Solubilization and purification of the Ni-stimulated arginine-vasopressin binding site of rat brain membranes.
Arginine vasopressin binding sites on rat brain membranes were solubilized and purified by affinity chromatography. Membrane protein solubilized with CHAPS bound arginine vasopressin (AVP) only in the presence of divalent cations. Specific binding to the solubilized tissue was maximally stimulated by Ni2+, and markedly stimulated by Co2+ (30% of maximal binding with Ni2+), Zn2+ (18%), and Fe2+ (11%), parallel to the effects of these ions on the binding of AVP to neural membranes. Binding to solubilized tissue was not stimulated by Mg2+, Cu2+, Mn2+, or Ca2+. In the presence of Ni2+, binding of AVP to solubilized tissue was reversible, and the dissociation constant (10.5 nM), pH optimum, and time course were virtually identical to those of the membrane-bound AVP binding site. Purification of solubilized AVP-binding proteins by affinity chromatography on AVP-sepharose followed by gel electrophoresis yielded a major band of 55 kdalton molecular weight when purified in the presence of 5 mM Mg2+, or a major band of 62 kdaltons when purified in the presence of 1-5 mM Ni2+ or 10 microM Zn2+. By means of a new binding assay involving conjugation of the 62 kdalton fraction to brain membranes, the extent of purification of AVP binding activity was 150-fold in the presence of Ni2+. We suggest that the 62 kdalton protein is a component of the Ni-stimulated AVP binding site.
Junig
J T
JT
Center for Brain Research, University of Rochester School of Medicine and Dentistry, NY 14642.
Abood
L G
LG
D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism*; D001923:Brain Chemistry; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D008297:Male; D008566:Membranes / Q000032:analysis; D009532:Nickel / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D011945:Receptors, Angiotensin / Q000302:isolation & purification* / Q000378:metabolism; D017483:Receptors, Vasopressin; D012995:Solubility
D002413:Cations, Divalent; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D001127:Arginine Vasopressin; D009532:Nickel