Human intestinal phenol sulfotransferase: assay conditions, activity levels and partial purification of the thermolabile form.
Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catecholamines and phenolic and catechol drugs. The small intestine is an important drug-metabolizing organ. Other human tissues including brain, liver, and platelet contain at least two forms of PST. A thermolabile (TL) form catalyzes the sulfate conjugation of dopamine and other monoamines whereas a thermostable (TS) form of the enzyme catalyzes the sulfate conjugation of "simple" phenols such as p-nitrophenol. In this study we found that human jejunal mucosa also contains at least two forms of PST with substrate specificities, sensitivities to inhibitors, and thermal stabilities similar to those of TL and TS PST in other human tissues. Optimal conditions were determined for the assay of TL and TS PST activities in jejunal mucosal preparations. Apparent Km values of TL PST for dopamine and of TS PST for p-nitrophenol were 10 microM and 0.92 microM, respectively. Jejunal mucosal TL PST, like TL PST in other human tissues, also catalyzed the sulfation of p-nitrophenol, but with a very high apparent Km of 1100 microM. PST activities were then assayed in 64 samples of jejunum obtained surgically, and average activities for TL and TS PST were 102 +/- 4.6 and 47.4 +/- 2.9 units per milligram of protein, (mean +/- SE), with 40 microM dopamine and 2 microM p-nitrophenol as substrates, respectively. Since jejunal mucosal TL PST had a much higher specific activity than that present in other human tissues, TL PST was partially purified from jejunal mucosa by ion exchange and Affi-Gel Blue chromatography. The substrate specificity, inhibitor sensitivity, and physical properties of partially purified jejunal TL PST were similar to those of TL PST in supernatants of jejunal mucosal homogenates. True Km values of the partially purified enzyme for dopamine and p-nitrophenol were 6.7 microM and 1400 microM, respectively. Human jejunal TL PST had an apparent molecular mass of 69 kDa with 35.5-kDa monomers. In summary, human jejunal mucosa contains forms of PST similar to those found in other human tissues, and human jejunal mucosa is a good source for the purification of TL PST.
Sundaram
R S
RS
Department of Pharmacology, Mayo Clinic/Mayo Foundation, Rochester, MN 55905.
Szumlanski
C
C
Otterness
D
D
van Loon
J A
JA
Weinshilboum
R M
RM
D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D066298:In Vitro Techniques; D007422:Intestines / Q000201:enzymology*; D008970:Molecular Weight; D009596:Nitrophenols / Q000494:pharmacology; D013431:Sulfates / Q000378:metabolism
D009596:Nitrophenols; D013431:Sulfates; D015239:Arylsulfotransferase