Evaluation of a novel aminopeptidase N inhibitor, in vitro, using two assay systems

Figures & data

Figure 1 The hydrolysis of Tyr-Gly in striatum membrane preparations. (A) Time course of the contents of Tyr-Gly (○) and of its hydrolysis product Tyr (•) with 0.5 mg/mL protein. Each point represents the mean with S.E.M. of 5 independent experiments. (B) Protein concentration- dependence of the change in the contents of Tyr-Gly (▪) and Tyr (▪) in 2 h incubation period. Each column represents the mean with S.E.M. of 5 independent experiments. When the error bars are not shown, they are smaller than the symbol.

Figure 2 Effects of amastatin and AHPA-Val on the amounts of Tyr-Gly and its hydrolysis product Tyr in striatum membrane preparations, respectively. Each point represents the mean and vertical bar indicates the S.E.M. of 6 ∼ 8 preparations. For the “veh”, we incubated Tyr-Gly in Tris-HCl buffer without protein and APNIs. Vs. respective concentration of amastatin, * p < 0.05, *** p < 0.001 (unpaired t-test).

Figure 3 Kinetic analyses of amastatin (A) and AHPA-Val (B) on the hydrolysis of Tyr-Gly using Lineweaver-Burk plot of 1/V against 1/[S]. The inhibitory potencies of AHPA-Val and amastatin were measured using Tyr-Gly as substrate with the concentrations indicated (0.05 mM, 0.1 mM and 0.2 mM). Each point represents the mean of five separate experiments.

Figure 4 Effects of amastatin (A) and AHPA-Val (B) on Met-enkephalin-induced twitch inhibition in guinea pig ileum preparations. Peptidase inhibitors were applied accumulatively. Each point represents the mean percentage inhibition with S.E.M of 9 independent experiments.

Table I.  Effects of amastatin or AHPA-Val on the IC₅₀ values of Met-enkephalin- induced twitch inhibition in guinea pig ileum preparations.

	Amastatin		AHPA-Val
Concentration (μM)	IC50(nM)	Ratio of potency	IC50(nM)	Ratio of potency
control	64.3 ± 0.83	1	64.3 ± 0.83	1
0.01	36.6 ± 1.41	1.78 ± 0.09	44.36 ± 1.34	1.45 ± 0.03
0.1	19.76 ± 0.63	3.28 ± 0.13	32.03 ± 1.09	2.01 ± 0.05
1	11.62 ± 0.92	5.64 ± 0.54	17.42 ± 0.06	3.72 ± 0.03
10	NE	NE	13.30 ± 0.58	4.92 ± 0.33

All preparations were pretreated with 10^{− 6} M of captopril and 10^{− 6} M of phosphoramidon before the amastatin or AHPA-Val application. The ratio of the potency shown in the Table was calculated from the following formula: ratio of potency = IC₅₀ of control treatment/ IC₅₀ after additional treatment. Each value represents the mean ± S.E.M. NE: not examined.

Figure 5 pA_1/2 analyses of amastatin (▪) and AHPA-Val (•). pA_1/2 value is defined as the negative log concentration of peptidase inhibitor that produced the effect to decrease the IC₅₀ of Met-enkephalin in half. pA_1/2 of amastatin and AHPA-Val is 7.79 and 7.08, respectively. Each point represents the mean of 9 independent experiments.

Figure 6 Stereoview of comparison between the binding modes of bestatin and AHPA-Val. The principal interactions between the inhibitor and active site are shown.