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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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Chromatographic patterns for purification of cathepsin B from horse mackerel meat. (A) SP-Sepharose cation-exchange chromatography. The crude enzyme (6732 mg protein) was applied to a column (2.64 × 100 cm), equilibrated with 50 mM acetate buffer (pH 4.5) containing 2 mM 2-mercaptoethanol, and eluted with a linear gradient of NaCl at the concentration of 0 to 1 M in the same buffer; (B) Superdex 75 gel filtration chromatography and the effect of CA-074 on the activities of pool A and B from Superdex 75. The enzyme solution (39 mg protein) was applied to a column (320 mL), equilibrated with 50 mM acetate buffer (pH 4.5), containing 2 mM 2-mercaptoethanol and 0.15 M NaCl.
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marinedrugs-13-06550-f001: Chromatographic patterns for purification of cathepsin B from horse mackerel meat. (A) SP-Sepharose cation-exchange chromatography. The crude enzyme (6732 mg protein) was applied to a column (2.64 × 100 cm), equilibrated with 50 mM acetate buffer (pH 4.5) containing 2 mM 2-mercaptoethanol, and eluted with a linear gradient of NaCl at the concentration of 0 to 1 M in the same buffer; (B) Superdex 75 gel filtration chromatography and the effect of CA-074 on the activities of pool A and B from Superdex 75. The enzyme solution (39 mg protein) was applied to a column (320 mL), equilibrated with 50 mM acetate buffer (pH 4.5), containing 2 mM 2-mercaptoethanol and 0.15 M NaCl.

Mentions: Cathepsin B was partially purified 3132-fold from 1.2 kg of horse mackerel meat with a yield of 1.7% (Table 1) using ammonium sulfate fractionation, cation-exchange chromatography, and gel filtration. The chromatographic profile on SP-Sepharose column is shown in Figure 1A. The active peak was eluted with a linear gradient of 0–0.6 M NaCl and could be separated from most of contaminating proteins. The active fractions were pooled and concentrated by ultrafiltration using YM-10 membrane. And then, the concentrated enzyme was applied to Superdex 75 gel filtration column. As shown in Figure 1B, Z-Arg-Arg-MCA hydrolyzing peak and Z-Phe-Arg-MCA hydrolyzing peaks were separately eluted on the gel filtration column, and two peaks were pooled as “pool A” and “pool B”, respectively. To determine which of the pools was suitable as cathepsin B fraction, the effects of cathepsin B specific inhibitors to both pools were investigated (Figure 1B). The activities of both pools were inhibited by E-64, cysteine protease inhibitor. On the other hands, CA-074, cathepsin B specific inhibitor, only suppressed the activity of pool B but not that of pool A. Hence, we decided that pool B was the cathepsin B fraction, and it was used for further purification. We tried to purify the protease from pool A separately; however, we could not identify it because of the decrease its activity due to freeze and thaw cycles.


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)

Chromatographic patterns for purification of cathepsin B from horse mackerel meat. (A) SP-Sepharose cation-exchange chromatography. The crude enzyme (6732 mg protein) was applied to a column (2.64 × 100 cm), equilibrated with 50 mM acetate buffer (pH 4.5) containing 2 mM 2-mercaptoethanol, and eluted with a linear gradient of NaCl at the concentration of 0 to 1 M in the same buffer; (B) Superdex 75 gel filtration chromatography and the effect of CA-074 on the activities of pool A and B from Superdex 75. The enzyme solution (39 mg protein) was applied to a column (320 mL), equilibrated with 50 mM acetate buffer (pH 4.5), containing 2 mM 2-mercaptoethanol and 0.15 M NaCl.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06550-f001: Chromatographic patterns for purification of cathepsin B from horse mackerel meat. (A) SP-Sepharose cation-exchange chromatography. The crude enzyme (6732 mg protein) was applied to a column (2.64 × 100 cm), equilibrated with 50 mM acetate buffer (pH 4.5) containing 2 mM 2-mercaptoethanol, and eluted with a linear gradient of NaCl at the concentration of 0 to 1 M in the same buffer; (B) Superdex 75 gel filtration chromatography and the effect of CA-074 on the activities of pool A and B from Superdex 75. The enzyme solution (39 mg protein) was applied to a column (320 mL), equilibrated with 50 mM acetate buffer (pH 4.5), containing 2 mM 2-mercaptoethanol and 0.15 M NaCl.
Mentions: Cathepsin B was partially purified 3132-fold from 1.2 kg of horse mackerel meat with a yield of 1.7% (Table 1) using ammonium sulfate fractionation, cation-exchange chromatography, and gel filtration. The chromatographic profile on SP-Sepharose column is shown in Figure 1A. The active peak was eluted with a linear gradient of 0–0.6 M NaCl and could be separated from most of contaminating proteins. The active fractions were pooled and concentrated by ultrafiltration using YM-10 membrane. And then, the concentrated enzyme was applied to Superdex 75 gel filtration column. As shown in Figure 1B, Z-Arg-Arg-MCA hydrolyzing peak and Z-Phe-Arg-MCA hydrolyzing peaks were separately eluted on the gel filtration column, and two peaks were pooled as “pool A” and “pool B”, respectively. To determine which of the pools was suitable as cathepsin B fraction, the effects of cathepsin B specific inhibitors to both pools were investigated (Figure 1B). The activities of both pools were inhibited by E-64, cysteine protease inhibitor. On the other hands, CA-074, cathepsin B specific inhibitor, only suppressed the activity of pool B but not that of pool A. Hence, we decided that pool B was the cathepsin B fraction, and it was used for further purification. We tried to purify the protease from pool A separately; however, we could not identify it because of the decrease its activity due to freeze and thaw cycles.

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