1. Controlling Sulfuryl-Transfer Biology
Ian Cook, Ting Wang, Wei Wang, Felix Kopp, Peng Wu, Thomas S. Leyh 
Cell Chemical Biology 
Volume 23, Issue 5, Pages (May 2016)
DOI: /j.chembiol
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2. Cell Chemical Biology 2016 23, 579-586DOI: (10. 1016/j. chembiol. 2016
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3. Figure 1 Cap Closure Sterically Restricts Active Site Access
(A) DHEA binds in a reactive conformation in the open and closed conformations of SUL2A1.
(B) The raloxifene (RAL) R group (see Figure 2A) prevents access to the active site when the cap is closed. The active-site cap of SULT2A1 is shown in the open (orange) and closed (blue) positions. With and without nucleotide bound, the enzyme spends 95% and ≤5% of its time in the closed conformation, respectively. Nucleotide is believed to be saturating in vivo.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2 Raloxifene Bound to the Estrogen Receptor
(A) The structure of raloxifene. Raloxifene is composed of a steroid-like base and a large R group inserted at C3 of the benzothiophene ring. The R group prevents binding to the closed form of SULT2A1 (see Figure 1B).
(B) Raloxifene (Ral) bound to the human estrogen receptor α.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 3
The Binding of Benzoyl-Ral to the E and E·PAP Forms of SULT2A1
Binding was monitored via intrinsic fluorescence of the enzyme (λex = 290 nm, λem = 340 nm). Data are plotted as a ratio of fluorescence intensity at a given ligand concentration to that in the absence of ligand (I/I0). Solution composition: SULT2A1 (0.50 μM), PAP (0 μM [gray], 125 μM [black]), MgCl2 (5.0 mM), KPO4 (50 mM) (pH 7.4), 25 ± 2°C. Titrations were performed in triplicate and averaged. Lines through the data represent the behavior predicted by a best-fit, single-site binding model. Ral, raloxifene.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 4 ER Activation in Ishikawa Cells
(A) ER activation in (−) SULT control cells. ER activation was monitored via alkaline phosphatase levels. EC50 values are similar (∼0.2 nM) for each of the five derivatives indicated (see Table 2). The Ishikawa cells were stably transfected with vector that did not contain a SULT coding region. SULT activity toward DHEA is not detected in extracts of these cells (Experimental Procedures).
(B) ER activation in (+) SULT2A1 cells. The Ishikawa cells were stably transfected with vector containing the SULT2A1 coding region. SULT activity in extracts of these cells was comparable with that seen in human liver extracts (Experimental Procedures). The EC50 values of cells too large to pass through the acceptor pore of SULT2A1 are not affected by 2A1 expression, while those of the compounds that can pass through the pore increase ∼104-fold.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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7. Figure 5
Sulfonation of Raloxifene and an Acetyl Derivative of the Raloxifene Base by SULT2A1 (+) and (−) Ishikawa Cells
Cells were grown to ∼70% confluency in MEM + 10% FBS, washed, and medium containing 25 μM radiolabeled acceptor ([3H]raloxifene or [14C]acetyl derivative, specific activity 0.65 and 0.13 Ci/mmol, respectively) was added. Formation of sulfonated acceptor in the medium was monitored via scintillation counting after chloroform extraction. Measurements were performed in duplicate and averaged. Acetyl-derivative data were fit using a single exponential. The acetyl derivative is rapidly sulfonated in the SULT2A1 (+) cell line and slowly in the (−) line. Raloxifene is not detectably sulfonated by either cell line. RAL, raloxifene.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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