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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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Effects of pH (A) and temperature (B) on the activity of the purified cathepsin B. (A) The activities of the purified cathepsin B were measured with Z-Phe-Arg-MCA at 50 °C using different buffers (pH 1.5~5.0, 0.2 M HCl-CH3COOH buffer; pH 5.0~6.0, 0.2 M CH3COOH-CH3COONa buffer; pH 6.0~8.0, 0.2 M KH2PO4-Na2HPO4 buffer; pH 8.0~10.0, 0.2 M boric acid + KCl-Na2CO3 buffer); and (B) the activities of the purified cathepsin B were measured at various temperatures at pH 5.0 using Z-Phe-Arg-MCA.
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marinedrugs-13-06550-f004: Effects of pH (A) and temperature (B) on the activity of the purified cathepsin B. (A) The activities of the purified cathepsin B were measured with Z-Phe-Arg-MCA at 50 °C using different buffers (pH 1.5~5.0, 0.2 M HCl-CH3COOH buffer; pH 5.0~6.0, 0.2 M CH3COOH-CH3COONa buffer; pH 6.0~8.0, 0.2 M KH2PO4-Na2HPO4 buffer; pH 8.0~10.0, 0.2 M boric acid + KCl-Na2CO3 buffer); and (B) the activities of the purified cathepsin B were measured at various temperatures at pH 5.0 using Z-Phe-Arg-MCA.

Mentions: Horse mackerel cathepsin B was examined the effects of pH and temperature using Z-Phe-Arg-MCA as a substrate (Figure 4). Optimum pH of the enzyme was 5.0 and the activity remained about 60% at pH 6.0 which was the similar pH as the minced meat of horse mackerel. Optimum temperature of the enzyme was 50 °C and the activity remained at least 40% at 60 °C, namely a temperature induced modori phenomenon on horse mackerel meat. These results supported our hypothesis that cathepsin B was involved in the deterioration of surimi-based products of the horse mackerel meat. While, in common mackerel [14] and blue scad [23], it has been reported that cathepsin L mainly participate in gel-weakening. Thus, the types of endogenous protease responsible for modori phenomenon vary depending on fish 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)

Effects of pH (A) and temperature (B) on the activity of the purified cathepsin B. (A) The activities of the purified cathepsin B were measured with Z-Phe-Arg-MCA at 50 °C using different buffers (pH 1.5~5.0, 0.2 M HCl-CH3COOH buffer; pH 5.0~6.0, 0.2 M CH3COOH-CH3COONa buffer; pH 6.0~8.0, 0.2 M KH2PO4-Na2HPO4 buffer; pH 8.0~10.0, 0.2 M boric acid + KCl-Na2CO3 buffer); and (B) the activities of the purified cathepsin B were measured at various temperatures at pH 5.0 using Z-Phe-Arg-MCA.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06550-f004: Effects of pH (A) and temperature (B) on the activity of the purified cathepsin B. (A) The activities of the purified cathepsin B were measured with Z-Phe-Arg-MCA at 50 °C using different buffers (pH 1.5~5.0, 0.2 M HCl-CH3COOH buffer; pH 5.0~6.0, 0.2 M CH3COOH-CH3COONa buffer; pH 6.0~8.0, 0.2 M KH2PO4-Na2HPO4 buffer; pH 8.0~10.0, 0.2 M boric acid + KCl-Na2CO3 buffer); and (B) the activities of the purified cathepsin B were measured at various temperatures at pH 5.0 using Z-Phe-Arg-MCA.
Mentions: Horse mackerel cathepsin B was examined the effects of pH and temperature using Z-Phe-Arg-MCA as a substrate (Figure 4). Optimum pH of the enzyme was 5.0 and the activity remained about 60% at pH 6.0 which was the similar pH as the minced meat of horse mackerel. Optimum temperature of the enzyme was 50 °C and the activity remained at least 40% at 60 °C, namely a temperature induced modori phenomenon on horse mackerel meat. These results supported our hypothesis that cathepsin B was involved in the deterioration of surimi-based products of the horse mackerel meat. While, in common mackerel [14] and blue scad [23], it has been reported that cathepsin L mainly participate in gel-weakening. Thus, the types of endogenous protease responsible for modori phenomenon vary depending on fish 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