Limits...
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.

Show MeSH

Related in: MedlinePlus

Nucleotide and deduced amino acid sequences of horse mackerel cathepsin B cDNA (GenBank accession number: LC093263). The N-terminal amino acid sequence of purified cathepsin B is underlined. The active site residues of cathepsin B are boxed. Asterisk indicates predicted N-glycosylation site. The cleavage site between light and heavy chains is indicated by an arrowhead.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663541&req=5

marinedrugs-13-06550-f005: Nucleotide and deduced amino acid sequences of horse mackerel cathepsin B cDNA (GenBank accession number: LC093263). The N-terminal amino acid sequence of purified cathepsin B is underlined. The active site residues of cathepsin B are boxed. Asterisk indicates predicted N-glycosylation site. The cleavage site between light and heavy chains is indicated by an arrowhead.

Mentions: We cloned a partial cDNA corresponding to cathepsin B from the horse mackerel muscle by RT-PCR. The nucleotide and deduced amino acid sequences of cathepsin B cDNA (647 nucleotides) are shown in Figure 5. The sequence consisted of a 647 bp nucleotide sequence encoding 215 amino acids. The cleavage site between the light and heavy chains of cathepsin B was confirmed by N-terminal amino acid sequence of the purified cathepsin B from horse makerel muscle (black arrowhead in Figure 5). This result shows slightly difference in other species, suggesting that there may be a variation in the enzyme responsible for cleaving between light and heavy chains.


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)

Nucleotide and deduced amino acid sequences of horse mackerel cathepsin B cDNA (GenBank accession number: LC093263). The N-terminal amino acid sequence of purified cathepsin B is underlined. The active site residues of cathepsin B are boxed. Asterisk indicates predicted N-glycosylation site. The cleavage site between light and heavy chains is indicated by an arrowhead.
© Copyright Policy
Related In: Results  -  Collection

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

marinedrugs-13-06550-f005: Nucleotide and deduced amino acid sequences of horse mackerel cathepsin B cDNA (GenBank accession number: LC093263). The N-terminal amino acid sequence of purified cathepsin B is underlined. The active site residues of cathepsin B are boxed. Asterisk indicates predicted N-glycosylation site. The cleavage site between light and heavy chains is indicated by an arrowhead.
Mentions: We cloned a partial cDNA corresponding to cathepsin B from the horse mackerel muscle by RT-PCR. The nucleotide and deduced amino acid sequences of cathepsin B cDNA (647 nucleotides) are shown in Figure 5. The sequence consisted of a 647 bp nucleotide sequence encoding 215 amino acids. The cleavage site between the light and heavy chains of cathepsin B was confirmed by N-terminal amino acid sequence of the purified cathepsin B from horse makerel muscle (black arrowhead in Figure 5). This result shows slightly difference in other species, suggesting that there may be a variation in the enzyme responsible for cleaving between light and heavy chains.

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