Zinc regulation of aminopeptidase B involved in neuropeptide production

Abstract

Aminopeptidase B (AP-B) is a metallopeptidase that removes basic residues from the N-termini of neuropeptide substrates in secretory vesicles. This study assessed zinc regulation of AP-B activity, since secretory vesicles contain endogenous zinc. AP-B was inhibited by zinc at concentrations typically present in secretory vesicles. Zinc effects were dependent on concentration, incubation time, and the molar ratio of zinc to enzyme. AP-B activity was recovered upon removal of zinc. AP-B with zinc became susceptible to degradation by trypsin, suggesting that zinc alters enzyme conformation. Zinc regulation demonstrates the metallopeptidase property of AP-B.

Figure 1. Zinc inhibition of aminopeptidase B (AP-B) at in vivo levels of zinc in secretory vesicles

AP-B activity was assessed at different concentrations of zinc of 5–80 μM ZnCl₂). AP-B (22 nM) was monitored with Arg-MCA substrate, with aminopeptidase activity detected by the formation of fluorescent AMC (aminomethylcoumarinamide). Results show inhibition of AP-B by zinc in a concentration-dependent manner.

Figure 2. Zinc regulation of AP-B

Aminopeptidase B (AP-B, 22 nM) was assessed at different concentrations of ZnCl₂ in time course assays that monitored activity at 10, 15, 30, and 45 minutes of incubation. AP-B activity was expressed as percent of control AP-B activity (100%) assayed in the absence of zinc.

Figure 3. Effects of different molar ratios of zinc to AP-B for aminopeptidase activity

AP-B was assayed in the presence of ZnCl₂ (0.5 μM) at different molar ratios of 80, 40, and 20 of zinc to AP-B at 37°C for 60 min. The percent inhibition of AP-B relative to AP-B control without zinc (0% inhibition) is shown. The mean of replicate assays (triplicate) with s.e.m. (standard error of the mean) is shown.

Figure 4. Analysis of zinc inhibition of AP-B by Lineweaver-Burk plot

Inhibition of AP-B (22 nM) by zinc (at 1, 2, 5, and 10 μM) was assessed by inverse Lineweaver-Burk plots [15], which indicated zinc as a mixed inhibitor. The Lineweaver-Burk plot also showed that AP-B (without zinc) has K_m value of 27 μM Arg-MCA and V_max of 1.2 μM/min. The K_i and K_i′ values for zinc inhibition of enzyme and enzyme/substrate complexes were calculated as 6 μM and 12 μM zinc, respectively.

Figure 5. Reversibility of zinc inhibition of AP-B

AP-B (22 nM) was incubated with ZnCl₂ (50 μM) for 5 min., and was then assayed for AP-B activity with different incubation times (2–75 min.). AP-B activity without removal of zinc (○), and after removal of ZnCl₂ by one (▲) or two (□) desalting column steps, was assessed. Recovery of AP-B activity is evident upon removal of ZnCl₂.

Figure 6. Zinc increases susceptibility of AP-B to degradation by trypsin

Purified AP-B (835 ng) was incubated without (lane 1) or with (lane 2) ZnCl₂ (250 μM) (for 5 min.), and was then subjected to incubation with trypsin (50 ng) at 30°C for 40 minutes. The integrity AP-B was then assessed by western blots with anti-AP-B, with equal amounts of AP-B (58 ng) present in each of lanes 1 and 2. Results show that zinc induced the susceptibility of AP-B to degradation by trypsin.