Characterization of a Main Extracellular Matrix Autoenzyme from the Dermis of Sea Cucumber Stichopus monotuberculatus: Collagenase

Figures & data

FIGURE 1 Light microscopy of the dermis from S. monotuberculatus stained with Van Gieson staining; a: outside edge of dermis; b: inner section of dermis; and c: inside edge of dermis. Scale bar = 50 um.

FIGURE 2 SDS-PAGE pattern of CCF and PSC from S. monotuberculatu.

Lane M: protein marker; Lane 1: CCF; Lane 2: PSC.

FIGURE 3 Zymograph for the analyses of autoenzyme activity on PSC.

Lane M, protein marker; lane 1: control (without inhibitor); lane 2: 1,10-phenathroline (10 mM); lane 3: EDTA (10 mM); lane 4: PMSF (1 mM); lane 5: TI (0.1 g/l); lane 6: Benzamidine (10 mM); lane 7: DTNB (10 mM); lane 8: Iodoacetic acid (10 mM); lane 9: E-64 (0.01 mM); lane 10: Leupeptin (0.1 mM); lane 11: Pepstatin A (0.1 mM); Lane M’: protein marker before staining.

FIGURE 4 SDS-PAGE of PSC digested by autoenzyme.

Lane M, protein marker; lane 1: control (without inhibitor); lane 2: 1,10-phenathroline (10 mM); lane 3: EDTA (10 mM); lane 4: PMSF (1 mM); lane 5: TI (0.1 g/l); lane 6: Benzamidine (10 mM); lane 7: DTNB (10 mM); lane 8: Iodoacetic acid (10 mM); lane 9: E-64 (0.01 mM); lane 10: Leupeptin (0.1 mM); lane 11: Pepstatin A (0.1 mM).

FIGURE 5 SDS-PAGE of CCF digested by autoenzyme.

Lane M, protein marker; Lane 1: PSC; lane 2: CCF; lane 3: crude enzyme extract; lane 4: CCF digested by autoenzyme; lane 5: CCF digested by autoenzyme with addition of 1,10-phenathroline (10 mM).

FIGURE 6 Effect of 1,10-phenanthroline on the soluble protein content during autolysis.

Control: absence of 1,10-phenanthroline; 1,10-phenanthroline: presence of 1,10-phenanthroline.

FIGURE 7 Collagenase activity of S. monotuberculatus with different temperatures.

FIGURE 8 Collagenase activity of S. monotuberculatus with different pHs.

TABLE 1 Effect of metal ions on collagenase activity

Supplement	Enzymatic hydrolyzation (%)
None	79.61 ± 1.46^bc
Ca^2+	80.27 ± 5.00^bcd
Cu^2+	64.13 ± 7.93^a
Mg^2+	84.10 ± 2.12^cde
Zn^2+	88.28 ± 3.74^de
Hg^2+	75.42 ± 3.43^b
Mn^2+	91.62 ± 3.50^e

Data are expressed as means ± SD of three determinations, and values followed by different superscript letters are significantly different (p < 0.05).

TABLE 2 Effect of metal ions on activity of EDTA-inhibited collagenase

Supplement	Enzymatic hydrolyzation (%)
EDTA	48.36 ± 5.36^ab
EDTA +Ca^2+	47.73 ± 4.84^ab
EDTA +Mg^2+	51.15 ± 5.83^b
EDTA +Zn^2+	39.27 ± 5.12^a
EDTA +Mn^2+	67.54 ± 4.67^c

Data are expressed as means ± SD of three determinations, and values followed by different superscript letters are significantly different (p < 0.05).