Sequence analysis, in vitro translation, and expression of the cDNA for rat liver minoxidil sulfotransferase.
A cDNA encoding minoxidil sulfotransferase (Mx-ST), a rat liver cytosolic sulfotransferase that catalyzes the 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfate conjugation of minoxidil and p-nitrophenol, has been isolated from a lambda gt11 cDNA library constructed from poly(A)+ RNA isolated from female Sprague-Dawley rat liver. The largest cDNA, designated Mx-STb, consists of 1245 base pairs and contains an open reading frame of 291 amino acids. The predicted size of the protein translated by Mx-STb is 33,909 Da; however, the molecular mass of the pure protein [Biochem. J. 270:721-728 (1990)] is estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 35,000 Da. The size of the protein obtained by in vitro translation of Mx-STb is identical to that of the pure protein. Results of initial studies of the expression of Mx-STb in COS-1 cells indicate that the expressed protein displays characteristic Mx-ST and p-nitrophenol sulfotransferase activity, is recognized by rabbit polyclonal antibodies raised against pure rat liver Mx-ST, and migrates at approximately 35,000 Da during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This paper presents the cloning and expression of a rat phenol sulfotransferase for which the physical, immunological, and kinetic properties are known. Isolation of the cDNA for Mx-ST will aid in the investigation of the heterogeneity, the tissue localization, and the characterization of the kinetic properties of this important drug-metabolizing enzyme, with respect to other similar phenol sulfotransferases present in rat liver cytosol.
Hirshey
S J
SJ
Department of Pharmacology, University of Rochester, New York 14642.
Dooley
T P
TP
Reardon
I M
IM
Heinrikson
R L
RL
Falany
C N
CN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D015870:Gene Expression / Q000235:genetics*; D007668:Kidney / Q000166:cytology / Q000201:enzymology / Q000502:physiology; D008099:Liver / Q000201:enzymology* / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D015238:Sulfotransferases / Q000235:genetics*; D014162:Transfection
D004247:DNA; D015238:Sulfotransferases; C066827:minoxidil sulfotransferase