressed in the midgut and hindgut. Real-time quantitative PCR analysis indicated that several Trypsin genes (OnTry5 and OnTry6) were significantly up-regulated in the gut of third-instar larvae after feeding on Cry1Ab protoxin from 2 to 24 h, whereas one Trypsin (OnTry2) was down-regulated at all time points. Four chymoTrypsin and chymoTrypsin homolog genes (OnCTP2, OnCTP5, OnCTP12 and OnCTP13) were up-regulated at least 2-fold in the gut of the larvae after feeding on Cry1Ab protoxin for 24 h. Our data represent the first in-depth study of gut transcripts encoding expanded families of protease genes in O. nubilalis larvae and demonstrate differential expression of protease genes that may be related to Cry1Ab intoxication and/or resistance.

  • fractionation of digestive proteinases from tenebrio molitor coleoptera tenebrionidae larvae and role in protein digestion
    Comparative Biochemistry and Physiology B, 2006
    Co-Authors: K S Vinokurov, Brenda Oppert, Y E Dunaevsky, D P Zhuzhikov, E N Elpidina, S Prabhakar, M A Belozersky
    Abstract:

    Abstract Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of “heavy” Trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy Trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.

  • digestive proteinases of yellow mealworm tenebrio molitor larvae purification and characterization of a Trypsin like proteinase
    Biochemistry, 2005
    Co-Authors: T A Tsybina, Brenda Oppert, Y E Dunaevsky, M A Belozersky, D P Zhuzhikov, E N Elpidina
    Abstract:

    A new Trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI7.4. The enzyme was also characterized by temperature optimum at 55°C, pH optimum at 8.5, and K m value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a Trypsin-like serine proteinase stable within the pH range of 5.0–9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N- terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50–72% identity with other insect Trypsin-like proteinases, and 44–50% identity to mammalian Trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.

M A Belozersky - One of the best experts on this subject based on the ideXlab platform.

  • fractionation of digestive proteinases from tenebrio molitor coleoptera tenebrionidae larvae and role in protein digestion
    Comparative Biochemistry and Physiology B, 2006
    Co-Authors: K S Vinokurov, Brenda Oppert, Y E Dunaevsky, D P Zhuzhikov, E N Elpidina, S Prabhakar, M A Belozersky
    Abstract:

    Abstract Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of “heavy” Trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy Trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.

  • digestive proteinases of yellow mealworm tenebrio molitor larvae purification and characterization of a Trypsin like proteinase
    Biochemistry, 2005
    Co-Authors: T A Tsybina, Brenda Oppert, Y E Dunaevsky, M A Belozersky, D P Zhuzhikov, E N Elpidina
    Abstract:

    A new Trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI7.4. The enzyme was also characterized by temperature optimum at 55°C, pH optimum at 8.5, and K m value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a Trypsin-like serine proteinase stable within the pH range of 5.0–9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N- terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50–72% identity with other insect Trypsin-like proteinases, and 44–50% identity to mammalian Trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.

Miklos Sahintoth - One of the best experts on this subject based on the ideXlab platform.

  • inactivation of mesoTrypsin by chymoTrypsin c prevents Trypsin inhibitor degradation
    Journal of Biological Chemistry, 2020
    Co-Authors: Vanda Toldi, Miklos Sahintoth, Andras Szabo
    Abstract:

    : MesoTrypsin is an unusual human Trypsin isoform with inhibitor resistance and the ability to degrade Trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesoTrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymoTrypsin C (CTRC) mitigates the harmful effects of mesoTrypsin by cleaving the autolysis loop. As human Trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesoTrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in Km , it digested β-casein poorly and bound soybean Trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesoTrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesoTrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesoTrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic Trypsin activity.

  • inactivation of mesoTrypsin by chymoTrypsin c prevents Trypsin inhibitor degradation
    Journal of Biological Chemistry, 2020
    Co-Authors: Vanda Toldi, Miklos Sahintoth, Andras Szabo
    Abstract:

    MesoTrypsin is an unusual human Trypsin isoform with inhibitor resistance and the ability to degrade Trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesoTrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymoTrypsin C (CTRC) mitigates the harmful effects of mesoTrypsin by cleaving the autolysis loop. As human Trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesoTrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in Km , it digested beta-casein poorly and bound soybean Trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesoTrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesoTrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesoTrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic Trypsin activity.

  • chymoTrypsin c caldecrin promotes degradation of human cationic Trypsin identity with rinderknecht s enzyme y
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Richard Szmola, Miklos Sahintoth
    Abstract:

    Digestive Trypsins undergo proteolytic breakdown during their transit in the human alimentary tract, which has been assumed to occur through Trypsin-mediated cleavages, termed autolysis. Autolysis was also postulated to play a protective role against pancreatitis by eliminating prematurely activated intrapancreatic Trypsin. However, autolysis of human cationic Trypsin is very slow in vitro, which is inconsistent with the documented intestinal Trypsin degradation or a putative protective role. Here we report that degradation of human cationic Trypsin is triggered by chymoTrypsin C, which selectively cleaves the Leu81-Glu82 peptide bond within the Ca2+ binding loop. Further degradation and inactivation of cationic Trypsin is then achieved through tryptic cleavage of the Arg122-Val123 peptide bond. Consequently, mutation of either Leu81 or Arg122 blocks chymoTrypsin C-mediated Trypsin degradation. Calcium affords protection against chymoTrypsin C-mediated cleavage, with complete stabilization observed at 1 mM concentration. ChymoTrypsin C is highly specific in promoting Trypsin degradation, because chymoTrypsin B1, chymoTrypsin B2, elastase 2A, elastase 3A, or elastase 3B are ineffective. ChymoTrypsin C also rapidly degrades all three human Trypsinogen isoforms and appears identical to enzyme Y, the enigmatic Trypsinogen-degrading activity described by Heinrich Rinderknecht in 1988. Taken together with previous observations, the results identify chymoTrypsin C as a key regulator of activation and degradation of cationic Trypsin. Thus, in the high Ca2+ environment of the duodenum, chymoTrypsin C facilitates Trypsinogen activation, whereas in the lower intestines, chymoTrypsin C promotes Trypsin degradation as a function of decreasing luminal Ca2+ concentrations.

D P Zhuzhikov - One of the best experts on this subject based on the ideXlab platform.

  • fractionation of digestive proteinases from tenebrio molitor coleoptera tenebrionidae larvae and role in protein digestion
    Comparative Biochemistry and Physiology B, 2006
    Co-Authors: K S Vinokurov, Brenda Oppert, Y E Dunaevsky, D P Zhuzhikov, E N Elpidina, S Prabhakar, M A Belozersky
    Abstract:

    Abstract Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of “heavy” Trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy Trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.

  • digestive proteinases of yellow mealworm tenebrio molitor larvae purification and characterization of a Trypsin like proteinase
    Biochemistry, 2005
    Co-Authors: T A Tsybina, Brenda Oppert, Y E Dunaevsky, M A Belozersky, D P Zhuzhikov, E N Elpidina
    Abstract:

    A new Trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI7.4. The enzyme was also characterized by temperature optimum at 55°C, pH optimum at 8.5, and K m value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a Trypsin-like serine proteinase stable within the pH range of 5.0–9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N- terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50–72% identity with other insect Trypsin-like proteinases, and 44–50% identity to mammalian Trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.