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Purification and Characterization of Cathepsin B from the Muscle of Horse Mackerel Trachurus japonicus.

Yoshida A, Ohta M, Kuwahara K, Cao MJ, Hara K, Osatomi K - Mar Drugs (2015)

Bottom Line: The active sites and an N-glycosylation site were conserved across species.We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B.Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852-8521, Japan. y-asami@nagasaki-u.ac.jp.

ABSTRACT
An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s), such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.

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Related in: MedlinePlus

Amino acid sequence alignment of horse mackerel and other cathepsin Bs. Identical residues with horse mackerel cathepsin B are shaded in gray. The active site residues of cathepsin B (Cys, His, Asn) are shaded in black with white letters. Asterisks indicate predicted N-glycosylation site. The cleavage site between light and heavy chains in other species is indicated by a black vertical line. Black arrowhead indicates N-terminal of heavy chain of cathepsin B from horse mackerel. Cathepsin B from common carp (GenBank accession number: AB215097.1), zebrafish (NP_998501.1), sea bream (AHZ34284.1), human (AAH95408.1), and bovine (NP_776456.1).
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marinedrugs-13-06550-f006: Amino acid sequence alignment of horse mackerel and other cathepsin Bs. Identical residues with horse mackerel cathepsin B are shaded in gray. The active site residues of cathepsin B (Cys, His, Asn) are shaded in black with white letters. Asterisks indicate predicted N-glycosylation site. The cleavage site between light and heavy chains in other species is indicated by a black vertical line. Black arrowhead indicates N-terminal of heavy chain of cathepsin B from horse mackerel. Cathepsin B from common carp (GenBank accession number: AB215097.1), zebrafish (NP_998501.1), sea bream (AHZ34284.1), human (AAH95408.1), and bovine (NP_776456.1).

Mentions: A sequence homology search of the deduced amino acid sequence of horse mackerel cathepsin B was performed on the NCBI Protein database with BLASTP and the multiple sequence alignment of the enzyme and cathepsin Bs from other species was shown in Figure 6. The deduced amino acid sequence was compared with cathepsin B from common carp (GenBank accession no.: AB215097.1), zebrafish (NP_998501.1), sea bream (AHZ34284.1), human (AAH95408.1), and bovine (NP_776456.1), which shared 84.2%, 83.3%, 86.0%, 75.8%, and 76.7% identities, respectively. The active sites and an N-glycosylation site of horse mackerel cathepsin B were conserved in cathepsin Bs from other species.


Purification and Characterization of Cathepsin B from the Muscle of Horse Mackerel Trachurus japonicus.

Yoshida A, Ohta M, Kuwahara K, Cao MJ, Hara K, Osatomi K - Mar Drugs (2015)

Amino acid sequence alignment of horse mackerel and other cathepsin Bs. Identical residues with horse mackerel cathepsin B are shaded in gray. The active site residues of cathepsin B (Cys, His, Asn) are shaded in black with white letters. Asterisks indicate predicted N-glycosylation site. The cleavage site between light and heavy chains in other species is indicated by a black vertical line. Black arrowhead indicates N-terminal of heavy chain of cathepsin B from horse mackerel. Cathepsin B from common carp (GenBank accession number: AB215097.1), zebrafish (NP_998501.1), sea bream (AHZ34284.1), human (AAH95408.1), and bovine (NP_776456.1).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663541&req=5

marinedrugs-13-06550-f006: Amino acid sequence alignment of horse mackerel and other cathepsin Bs. Identical residues with horse mackerel cathepsin B are shaded in gray. The active site residues of cathepsin B (Cys, His, Asn) are shaded in black with white letters. Asterisks indicate predicted N-glycosylation site. The cleavage site between light and heavy chains in other species is indicated by a black vertical line. Black arrowhead indicates N-terminal of heavy chain of cathepsin B from horse mackerel. Cathepsin B from common carp (GenBank accession number: AB215097.1), zebrafish (NP_998501.1), sea bream (AHZ34284.1), human (AAH95408.1), and bovine (NP_776456.1).
Mentions: A sequence homology search of the deduced amino acid sequence of horse mackerel cathepsin B was performed on the NCBI Protein database with BLASTP and the multiple sequence alignment of the enzyme and cathepsin Bs from other species was shown in Figure 6. The deduced amino acid sequence was compared with cathepsin B from common carp (GenBank accession no.: AB215097.1), zebrafish (NP_998501.1), sea bream (AHZ34284.1), human (AAH95408.1), and bovine (NP_776456.1), which shared 84.2%, 83.3%, 86.0%, 75.8%, and 76.7% identities, respectively. The active sites and an N-glycosylation site of horse mackerel cathepsin B were conserved in cathepsin Bs from other species.

Bottom Line: The active sites and an N-glycosylation site were conserved across species.We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B.Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852-8521, Japan. y-asami@nagasaki-u.ac.jp.

ABSTRACT
An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s), such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.

Show MeSH
Related in: MedlinePlus