Purification and Characterization of Proteinases Identified as Cathepsins L and L-like (58 kDa) Proteinase from Mackerel (<i>Scomber australasicus</i>)

A new and simple method to distinguish between cathepsin B and cathepsin L in crude extracts of herring (Clupea harengus) muscle has been established. An acid treatment of crude extracts (exposed to pH 3 for 5 min) activated a latent form of cathepsin L and inactivated cathepsin B. Furthermore, in neutral crude extract, the hydrolysis of benzyloxycarbonyl-l-phenylalanyl-l-arginyl-4-methylcoumarine (Z-Phe-Arg-MCA) (cathepsin B and cathepsin L substrates) was between 0% and 15% of the hydrolysis of benzyloxycarbonyl-l-arginyl-l-arginyl-7-amino-4-methylcoumarine (Z-Arg-Arg-MCA; cathepsin B substrate). Cathepsin B activity is measured in neutral extract using the specific cathepsin B substrate Z-ArgArg-MCA and cathepsin L activity is measured in acid-treated extract with Z-Phe-Arg-MCA as substrate. The specific cathepsin B inhibitor, CA-074, did not inhibit the Z-Arg-Arg-MCA significantly without affecting the Z-Phe-Arg-MCA activity. An acid treatment of the crude extract is therefore a more advantageous approach to discriminate between cathepsin B and cathepsin L activities.

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“…, 2000). This is much higher than a pH optimum of about 5 for cathepsin L found in fish muscle (Lee et al. , 1993; Aoki et al.…”

Section: Resultsmentioning

confidence: 69%

New method to discriminate between cathepsin B and cathepsin L in crude extracts from fish muscle based on a simple acidification procedure

Godiksen

Nielsen

2006

Int J of Food Sci Tech

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“…It has also been reported that cathepsin L is highly up-regulated in haemopoietic tissues including liver, kidney and blood during bacterial and viral infection in fishes (Aranishi et al 1997;Yeh & Klesius 2009;Ahn et al 2010;Chen et al 2011), which clearly indicates the immunological role of cathepsin L in fishes. Cathepsin L in fish shows a strong proteolytic activity for several proteins including myofibrillar proteins in muscles of Oncorhynchus keta (Yamashita & Konagaya 1990), Scomber japonicas (Lee et al 1993), Engraulis japonica and Atheresthes stomias (Visessanguan et al 2003) suggesting its participation in intracellular and extracellular protein catabolism in fish (Aranishi et al 1997). Cathepsin L is present in fish mucus and it is reported to produce antimicrobial peptides during infection (Lee et al 1993).…”

Section: Introductionmentioning

confidence: 99%

“…Cathepsin L in fish shows a strong proteolytic activity for several proteins including myofibrillar proteins in muscles of Oncorhynchus keta (Yamashita & Konagaya 1990), Scomber japonicas (Lee et al 1993), Engraulis japonica and Atheresthes stomias (Visessanguan et al 2003) suggesting its participation in intracellular and extracellular protein catabolism in fish (Aranishi et al 1997). Cathepsin L is present in fish mucus and it is reported to produce antimicrobial peptides during infection (Lee et al 1993).…”

Section: Introductionmentioning

confidence: 99%

A murrel cysteine protease, cathepsin L: bioinformatics characterization, gene expression and proteolytic activity

et al. 2014

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Cathepsin L, a lysosomal endopeptidase, is a member of the peptidase C1 family (papain-like family) of cysteine proteinases that cleave peptide bonds of lysosomal proteins. In this study, we report a cathepsin L sequence identified from the constructed cDNA library of striped murrel Channa striatus (designated as CsCath L) using genome sequencing FLX TM technology. The full-length CsCath L contains three eukaryotic thiol protease domains at positions 134-145, 278-288 and 299-318. Phylogenetic analysis revealed that the CsCath L was clustered together with other cathepsin L from teleosts. The three-dimensional structure of CsCath L modelled by the I-Tasser program was compared with structures deposited in the Protein Data Bank to find out the structural similarity of CsCath L with experimentally identified structures. The results showed that the CsCath L exhibits maximum structural identity with pro-cathepsin L from human. The RNA fold structure of CsCath L was predicted along with its minimum free energy (-471.93 kcal/mol). The highest CsCath L gene expression was observed in liver, which was also significantly higher (P < 0.05) than that detected in other tissues taken for analysis. In order to investigate the mRNA transcription profile of CsCath L during infection, C. striatus were injected with fungus (Aphanomyces invadans) and bacteria (Aeromonas hydrophila) and its expression was up-regulated in liver at various time points. Similar to gene expression studies, the highest CsCath L enzyme activity was also observed in liver and its activity was up-regulated by fungal and bacterial infections.

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“…Cathepsin L is capable of hydrolyzing a broad range of proteins including myosin, actin, nebulin, cytosolic proteins, collagen, and elastin [18]. Cathepsin L is a major proteinase that degrades myofibrillar proteins in antemortem or postmortem muscle of chum salmon (Oncorhynchus keta) [19], spotted mackerel (Scomber japonicus) [20], and Pacific whiting (Merluccius productus) [21].…”

Section: Introductionmentioning

confidence: 99%

Molecular Cloning, Expression, and Characterization of Cathepsin L from Mud Loach (Misgurnus mizolepis)

Ahn

Sung

Kim

et al. 2010

Appl Biochem Biotechnol

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Cathepsin L is an important protease in the initiation of protein degradation and one of the most powerful endopeptidases. In this study, we cloned mud loach (Misgurnus mizolepis) cathepsin L (MlCtL) cDNA, and the pro-mature enzyme of MlCtL (proMlCtL) was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in a pGEX-4 T-1 vector. The recombinant proMlCtL was overexpressed in E. coli DH5αMCR as a 62-kDa protein. Its activity was quantified by measuring the cleavage of synthetic fluorogenic peptide substrates, and the protease activity of proMlCtL was also demonstrated by gelatin zymography. Antipain and leupeptin were shown to inhibit the protease activity of proMlCtL. Our results suggest that the structural features and evolutionary relationship of the mud loach cathepsin L gene were similar to that of the other mammalian cathepsin Ls; however, the proMlCtL protein was more stable at neutral and alkaline pH. The optimum temperature for the proMlCtL enzyme was found to be 40 °C. In addition, proMlCtL activity was dependent upon the presence of several metal ions and detergents.

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Purification and Characterization of Proteinases Identified as Cathepsins L and L-like (58 kDa) Proteinase from Mackerel (Scomber australasicus)

Cited by 51 publications

References 8 publications

New method to discriminate between cathepsin B and cathepsin L in crude extracts from fish muscle based on a simple acidification procedure

New method to discriminate between cathepsin B and cathepsin L in crude extracts from fish muscle based on a simple acidification procedure

A murrel cysteine protease, cathepsin L: bioinformatics characterization, gene expression and proteolytic activity

Molecular Cloning, Expression, and Characterization of Cathepsin L from Mud Loach (Misgurnus mizolepis)

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