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Enzymatic Properties, Tissue-Specific Expression, and Lysosomal Location of Two Highly Homologous Rat SULT1C2 Sulfotransferases☆,☆☆

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Abstract

We have isolated two highly homologous but distinct rat sulfotransferase cDNAs termed ratSULT1C2 and ratSULT1C2A encoding polypeptides of 297 amino acids each. The amino acid sequence of ratSULT1C2 is 84% identical to the human SULT1C2 and 81% identical to a rabbit SULT1C2 sulfotransferase. ratSULT1C2 and ratSULT1C2A are 92% identical but differ in 22 amino acids. The majority of these amino acid substitutions in ratSULT1C2A is not found in the human and rabbit SULT1C2, which identifies ratSULT1C2 as the orthologue of these sulfotransferases, whereas SULT1C2A is a closely related but distinct enzyme. ratSULT1C2 and 2A sulfotransferases do not sulfonate steroids, dopamine, acetaminophen, or α-naphthol, but only p-nitrophenol. Prokaryotically expressed ratSULT1C2A is less active than ratSULT1C2. ratSULT1C2/2A mRNAs are abundant in kidney and less abundant in stomach and liver. The enzymes are expressed as 34-kDa polypeptides in rat kidney, liver, and stomach. In addition, a 28-kDa cross-reacting polypeptide is found in kidney only. Immunohistochemistry revealed expression of ratSULT1C2/2A in the epithelial cells of the proximal tubules of the kidney, bile duct epithelia, hepatocytes, and the epithelium of the gastric mucosal glands. Although the cDNA predicted amino acid sequence identifies both sulfotransferases as cytosolic enzymes, in tissue sections, in the kidney cell line NRK 52, and in transiently transfected BHK cells a considerable fraction of the enzyme was found in a granular perinuclear compartment. Costaining with a lysosomal marker in gastric mucosa tissue sections and cultured cells identifies these structures as lysosomes.

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☆

GenBank accession numbers for sulfotransferase cDNA sequences: ratSULT1C2, AJ238391; ratSULT1C2A, AJ238392.

☆☆

Abbreviations used: SULT, sulfotransferase; GST, glutathione transferase; ORF, open reading frame; PAPS, 3′-phosphoadenosine-5′-phosphosulfate; IPTG, isopropylthiogalactoside; TBS, Tris-buffered saline; EDTA, ethylenediaminetetraacetic acid; BHK, baby hamster kidney.

1

These authors contributed equally to the results.

2

Present address: Department of Nutrition, Zhejiang Medical University, Hangzhou, Zhejiang 310031, People's Republic of China.

3

Present address: Arimedes Biotechnology GmbH, Berlin–Buch, Germany.

4

To whom correspondence should be addressed at present address: Institut für Physiologische Chemie, Rheinische Friedrich Wilhelms Universität, Nuβallee 11, 53115 Bonn, Germany. Fax: 0049-228-73-2416.

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