Human liver thermolabile phenol sulfotransferase: cDNA cloning, expression and characterization.
The sulfate conjugation of phenolic biogenic amines in humans is catalyzed by the thermolabile (TL) form of phenol sulfotransferase (PST). As a first step toward cloning a cDNA for TL PST, the enzyme was purified from jejunal mucosa, the human tissue with the highest known specific activity, and purified TL PST was subjected to limited proteolysis and amino acid sequencing. The PCR was then performed with human liver cDNA as template and primers designed on the basis of an 18 amino acid stretch of TL PST sequence to obtain a probe for screening cDNA libraries. When cDNA library screening proved unsuccessful, PCR primers were designed based on the nucleotide sequence of a functionally uncharacterized human brain cDNA that had been speculated to represent an aryl sulfotransferase. This brain cDNA encoded the 18 amino acid sequence that we had determined in TL PST. With human liver cDNA as template, these primers amplified a PCR product that contained an 885 nucleotide open reading frame that encoded 295 amino acids--including the 18 amino acids of TL PST sequence. In vitro transcription and translation of this human liver cDNA resulted in the synthesis of a 35.5 kDa protein. The human liver cDNA was also expressed in COS-1 cells, and the biochemical and physical characteristics of the encoded enzyme were identical with those of human liver TL PST. The cloning of a cDNA for human liver TL PST represents an important step toward understanding the molecular basis for the regulation of this enzyme in humans.
Wood
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Department of Pharmacology, Mayo Medical School/Mayo Clinic, Rochester, MN 55905.
Aksoy
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Aksoy
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Weinshilboum
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D000595:Amino Acid Sequence; D000818:Animals; D015239:Arylsulfotransferase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D006358:Hot Temperature; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology; D007583:Jejunum / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013816:Thermodynamics; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D015239:Arylsulfotransferase