Structural and functional characterization of estrogen sulfotransferase isoforms: distinct catalytic and high affinity binding activities.
Estrogen sulfotransferase (EST) purified from the guinea pig (gp) adrenal gland consists of multiple charge isoforms with isoelectric points (pls) ranging from 6.5 to 5.2. Four individual isoforms were isolated for use in antibody production, as well as for tryptic fragmentation analysis. The multiple charge isoforms manifested a high degree of relatedness as evidence by complete immunocross-reactivity. This relatedness was further demonstrated by peptide mapping analysis, which revealed essentially identical elution profiles for three of the isoforms, whereas the fourth most acidic isoform, although similar to the other three isoforms, demonstrated some differences. To further explore the nature of the charge isoforms, native gpEST and EST expressed by CHO-K1 cells transfected with the gpEST cDNA were subjected to additional biochemical characterization. An interesting finding was that, in addition to EST activity, gpEST bound estradiol with a high affinity [dissociation constant (Kd) 10 nM]; furthermore, the binding activity was absolutely dependent on the cofactor, adenosine-3',5'-diphosphate (3',5'-ADP). An unexpected and novel finding was that only the most basic pl 6.5 isoform was catalytically active, while estradiol-binding activity was associated with a more acidic isoform (pl 5.5-5.2); however, it has not as yet been possible to definitively assign the binding activity to a specific isoform.(ABSTRACT TRUNCATED AT 250 WORDS)
Lee
Y C
YC
Section on Steroid Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Komatsu
K
K
Driscoll
W J
WJ
Strott
C A
CA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D016466:CHO Cells; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003488:Cyanogen Bromide; D015180:Electrophoresis, Gel, Two-Dimensional; D004958:Estradiol / Q000378:metabolism; D006168:Guinea Pigs; D015151:Immunoblotting; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008297:Male; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004958:Estradiol; D000244:Adenosine Diphosphate; D015238:Sulfotransferases; C020122:estrone sulfotransferase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003488:Cyanogen Bromide