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Fig. 4. The stem region is important for HS6ST sulfotransferase activity and oligomer formation. (A) Sulfotransferase activities of stem-deleted m6ST1 proteins (m6ST1
stem20-EGFP and m6ST1stem55-EGFP) were compared to that of m6ST1-EGFP. To analyze sulfotransferase activity, CHO-K1 cells were transfected with control vector, pm6ST1-EGFP, pm6ST1stem20-EGFP, or pm6ST1stem55-EGFP and 24 hours later cell extracts were prepared with buffer A for at least 1 hour and then centrifuged. Crude cell extracts were used for the sulfotransferase assay as described in the Materials and Methods. The amount of the cell lysate used in the experiment was normalized using the EGFP fluorescence intensity. The sulfotransferase activity of m6ST1-EGFP was set at 100%. The sulfotransferase activities of the stem-deleted proteins were greatly reduced when compared to that of the wild type. The results shown are the mean±s.d. of three experiments. (B) Oligomerization of m6ST involves the stem region. CHO-K1 cells were transfected with pm6ST1YQY-EGFP, pm6ST2LQY-EGFP, pm6ST3YQY-EGFP, pm6ST1C2-EGFP, pm6ST2C2-EGFP or pm6ST3C2-EGFP and 24 hours later the cells were treated with buffer A for at least 1 hour and then centrifuged. The cell lysates were fractionated by HR10/30 SuperoseTM 6 chromatography (Amersham Pharmacia Biotech FPLC system), 0.5 ml fractions were collected, and the GFP fluorescence of each fraction was measured by using a fluorescent spectrophotometer (Hitachi) at the excitation wavelength 488 nm and the emission wavelength 507 nm. The position at which the molecular mass markers eluted, in addition to Vo and Vt, are indicated above the figure.
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