The proton-driven dissociation of oestradiol-receptor dimers as a preparative tool. Isolation of a 32 kDa fragment from porcine uteri and assignment of C-terminal origin by partial sequencing.
Homodimers of the porcine oestradiol receptor dissociated at pH 6.4. The monomers reassociate after neutralization. This property is retained in a 32 kDa receptor fragment generated by co-adsorbed endopeptidases from cytosolic receptor bound to heparin-Sepharose. The fragment was purified by successive gel filtrations in the dimer and monomer states. Precipitations with ethanol and (NH4)2SO4 respectively served as concentrating steps. In all, 10-15 nmol of the homogeneous fragment were recovered from 8 kg batches of porcine uteri with a approximately 10(5)-fold enrichment and in approximately 20% yields. Its oestradiol-binding capacity was identical with that of the intact receptor. The N-terminus was blocked. Two decapeptides from a tryptic digest were sequenced. One of them corresponded to amino acids 353-362 of the human receptor, a sequence fully conserved in all species investigated. The second peptide differed in positions 553, 554 and 557 from the 549-558 sequence of the human protein.
Thole
H H
HH
Max-Planck-Institut für experimentelle Endokrinologie, Hannover, Federal Republic of Germany.
Jungblut
P W
PW
Jakob
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D011959:Receptors, Estradiol / Q000737:chemistry / Q000302:isolation & purification*; D016415:Sequence Alignment; D013552:Swine; D014599:Uterus / Q000737:chemistry* / Q000378:metabolism; D014981:Xenopus
D011959:Receptors, Estradiol