High-performance hydrophobic interaction chromatography of estrogen receptors and magnesium dependent protein kinase(s): detection of two molecular forms of estrogen receptors in the presence and absence of sodium molybdate.
The separation characteristics of estrogen receptors (ER) from human breast cancer were evaluated based on their hydrophobic properties. Results show that (1) two distinct hydrophobic isoforms of ER exist either in the presence of sodium molybdate (peaks MI and MII with retention times of 15-17 min and 24-26 min) or in its absence (peaks I and II with retention times of 25-27 min and 34-36 min respectively); (2) this is observed whether molybdate (MoO2-4) is added to prepared cytosol or to the buffer prior to homogenization; (3) isoform MII and I separated with similar retention times suggesting they are the same ER species; and (4) isoform MI (Rt = 15-17 min) is a distinct ER species from either MII/I (Rt = 25-28 min) or II (Rt = 34-36 min). The latter isoform represents a highly hydrophobic species seen only in the absence of MoO2-4. Finally, (5) MoO2-4 ions appear to interconvert the most hydrophobic species (II) into the least hydrophobic isoform (MI) with virtually no change in the quantity of isoform(s) MII/I. However, it cannot be ascertained if the II----MI interconversion proceeds via isoform MII/I. Isoform II may result from the interaction with the stationary phase via its DNA binding site since MoO2-4, which is suggested to directly interact with this site, selectively interacts with peak II. These results imply the usefulness of inclusion of receptor stabilizing reagents in the mobile phase for preserving receptor integrity and in elucidating the interrelationships of ER isoforms and associated macromolecules.
Hyder
S M
SM
Department of Biochemistry, University of Louisville, KY 40292.
Sato
N
N
Hogancamp
W
W
Wittliff
J L
JL
D001943:Breast Neoplasms / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D005260:Female; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008982:Molybdenum; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011960:Receptors, Estrogen / Q000302:isolation & purification* / Q000378:metabolism
D011960:Receptors, Estrogen; D008982:Molybdenum; C024687:sodium molybdate(VI); D011494:Protein Kinases; D008274:Magnesium