FIGURE 8.

Effects of NEM on hPLCβ3 activity and U73122-mediated activation of the enzyme in DDM-mixed micelles. A, structure of NEM. B, effect of NEM on hPLCβ3 activity in DDM-mixed micelles. PIP₂, reconstituted in DDM, was combined with assay buffer and hPLCβ3 in a final volume of 100 μl. NEM or U73122, at indicated concentrations, was added to the assay mixture prior to the addition of enzyme. Assays were initiated by moving samples to a 37 °C water bath. Incubation times were adjusted to ensure that less than 15% PIP₂ was hydrolyzed in all cases. Data represent mean ± S.D. from triplicate determinations. C, effect of NEM on the U73122-mediated activation of hPLCβ3 in DDM-mixed micelles. PIP₂, reconstituted in DDM, was combined with assay buffer and hPLCβ3 in a final volume of 100 μl. NEM, at indicated concentrations, and U73122 (40 μm) were added to the assay mixture prior to the addition of enzyme. * indicates that NEM was preincubated with enzyme prior to adding the enzyme to the assay mixture. Assays were initiated by moving samples to a 37 °C water bath. Incubation times were adjusted to ensure that less than 15% PIP₂ was hydrolyzed in all cases. Data represent mean ± S.D. for triplicate determinations from one representative experiment.