Purification and characterization of a Cys-Gly hydrolase from the gastropod mollusk, Patella caerulea

Figures & data

Figure 1. Gel chromatography on Sephacryl S-300 column. See materials and methods for details. Inset: SDS/PAGE analysis of Cys-Gly hydrolase activity purified from P. caerulea.

Table 1. Purification of P. caerulea Cys-Gly hydrolase.

Fraction	Total volume (mL)	Total activity (UE)	Specific activity (UE/mg)	Recovery %	Purification
Extract	10.2	6.1	0.11
DE-52 Batch	4.8	3.3	0.14	54	1.3
Chloroform precipitation	3.5	1.0	3.2	16	29
Sephacryl S-300	13.5	0.8	15.5	13	140

Figure 2. Effect of EDTA and bestatin on Cys-Gly hydrolase activity. Panel A: Purified enzyme was incubated both in the absence and in the presence of different concentrations of EDTA ranging from 0.25 to 10 mM. Panel B: Purified enzyme was incubated both in the absence and in the presence of different concentrations of bestatin ranging from 1 nm to 20 mM. Data are reported as percentage residual activity.

Table 2. Kinetic parameters of P. caerulea Cys-Gly hydrolase activity.

pH	KM (mM)	kcat (min^−1)	kcat/KM (mM^−1 min^−1)
6.5	0.30 ± 0.11	1300 ± 150	4333 ± 1718
7.0	0.38 ± 0.12	1800 ± 250	4737 ± 1669
7.5	0.33 ± 0.11	2300 ± 300	6972 ± 2557
8.0	0.40 ± 0.12	2850 ± 300	7130 ± 2312
8.5	0.42 ± 0.06	3100 ± 150	7380 ± 1120

Kinetic parameters, determined at 30 °C with Cys-Gly as substrate, are expressed as measured value ± SEM.

Figure 3. Multiple sequence alignment relative to the six aminopeptidase identified by MS/MS.