Functional Characterization of a High-Affinity Choline Transport System in Human Keratinocytes

The present paper describes the functional characterization of two human aryl sulphotransferase (HAST) cDNAs, HAST1 and HAST3, previously isolated by us from liver and brain, respectively [Zhu, Veronese, Sansom, and McManus (1993) Biochem. Biophys. Res. Commun. 192, 671-676; Zhu, Veronese, Bernard, Sansom and McManus (1993) Biochem. Biophys. Res. Commun. 195, 120-127]. These appear to encode the two major forms of phenol sulphotransferase (PST) characterized in a number of human tissue cytosols, these being the phenolsulphating (P-PST) and monoamine-sulphating (M-PST) forms of phenol sulphotransferase. HAST1 and HAST3 cDNAs were functionally expressed in COS-7 cells and kinetically characterized using the model substrates for P-PST and M-PST, p-nitrophenol and dopamine (3,4-dihydroxyphenethylamine) respectively. COS-expressed HAST1 was shown to be enzymatically active in sulphating p-nitrophenol with high affinity (Km 0.6 microM), whereas dopamine was the preferred substrate for HAST3 (Km 9.7 microM). HAST1 could also sulphate dopamine, as could HAST3 sulphate p-nitrophenol, but the Km for these reactions were at least two orders of magnitude greater than for the preferred substrates. COS-expressed HAST1 and HAST3 displayed inhibition profiles with the ST inhibitor 2,6-dichloro-4-nitrophenol (DCNP), identical with human liver cytosolic P-PST and M-PST activities respectively. Thermal-stability studies with the expressed enzymes showed that HAST1 was considerably more thermostable (TS) than HAST3, which is consistent with P-PST being termed the TS PST and M-PST being termed the thermolabile (TL) PST. Western immunoblot analyses of the expressed PST proteins using an antibody generated to a bacterially expressed rat liver aryl/phenol ST showed that HAST1 and HAST3 migrated as single proteins with different electrophoretic mobilities (32 versus 34 kDa). This is consistent with the differences in electrophoretic mobilities observed for P-PST and M-PST in a variety of tissues reported by other workers. This report on the functional characterization of P-PST and M-PST cDNAs provides important information on the structural as well as functional relationships of human PSTs, which sulphate a vast array of exogenous and endogenous compounds.

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Functional characterization of PCFT/HCP1 as the molecular entity of the carrier-mediated intestinal folate transport system in the rat model

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00309.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. G660-G668 ◽

Cited By ~ 67

Author(s):

Katsuhisa Inoue ◽

Yasuhiro Nakai ◽

Sayaka Ueda ◽

Shunsuke Kamigaso ◽

Kin-ya Ohta ◽

...

Keyword(s):

Small Intestine ◽

Transport System ◽

Functional Characterization ◽

Cellular Membrane ◽

Molecular Entity ◽

Xenopus Laevis Oocytes ◽

Rat Small Intestine ◽

Distribution Profile ◽

Folate Transport

Proton-coupled folate transporter/heme carrier protein 1 (PCFT/HCP1) has recently been identified as a transporter that mediates the translocation of folates across the cellular membrane by a proton-coupled mechanism and suggested to be the possible molecular entity of the carrier-mediated intestinal folate transport system. To further clarify its role in intestinal folate transport, we examined the functional characteristics of rat PCFT/HCP1 (rPCFT/HCP1) expressed in Xenopus laevis oocytes and compared with those of the carrier-mediated folate transport system in the rat small intestine evaluated by using the everted tissue sacs. rPCFT/HCP1 was demonstrated to transport folate and methotrexate more efficiently at lower acidic pH and, as evaluated at pH 5.5, with smaller Michaelis constant ( Km) for the former (2.4 μM) than for the latter (5.7 μM), indicating its characteristic as a proton-coupled folate transporter that favors folate than methotrexate as substrate. rPCFT/HCP1-mediated folate transport was found to be inhibited by several but limited anionic compounds, such as sulfobromophthalein and sulfasalazine. All these characteristics of rPCFT/HCP1 were in agreement with those of carrier-mediated intestinal folate transport system, of which the Km values were 1.2 and 5.8 μM for folate and methotrexate, respectively, in the rat small intestine. Furthermore, the distribution profile of the folate transport system activity along the intestinal tract was in agreement with that of rPCFT/HCP1 mRNA. This study is the first to clone rPCFT/HCP1, and we successfully provided several lines of evidence that indicate its role as the molecular entity of the intestinal folate transport system.

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Functional characterization of high-affinity Na+/dicarboxylate cotransporter found in Xenopus laevis kidney and heart

AJP Cell Physiology ◽

10.1152/ajpcell.00295.2004 ◽

2005 ◽

Vol 289 (5) ◽

pp. C1159-C1168 ◽

Cited By ~ 9

Author(s):

Naomi Oshiro ◽

Ana M. Pajor

Keyword(s):

Xenopus Laevis ◽

Cdna Sequence ◽

Citric Acid Cycle ◽

Functional Characterization ◽

Activation Kinetics ◽

Electrophysiological Properties ◽

High Affinity ◽

The Family ◽

Kidney Liver

The SLC13 gene family includes sodium-coupled transporters for citric acid cycle intermediates and sulfate. The present study describes the sequence and functional characterization of a SLC13 family member from Xenopus laevis, the high-affinity Na+/dicarboxylate cotransporter xNaDC-3. The cDNA sequence of xNaDC-3 codes for a protein of 602 amino acids that is ∼70% identical to the sequences of mammalian NaDC-3 orthologs. The message for xNaDC-3 is found in the kidney, liver, intestine, and heart. The xNaDC-3 has a high affinity for substrate, including a Km for succinate of 4 μM, and it is inhibited by the NaDC-3 test substrates 2,3-dimethylsuccinate and adipate. The transport of succinate by xNaDC-3 is dependent on sodium, with sigmoidal activation kinetics, and lithium can partially substitute for sodium. As with other members of the family, xNaDC-3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC-3 are unique and involve large leak currents, possibly mediated by anions, that are activated by binding of sodium or lithium to a single site.

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