Figure 8.

Enzyme Activity Analysis of Recombinant LH3 with and without Mutation 1 or Mutation 2

The 15,000 × g supernatant of insect cell homogenate was used for activity assays. LH, GT, and GGT activities were assayed,²⁸ and an amount of LH3 protein was measured with monoclonal anti c-myc antibody 9E10 (Santa Cruz), anti-mouse-IgG-AlexaFluor488 (Molecular Probes) and Typhoon 9400 (GE Healthcare) for fluorescence detection of immunoblot analysis. Quantification of immunostained LH3 protein levels was performed with ImageQuant TL 7.0 software, GE Healthcare. Relative specific activities of mutation 1 (LH3mut1) and mutation 2 (LH3mut2) were calculated and compared to activities of nonmutated LH3. The data show dramatic reduction of glycosyltransferase activities of LH3mut1 and LH activity of LH3mut2. Activities are expressed as the percentage from activity of nonmutated LH3. n = 4 independent measurements, except n = 3 in GT of LH3mut1. The SD indicated by the bars. The asterisk represents activity below detection range.