ABCC3 p.Ser17Gly
| Predicted by SNAP2: | A: N (82%), C: N (78%), D: N (78%), E: N (72%), F: N (87%), G: N (87%), H: N (97%), I: N (78%), K: N (66%), L: N (87%), M: N (78%), N: N (97%), P: N (87%), Q: N (87%), R: N (82%), T: N (93%), V: N (82%), W: N (82%), Y: N (93%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: N, Y: N, |
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[hide] Increased estrogen sulfation of estradiol 17beta-D... J Pharmacol Exp Ther. 2006 Nov;319(2):818-31. Epub 2006 Aug 8. Sun H, Liu L, Pang KS
Increased estrogen sulfation of estradiol 17beta-D-glucuronide in metastatic tumor rat livers.
J Pharmacol Exp Ther. 2006 Nov;319(2):818-31. Epub 2006 Aug 8., [PMID:16895976]
Abstract [show]
Changes in the disposition of estradiol 17beta-d-glucuronide (E(2)17G), a substrate of the organic anion-transporting polypeptide family (Oatp) and multidrug resistance-associated protein 2 (Mrp2), were examined in livers of male Wag/Rij rats that were injected with CC531 cells intraportally to induce metastatic tumors (n = 5) or with phosphate-buffered saline for sham-operated controls (n = 4). Multiple indicator dilution, single-pass liver perfusions revealed extremely high influx clearances of [(3)H]E(2)17G (>190 ml/min) in both groups. In recirculating liver perfusions, [(3)H]E(2)17G decayed monoexponentially in the reservoir perfusate, and the total (9.19 +/- 1.33 versus 8.18 +/- 0.94 ml/min) and biliary (4.94 +/- 1.07 versus 4.60 +/- 0.86 ml/min) clearances were similar in both groups (P > 0.05). The metabolic clearance of E(2)17G was higher in the tumor group (4.60 +/- 0.64 versus 3.23 +/- 0.23 ml/min, P < 0.05). E(2)3S17G, the 3-sulfate metabolite, whose identity was confirmed by mass spectrometry, appeared only in bile and not perfusate. Liver microsomal incubations of E(2)3(35)S17G and [(3)H]estrone sulfate revealed similar sulfatase activities between the tumor and sham livers, albeit the activities were much lower for E(2)3(35)S17G. Oatp1a1 and Oatp1b2 protein expression in liver membrane fragments was reduced by 42% and 38%, respectively, whereas that of cytosolic estrogen sulfotransferase (Sult1e1) was significantly increased (41%) with tumor (P < 0.05). All of the observations were captured by modeling. From modeling, we showed that reduction of the high influx clearance (546 to 283 ml/min) failed to lower the total clearance of E(2)17G, whereas up-regulation of Sult1e1 increased the E(2)17G sulfation clearance (2.56 to 3.69 ml/min) in livers with metastatic tumors.
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No. Sentence Comment
38 E2335 S17G was biosynthesized by recirculation of 0.5 M E217G in oxygenated Krebs-Henseleit bicarbonate (KHB) at 40 ml/min through the liver, with Na2 35 SO4 (18 Ci/min for 30 min) that was infused directly into the portal vein cannula.
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ABCC3 p.Ser17Gly 16895976:38:6
status: NEW39 The pooled bile was lyophilized, reconstituted, and injected into HPLC running gradient program I for isolation (see HPLC Methods); the eluant corresponding to E2335 S17G was desalted by a reverse phase Sep-Pak cartridge (Waters, MA), and the purified content was stored at -20°C.
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ABCC3 p.Ser17Gly 16895976:39:166
status: NEW40 The radiochemical purities of [3 H]E217G, [3 H]E1S, and E2335 S17G were 98, 99, and 95%, respectively, as evaluated by HPLC.
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ABCC3 p.Ser17Gly 16895976:40:62
status: NEW128 Sulfatase Activity in Subcellular Fractions An aliquot (75 l) of microsomal (0.3 mg) or cytosolic (0.7 mg) fraction was mixed with 75 l of E2335 S17G (ϳ50,000 dpm) in Tris-HCl (25 mM, pH 7.4).
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ABCC3 p.Ser17Gly 16895976:128:161
status: NEW132 The reconstituted solution was injected into HPLC running program III (see under HPLC Methods) for desulfation of E2335 S17G.
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ABCC3 p.Ser17Gly 16895976:132:120
status: NEW135 Hence, parallel studies were conducted to estimate sulfatase activity by formation of 35 SO4 2- from E2335 S17G and [3 H]E1 from [3 H]E1S.
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ABCC3 p.Ser17Gly 16895976:135:107
status: NEW136 The desulfation intrinsic clearances were estimated as the ratio of desulfation rate to E2335 S17G or [3 H]E1S concentration at time 0.
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ABCC3 p.Ser17Gly 16895976:136:94
status: NEW153 Program III was capable of separating 35 SO4 2- (RT ϳ3 min) and E2335 S17G (RT ϳ18 min) well.
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ABCC3 p.Ser17Gly 16895976:153:76
status: NEW225 Futile Cycling: Sulfatase Activity in Subcellular Fractions The retention time of the E2335 S17G deviated slightly from that of the E2335 S17G standard in buffer because of the presence of different matrices during workup from the microsomal and cytosolic incubation samples (Fig. 4).
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ABCC3 p.Ser17Gly 16895976:225:92
status: NEWX
ABCC3 p.Ser17Gly 16895976:225:138
status: NEW226 Only the microsomal but not the cytosolic fraction demonstrated desulfation activities toward E2335 S17G to form the product, 35 SO4 2- , as shown in the HPLC tracings.
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ABCC3 p.Ser17Gly 16895976:226:100
status: NEW227 However, the desulfation activities toward E2335 S17G as well as [3 H]E1S, a model substrate to demonstrate microsomal activity of aryl- sulfatases, were not significantly different in microsomal fractions prepared from peritumor liver and sham-operated liver tissues (Table 2).
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ABCC3 p.Ser17Gly 16895976:227:49
status: NEW228 The desulfation activity toward E2335 S17G was low in relation to that for E1S.
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ABCC3 p.Ser17Gly 16895976:228:38
status: NEW237 Fig. 4. Radioelution chromatograms of products derived from incubation of E2335 S17G with Tris-HCl buffer (Ⅺ), and cytosolic (F) or microsomal (E) fractions.
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ABCC3 p.Ser17Gly 16895976:237:80
status: NEW238 The retention times of E23S17G (ϳ18 min, f) and 35 SO4 2- (ϳ3 min) were coincident with those obtained upon injection of the standards, E2335 S17G and 35 SO4 2- , into the HPLC column.
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ABCC3 p.Ser17Gly 16895976:238:154
status: NEW239 Desulfation of E2335 S17G in microsomes but not cytosol nor buffer, albeit of low activity, was observed.
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ABCC3 p.Ser17Gly 16895976:239:21
status: NEW269 Values of CLin in sham and tumor groups were 20 to 40 times the value of the blood flow TABLE 2 Desulfation activities or intrinsic clearances toward E2335 S17G and ͓3 H͔E1S (for confirmation of desulfation activity) in microsomal fractions, prepared from sham-operated and peritumor tissue at 4 weeks postinoculation Data are means Ϯ S.D. Desulfation Intrinsic Clearance Sham-Operated Liver Tissue Peritumor Tissue l/s/mg microsomal protein E23S17G (CLint,desult,E23S17G) 0.16 Ϯ 0.04 0.12 Ϯ 0.02 E1S (CLint,desult, E1S) 7.88 Ϯ 0.76 8.09 Ϯ 1.29 Fig. 5.
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ABCC3 p.Ser17Gly 16895976:269:156
status: NEW310 The identity of the metabolite, E23S17G, was confirmed unequivocally with mass spectrometry and microsomal incubation studies with the radiolabeled E2335 S17G that was deconjugated by arylsulfatase C (also known as estrogen sulfatase) to reform E217G and 35 SO4 2- (Fig. 4), providing direct evidence of futile cycling between E23S17G and E217G.
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ABCC3 p.Ser17Gly 16895976:310:154
status: NEW