Abstract
A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps (hydroxylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATP- agarose II). The minimum molecular weight of the purified enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be ~33,000. Gel filtration chromatography revealed a native molecular weight of 34,000, indicating the enzyme being present in the monomeric form. The purified sulfotransferase displayed enzymatic activities, with a pH optimum of 9.25, toward various tyrosine and 3,4-dihydroxyphenyl-alanine (Dopa) isomers, except DL-ortho-tyrosine. Thyroid hormones, as well as dopamine and p-nitrophenol, could also be used as substrates. The apparent K(m) value of the enzyme (designated the Dopa/tyrosine sulfotransferase) for L-Dopa, determined at a constant 14 μM of 3'-phosphoadenosine 5'-phosphosulfate, was 0.76 mM. The intact enzyme was found to be N-blocked when subjected to N- terminal sequencing. Three internal partial amino acid sequences, obtained by analyzing its proteolytic fragments, were found to be distinct from the homologous sequences of other known rat liver sulfotransferases. The deduced amino acid sequence of a full-length cDNA isolated from a rat liver cDNA library confirmed the identity of the Dopa/tyrosine sulfotransferase as a new type of aryl sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pMSG-CMV) harboring the full-length cDNA, a 33-kDa protein displaying enzymatic and immunological properties similar to those of the purified Dopa/tyrosine sulfotransferase was expressed.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 30470-30478 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 270 |
| Issue number | 51 |
| DOIs | |
| State | Published - Dec 22 1995 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Purification, characterization, and molecular cloning of a novel rat liver Dopa/tyrosine sulfotransferase. / Sakakibara, Yoichi; Takami, Yasunari; Zwieb, Christian W; Nakayama, Tatsuo; Suiko, Masahito; Nakajima, Hiroshi; Liu, Ming Cheh.
In: Journal of Biological Chemistry, Vol. 270, No. 51, 22.12.1995, p. 30470-30478.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Purification, characterization, and molecular cloning of a novel rat liver Dopa/tyrosine sulfotransferase
AU - Sakakibara, Yoichi
AU - Takami, Yasunari
AU - Zwieb, Christian W
AU - Nakayama, Tatsuo
AU - Suiko, Masahito
AU - Nakajima, Hiroshi
AU - Liu, Ming Cheh
PY - 1995/12/22
Y1 - 1995/12/22
N2 - A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps (hydroxylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATP- agarose II). The minimum molecular weight of the purified enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be ~33,000. Gel filtration chromatography revealed a native molecular weight of 34,000, indicating the enzyme being present in the monomeric form. The purified sulfotransferase displayed enzymatic activities, with a pH optimum of 9.25, toward various tyrosine and 3,4-dihydroxyphenyl-alanine (Dopa) isomers, except DL-ortho-tyrosine. Thyroid hormones, as well as dopamine and p-nitrophenol, could also be used as substrates. The apparent K(m) value of the enzyme (designated the Dopa/tyrosine sulfotransferase) for L-Dopa, determined at a constant 14 μM of 3'-phosphoadenosine 5'-phosphosulfate, was 0.76 mM. The intact enzyme was found to be N-blocked when subjected to N- terminal sequencing. Three internal partial amino acid sequences, obtained by analyzing its proteolytic fragments, were found to be distinct from the homologous sequences of other known rat liver sulfotransferases. The deduced amino acid sequence of a full-length cDNA isolated from a rat liver cDNA library confirmed the identity of the Dopa/tyrosine sulfotransferase as a new type of aryl sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pMSG-CMV) harboring the full-length cDNA, a 33-kDa protein displaying enzymatic and immunological properties similar to those of the purified Dopa/tyrosine sulfotransferase was expressed.
AB - A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps (hydroxylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATP- agarose II). The minimum molecular weight of the purified enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be ~33,000. Gel filtration chromatography revealed a native molecular weight of 34,000, indicating the enzyme being present in the monomeric form. The purified sulfotransferase displayed enzymatic activities, with a pH optimum of 9.25, toward various tyrosine and 3,4-dihydroxyphenyl-alanine (Dopa) isomers, except DL-ortho-tyrosine. Thyroid hormones, as well as dopamine and p-nitrophenol, could also be used as substrates. The apparent K(m) value of the enzyme (designated the Dopa/tyrosine sulfotransferase) for L-Dopa, determined at a constant 14 μM of 3'-phosphoadenosine 5'-phosphosulfate, was 0.76 mM. The intact enzyme was found to be N-blocked when subjected to N- terminal sequencing. Three internal partial amino acid sequences, obtained by analyzing its proteolytic fragments, were found to be distinct from the homologous sequences of other known rat liver sulfotransferases. The deduced amino acid sequence of a full-length cDNA isolated from a rat liver cDNA library confirmed the identity of the Dopa/tyrosine sulfotransferase as a new type of aryl sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pMSG-CMV) harboring the full-length cDNA, a 33-kDa protein displaying enzymatic and immunological properties similar to those of the purified Dopa/tyrosine sulfotransferase was expressed.
UR - http://www.scopus.com/inward/record.url?scp=0029559445&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029559445&partnerID=8YFLogxK
U2 - 10.1074/jbc.270.51.30470
DO - 10.1074/jbc.270.51.30470
M3 - Article
C2 - 8530477
AN - SCOPUS:0029559445
VL - 270
SP - 30470
EP - 30478
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 51
ER -