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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 cysteine protease inhibitors on modori gel prepared from horse mackerel meat. 1, one-step heated gel at 90 °C for 20 min; 2, modori gel without protease inhibitor; 3, modori gel with 0.01 mM E-64; and 4, modori gel with 0.01 mM CA-074.
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marinedrugs-13-06550-f007: Effects of cysteine protease inhibitors on modori gel prepared from horse mackerel meat. 1, one-step heated gel at 90 °C for 20 min; 2, modori gel without protease inhibitor; 3, modori gel with 0.01 mM E-64; and 4, modori gel with 0.01 mM CA-074.

Mentions: We prepared modori gel of horse mackerel meat with or without cathepsin B inhibitors (Figure 7). Breaking force of modori gel was significantly lower than that of control gel. A cysteine protease inhibitor, E-64, and a cathepsin B specific inhibitor, CA-074, dramatically improved breaking force of modori gels. Therefore, it was suggested that cathepsin B caused the modori phenomenon in horse mackerel thermal gel. Since we confirmed that cathepsin B was the target endogenous protease to avoid disintegration of horse mackerel gel, it is possible to use oryzacystatin as a food additive upon thermal gel processing. Oryzacystatin is a natural cysteine protease inhibitor from rice [25] and is studied about its utilization as a food additive on thermal gel of walleye pollock [26]. The effect of oryzacystatin on modori gel of horse mackerel should be clarified for the further study. On the other hand, the endogenous proteases which have high activity and thermal stability, can be used for soft food processing without chemical additive. In this case, the autolytic activity of cathepsin B in horse mackerel muscle has a possibility for application to make soft foods, such as weaning meal and food for the elderly.


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 cysteine protease inhibitors on modori gel prepared from horse mackerel meat. 1, one-step heated gel at 90 °C for 20 min; 2, modori gel without protease inhibitor; 3, modori gel with 0.01 mM E-64; and 4, modori gel with 0.01 mM CA-074.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06550-f007: Effects of cysteine protease inhibitors on modori gel prepared from horse mackerel meat. 1, one-step heated gel at 90 °C for 20 min; 2, modori gel without protease inhibitor; 3, modori gel with 0.01 mM E-64; and 4, modori gel with 0.01 mM CA-074.
Mentions: We prepared modori gel of horse mackerel meat with or without cathepsin B inhibitors (Figure 7). Breaking force of modori gel was significantly lower than that of control gel. A cysteine protease inhibitor, E-64, and a cathepsin B specific inhibitor, CA-074, dramatically improved breaking force of modori gels. Therefore, it was suggested that cathepsin B caused the modori phenomenon in horse mackerel thermal gel. Since we confirmed that cathepsin B was the target endogenous protease to avoid disintegration of horse mackerel gel, it is possible to use oryzacystatin as a food additive upon thermal gel processing. Oryzacystatin is a natural cysteine protease inhibitor from rice [25] and is studied about its utilization as a food additive on thermal gel of walleye pollock [26]. The effect of oryzacystatin on modori gel of horse mackerel should be clarified for the further study. On the other hand, the endogenous proteases which have high activity and thermal stability, can be used for soft food processing without chemical additive. In this case, the autolytic activity of cathepsin B in horse mackerel muscle has a possibility for application to make soft foods, such as weaning meal and food for the elderly.

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