Functional characterization and specific effects of various peptides on enzymatic activity of a DPP-III homologue from goat brain

Figures & data

Figure 1 Effect of thiol compounds on (a) untreated and (b) DTNB- pretreated DPP-III.

Figure 2 Effect of different divalent metal ions on (a) untreated and (b) o-phenanthroline pretreated DPP-III.

Table I.  Effect of metal ions in combination with other metal ions and EDTA on the activity of DPP-III.

Inhibitor	% activity
Control (No inhibitor)	100
1 mM EDTA +0.1 mM ZnCl2	50.3
0.1 mM ZnCl2	46.8
1 mM EDTA	49.2
0.1 mM CoCl2	174.4
0.1 mM NiCl2	12.4
0.01 mM ZnCl2	62.6
0.1 mM CoCl2+0.01 mM ZnCl2	107.7
0.1 mM CoCl2+0.1 mM ZnCl2	54.5
0.1 mM NiCl2+0.1 mM CoCl2	53.6
0.1 mM HgCl2	11.1
0.1 mM KCl	82.0
0.1 mM LiCl	73.0

Figure 3 Effect of DMSO and ethanol on DPP-III activity.

Table II.  Stability of DPP-III under storage conditions.

Time (in months)	% residual activity in the presence (or absence) of glycerol
Control	100
After 1 month	73.5 (61.85)
After 2 months	59 (21.97)
After 3 months	50 (13.32)
After 4 months	40.3 (7.01)
After 5 months	18 (1.72)
After 6 months	8 (0.25)

The purified DPP-III was stored in 50 mM Tris-HCl buffer pH 7.5 at − 20°C in the presence of 10% glycerol. The table shows % relative activity compared with the activity at the time of purification as 100. Values in parentheses show the activity in the absence of glycerol.

Figure 4 Effect of urea on DPP-III activity.

Table III.  Effect of different dipeptides on the activity of goat brain DPP-III.

Dipeptide	Concentration (mM)	% inhibition
Phe-Met	0.5	78
	1.0	85
Pro-Arg-OH	0.5	41
	1.0	54
His-Lys	0.5	36
	1.0	41
Ser-Met	0.5	32
	1.0	32
Pro-Phe	0.5	40
	1.0	56
Pro-Glu	0.5	35
	1.0	34
Leu-Trp	0.5	85
	1.0	90
Leu-Arg	0.5	100
	1.0	100
Asp-Ala	0.5	25
	1.0	25
Phe-Leu	0.5	40
	1.0	58
His-Leu	0.5	57
	1.0	76
Arg-Phe	0.5	46
	1.0	56
Trp-Met	0.5	76
	1.0	94
His-Phe	0.5	42
	1.0	46
Arg-Val	0.5	33
	1.0	49
Gly-Glu	0.5	39
	1.0	36
Phe-Ala	0.5	30
	1.0	41
Met-Arg	0.5	95
	1.0	99
Ile-His	0.5	37
	1.0	34
Lys-Ala	0.5	20
	1.0	23
Val-Tyr	0.5	63
	1.0	84
Tyr-Gly	0.5	35
	1.0	31
Tyr-Ile	0.5	60
	1.0	72
His-Pro	0.5	18
	1.0	16
Gly-Phe	0.5	32
	1.0	46
Phe-Arg	0.5	68
	1.0	75
Arg-Trp	0.5	40
	1.0	46

The enzyme was incubated at 37°C in the standard reaction mixture with the substrates at a final concentration of 0.15 mM. The relative activity was calculated in relation to the most hydrolyzed substrate, Arg-Arg-4mβNA (100%).

Table IV.  The effect of different tripeptides/tetrapeptides/oligopeptides on the activity of goat brain DPP-III.

Peptide	Concentration (mM)	% inhibition
Arg-Phe-Ala.	0.5	93
	1.0	99
Leu-Trp-Met-Arg-Phe-Ala	0.5	99
	1.0	100
Leu-Trp-Met	0.5	90
	1.0	92
Ala-Ala-Ala-Ala	0.5	58
	1.0	49
Tyr-Gly-Gly-Phe	0.5	40
	1.0	53
Trp-Met-Asp-Phe	0.5	80
	1.0	82
Ala-Ala-Ala	0.5	No inhibition
	1.0	4
Gly-Phe-Leu	0.5	79
	1.0	82
Tyr-Gly-Gly-Phe-Leu
(Leu-enkephlin)	0.5	56
	1.0	92
Tyr-Gly-Gly-Phe-Met
(Met-enkephlin)	0.5	61

The enzyme was incubated at 37°C in the standard reaction mixture with the substrates at final concentration of 0.15 mM. The relative activity was calculated in relation to the most hydrolyzed substrate, Arg-Arg-4mβNA (100%).

Figure 5 Dixon plot for determination of (a) apparent inhibition constant K_i and (b) Cornish-Bowden plot for DPP-III with Arg-Phe-Ala.

Table V.  Inhibition of DPP-III from goat brain with different peptides.

Peptide	Ki ((M)	Type of inhibition
Leu-Trp-Met-Arg-Phe-Ala	7	Competitive
Arg-Phe-Ala	4	Competitive
Gly-Phe-Leu	15	Non-competitive
Leu-Trp-Met	31	Competitive
Leu-Trp	39	Competitive
Gastrin tetra peptide-NH2	55	Competitive
Leu-enkephalin	90	Competitive
Met- enkephalin	130	Competitive
Val-Tyr	250	Competitive
Trp-Met	380	Competitive