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. 2012 Jul 9;3(10):767–781. doi: 10.1021/cn3000718

Figure 3.

Figure 3

Characterization of the L531A/V532A CHT (LV-AA) mutant in transiently transfected cell lines. (A) Comparison of cell surface residence among CHT, L531A, V532A, and LV-AA CHT in transiently transfected COS-7 cells. Following cell surface biotinylation, total (T) or surface (S) fractions or the wild type and each mutant CHT were examined. The total fractions represent 10% of the input into the biotinylated protein extractions. (B) Saturation kinetic analysis of CHT LV-AA compared to WT CHT was performed in transiently transfected COS-7 cells. CHT LV-AA has a significant increase in activity compared to WT CHT (two way ANOVA interaction P-value <0.001 to 0.003). (C) The measured Vmax was 0.0676 ± 00841 pmol choline/min·mg for WT CHT versus 0.463 ± 00.119 pmol choline/min·mg for CHT LV-AA (p = 0.0006, n = 5, two way ANOVA). The KM was not significantly altered by the mutation (data not shown), 10.58 ± 4.287 μM for WT CHT versus 7.484 ± 2.581 μM for CHT LV-AA (Student’s t test). (D) Biotinylation-stripping endocytosis assay by treatment with MesNa after incubation at 37 °C for 5–15 min shows a greater level CHT LV-AA maintained on the cell surface than WT CHT. (E) Quantification of internalization at 5 min finds an impact of the LV-AA CHT mutation as a reduction in the percent internalized protein compared to the WT CHT (p = 0.03, n = 3, Student’s t-test).