Characteristics of carboxypeptidase B from pyloric ceca of the starfish Asterina pectinifera

Abstract

Carboxypeptidase B was purified from the pyloric ceca of the starfish Asterina pectinifera. The final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 34,000. The value of the specificity constant (kcat/Km) for hydrolysis of benzoyl-glycyl-L-arginine by the purified enzyme was 1.72×10 5 M -1 sec -1 . The optimal pH and the optimal temperature of the enzyme were pH 7.5 and 55 ℃, respectively. The enzyme was unstable above 50 ℃ and below pH 5.0. The enzyme was activated by Co 2+ , and inhibited by EDTA. The N-terminal amino acid sequence of the enzyme was determined as ATFDYNKYHSYQEIMDWVTN.

Isolation and Characteristics of Carboxypeptidase B from Zebra Blenny (Salaria basilisca) Viscera

“…The procedure applied in the purification of CPB from zebra blenny is more efficient than that applied in the purification of CPB from ostrich S. camelus (Bradley et al, 1996) which yielded 3.4% after two steps of purification. However, it was similar to that applied in the purification of CPB from camel (Al-Ajlan and Bailey, 1999) and starfish Asterina pectinifera (Kishimura et al, 2006), which yielded 29.3 and 23% after four and three steps of purification, respectively. The purified enzyme gave a single band on SDS-PAGE, and its molecular weight was estimated to be 34.5 kDa (Figure 2), corresponding to that determined by gel filtration.…”

“…The enzyme is highly stable over a broad pH range, maintaining over 100% of its original activity between pH 6.0 and 10.0, after incubation for 30 min at 25 • C, and more than 95 and 85% of its activity was retained at pH 6.0 and 11.0, respectively. The pH stability profile of S. basilisca CPB was similar to those of the starfish A. pectinifera (Kishimura et al, 2006) and A. amurensis (Kishimura and Hayashi, 2002).…”

“…The purified enzyme gave a single band on SDS-PAGE, and its molecular weight was estimated to be 34.5 kDa (Figure 2), corresponding to that determined by gel filtration. The molecular weight of the S. basilisca CPB was similar to those from human (Marinkovic et al, 1977), starfish A. pectinifera (Kishimura et al, 2006), and starfish Asterias amurensis (Kishimura and Hayashi, 2002); and was higher than those of CPBs from camel (Al-Ajlan and Bailey, 1999), dogfish S. canicula (Hajjou et al, 1995), white shrimp P. setiferus (Gates and Travis, 1973), and cod (Overnell, 1973); and was lower than that of ostrich S. camelus CPB (Bradley et al, 1996), which had a molecular weight of about 35 kDa.…”

Characterization of protease activities in a crude extract of germinated cacao

“…However, in the presence of CoCl 2 at 1 mM, APE activity became stabilized, with 1.0 and 1.2 U/mg protein at 60 and 80°C, respectively (Figure 4b). Increases of 1.7-to 50-fold the original activity of CP (Cheng, Ramakrishnan & Chan, 1999;Kishimura, Hayashi & Ando, 2006) and of APE enzymes (Bolumar, Sanz, Aristoy, & Toldrá, 2003;Dong et al, 2005), activated by cobalt, were attained using 4 µM to 1 mM CoCl 2 . Nevertheless, inhibition of APE by Co 2+ at 10 mM has also been detected (MercadoFlores et al, 2004;Mohamed et al, 2009).…”

Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber Stichopus japonicus

“…Studies on starfish proteases were initiated by Camacho et al (1970) who reported that the purified trypsin-like proteases from the pyloric caeca of the starfish Dermasterias imbricata were capable of hydrolyzing fibrin under alkaline conditions. Kishimura and Hayashi (2002a) and Kishimura et al (2006a) isolated the trypsin from the pyloric caeca of the starfish Asterina pectinifera and carboxypeptidase B from A. amurensis (Kishimura and Hayashi 2002b) and subsequently examined the characteristics and N-terminal amino acid sequences of the two enzymes. The proteases from the digestive tract of the sea cucumber Stichopus japonicus have been purified and characterized as alkaline proteases with optimal activities at pH 8.0 and 13.5.…”