Comparison of the kinetic properties of IDE and cysteine-free IDE with Abz-GGFLRKHGQ-EDDnp as substrate.

<p>Kinetic constants were determined by measuring rates at variable concentration of Abz-GGFLRKHGQ-EDDnp as substrate. Data were fit to the Hill equation.</p

Effect of converting individual cysteine residues to serine on IDE kinetics.

<p>The activity of IDE and its cysteine mutants was determined using a fixed concentration of 10 µM Abz–GGFLRKHGQ-EDDnp as substrate. K_A was determined by varying [ATP] at a fixed substrate concentration of 10 µM. Data is expressed as the mean plus or minus the standard deviation. Each value represents measurements made two to four times.</p

IQ and selected analogues reverse Aβ40 inhibition of nAChRs in PC12 cells.

<p>(A) Current responses (normalized by the maximal current evoked by 0.2 mM CCh) of neuronal-differentiated PC12 cells exposed for 2 s to 0.2 mM CCh plus 200 nM Aβ40 in all experimental conditions, except for the control measurement with CCh alone, and, as indicated, 500 nM of different IQ analogues. Bars represent means ± S.D. of at least 3 replicate measurements performed in 4–6 different cells (**, p<0.01; *** p<0.001 in the comparison with the control current evoked by CCh alone).</p

Activity of IDE and its mutants with amyloid beta peptide and insulin as substrates.

<p>Reactions containing 10 µM Aß_1–40 or 10 µM insulin as substrates in 50 mM Tris-HCl buffer, pH 7.4, were incubated at 37°C for 30 to 60 min. Rates were determined by measuring the disappearance of the substrate peak by HPLC as described in Methods. IDE^C812S and IDE^C819S were used as controls to demonstrate the specificity of effects resulting from mutating Cys 904 and Asn 575. Data is expressed as the mean plus or minus the standard deviation. Each value represents measurements made two to five times.</p

Structure of IDE showing the relative positions of Cys904, Cys573, and Asn575.

<p>The N- and C-terminal halves of the molecule are colored yellow and blue, respectively. Bound ATP is shown in stick representation.</p

Ribbons representation of the IDE crystal structure.

<p>The position of cysteine residues, and other key features of IDE are indicated.</p

Comparison of the kinetics of IDE to C904S/A and to N575T/L.

<p>Reaction mixtures contained 50 mM Tris-HCl buffer, pH 7.4, 10 µM Abz-GGFLRKHGQ-EDDnp and either 1 µg of IDE (closed circles), 4 µg of IDE C904S (closed inverted triangle), 4 µg of IDE C904A (closed diamonds), 5 µg of IDE N575T (closed squares), or 2 µg of IDE N575L (closed triangles). The insert shows a comparison of the IDE mutants. Shown are representative data from experiments that were replicated with different enzyme preparations.</p

IQ makes Aβ40 inhibition of α3β4 nAChR currents in transformed HEK cells reversible.

<p>HEK cells expressing α3β4 nAChRs received consecutive shots (at 5 min intervals) of 0.2 mM CCh plus 200 nM Aβ, in the absence or presence of IQ (0.5 µM) as indicated. Shots 1–3 contained 0.2 mM CCh alone (white bars), 0.2 mM CCh plus 200 nM Aβ (light grey bars), 0.2 mM CCh plus 200 nM Aβ and 0.5/2 µM IQ (grey bars) or 0.5 µM SQI (black bars), used as an inactive control. Shots 4–6 contained 0.2 mM CCh alone for evaluation of reversibility of receptor inhibition. Bars represent mean values ± S.D. of at least 3 replicate measurements (normalized by the maximal current evoked by 0.2 mM CCh) obtained from 4–6 different cells. (***, p<0.001, in comparison with 0.2 mM CCh plus 200 nM Aβ).</p

Comparison of the activation of IDE mutants by ATP.

<p>Reaction mixtures were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046790#pone-0046790-g003" target="_blank">Figure 3</a> with ATP varied as indicated. Shown are cysteine-free IDE (2 µg - closed circles), IDE Cys904Ser (8 µg - closed squares), and IDE Cys904Ala (2 µg - closed triangles). The activity is plotted as the activity observed at the indicated ATP concentration relative to the activity in the absence of added ATP. Shown are representative data from experiments that were replicated with different enzyme preparations.</p

Comparison of the activation of IDE to cysteine-free IDE by ATP and TNP-ATP.

<p>Reactions of 200 µl contained 50 mM Tris-HCl buffer, pH 7.4, 10 µM Abz-GGFLRKHGQ-EDDnp and 1 µg of IDE (closed circles) or 2 µg cysteine-free IDE (closed squares) and the indicated amount of ATP (left) or TNP-ATP (right). Relative activity is defined as the activity observed at the indicated ATP or TNP concentration relative to the activity in their absence. A higher amount of cysteine-free enzyme was used as noted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046790#pone-0046790-g002" target="_blank">Figure 2</a>. Shown are representative data from experiments that were repeated twice using different enzyme preparations.</p