FIGURE 3.
N-Glycosylation is not required the catalytic activity of folded AChET protein. A, HEK293T cells were transfected with cDNAs encoding AChET with or without cDNA encoding PRiMA. Equal amounts of protein from the cell lysates were incubated with or without Endo H or PNGase F under the non-denaturing conditions. The cell lysates were analyzed by SDS-PAGE and Western blotting with anti-AChE antibody (upper panel). The amount of the Endo H-sensitive or PNGase F-sensitive AChET was quantified by calibrated densitometry (lower panel). The data are expressed as % of total AChET protein. B, AChE enzymatic activity of the cell lysate from A was determined by Ellman assay. The data are expressed as % of total AChE activity before the treatment. C, the cell lysates from A were subjected to sucrose density gradient analysis. The enzymatic activities are plotted as a function of the S value, estimated from the position of the sedimentation markers. The enzymatic activities are expressed in arbitrary units. Representative gradient profiles and gels from four independent experiments are shown. Values are the means ± S.E. obtained in four independent experiments (n = 4). **, p < 0.01 compared with undigested control.