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. 2013 Jul 9;2:e00691. doi: 10.7554/eLife.00691

Figure 2. Soluble substrate activity and Interfacial kinetic analysis of semisynthetic PTEN.

(A) PTEN activity to a soluble substrate, diC6-PIP3. (n-PTEN: kcat = 2.6 ± 0.1 min−1, Km = 67 ± 4.2 μM, kcat/Km = 0.038 ± 0.001 min−1μM−1; 4p-PTEN: kcat/Km = 0.005 ± 0.0002 min−1μM−1) (B and C) PTEN activity to palmitoyl PIP3 incorporated into phosphatidylcholine vesicles. In the bulk dilution experiment (B) enzymatic activity for n-PTEN and 4p-PTEN was measured at a fixed surface concentration of 1% PIP3 while the bulk concentration was varied. In the surface dilution experiment (C) activity was measured at a fixed bulk concentration of 50 μM PIP3 while the surface concentration was varied. (D) 4p-PTEN has lower activity than n-PTEN only at low PIP3 concentrations. (E) Summary of the interfacial kinetic analysis of n-PTEN and 4p-PTEN. Data are reported as the mean ± the SEM from three experiments performed in duplicate. Apparent Vmax values were obtained from the best fit curves from the first four points of the surface dilution experiments.

DOI: http://dx.doi.org/10.7554/eLife.00691.007

Figure 2.

Figure 2—figure supplement 1. Interfacial kinetic analysis of semisynthetic PTENs.

Figure 2—figure supplement 1.

(A) 32P labeled PIP3 was generated as outlined in the ‘Materials and methods’ section. (B) PTEN activity to radiolabled palmitoyl PIP3 incorporated into phosphatidylcholine vesicles is linear with respect to enzyme concentration. (C) Apparent kcat and apparent Km values were obtained by nonlinear regression analysis for each form of PTEN for surface dilution experiments (top row) and bulk dilution experiments (bottom row). Apparent kcat and apparent Km values were then fit to the equations in the ‘Materials and methods’ section to obtain the interfacial kinetic parameters in tabulated in Figure 2E and Figure 2—figure Supplement 2
DOI: http://dx.doi.org/10.7554/eLife.00691.008
Figure 2—figure supplement 2. Bulk and surface dilution curves of t-PTEN.

Figure 2—figure supplement 2.

(A) In the bulk dilution experiment enzymatic activity for t-PTEN was measured at a fixed surface concentration of 1% PIP3 while the bulk concentration was varied. (B) In the surface dilution experiment activity was measured at a fixed bulk concentration of 50 µM PIP3 while the surface concentration was varied. Data a represented as the mean ± the SEM of three experiments performed in duplicate. (C) Summary of the interfacial kinetic analysis of t-PTEN. Data are reported as the mean ± the SEM from three experiments performed in duplicate. Apparent Vmax values were obtained from the best fit curves from the first four points of the surface dilution experiments.
DOI: http://dx.doi.org/10.7554/eLife.00691.009
Figure 2—figure supplement 3. Anionic lipid stimulation of n-PTEN and 4p-PTEN.

Figure 2—figure supplement 3.

At a low surface concentration of PIP3 (0.01%) incorporated vesicles, (A) PIP2 and (B) Phosphatidylserine stimulate the enzymatic activity of n-PTEN and 4p-PTEN. Data are reported as the mean ± the SEM of three experiments performed in duplicate.
DOI: http://dx.doi.org/10.7554/eLife.00691.010