PURIFICATION OF RAT-BRAIN, RABBIT AORTA, AND HUMAN PLATELET THROMBOXANE A(2) PROSTAGLANDIN H-2 RECEPTORS BY IMMUNOAFFINITY CHROMATOGRAPHY EMPLOYING ANTIPEPTIDE AND ANTIRECEPTOR ANTIBODIES

Citation
C. Borg et al., PURIFICATION OF RAT-BRAIN, RABBIT AORTA, AND HUMAN PLATELET THROMBOXANE A(2) PROSTAGLANDIN H-2 RECEPTORS BY IMMUNOAFFINITY CHROMATOGRAPHY EMPLOYING ANTIPEPTIDE AND ANTIRECEPTOR ANTIBODIES, The Journal of biological chemistry, 269(8), 1994, pp. 6109-6116
Citations number
35
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0021-9258
Volume
269
Issue
8
Year of publication
1994
Pages
6109 - 6116
Database
ISI
SICI code
0021-9258(1994)269:8<6109:PORRAA>2.0.ZU;2-Q
Abstract
In the present study, a new polyclonal antibody (TxAb) was raised agai nst native thromboxane A(2) (TXA(2))/prostaglandin H-2 (PGH(2)) recept or protein. Previously developed anti-peptide antibodies (P(1)Ab, P(2) Ab) and TxAb were then used to prepare immunoaffinity columns to purif y TXA(2)/PGH(2) receptors from platelets, brain, and aorta. In platele ts, SDS-polyacrylamide gel electrophoresis revealed the purification o f a 55-kDa protein by each affinity column. Identification of this pro tein as the TXA(2)/PGH(2) receptor was based on: 1) an identical elect rophoretic mobility to authentic receptor; 2) immunoblotting of TxAb a gainst P(1)Ab and P(2)Ab-purified protein; 3) immunoblotting of P(1)Ab /P(2)Ab against TxAb-purified protein; and 4) specific [H-3]SQ29,548 b inding to TxAb-purified protein. P(1)Ab/TxAb purification of receptors from brain revealed a major protein band at 55 kDa. Furthermore, the eluates from ligand affinity chromatography confirmed the presence of this 55-kDa protein in brain (which was immunoblotted with TxAb), and contained specific [H-3]SQ29,548 binding. In addition to the 55-kDa pr otein, P(1)Ab/TxAb also purified a minor protein in brain at 52 kDa, w hich when concentrated, cross-blotted with TxAb and P(1)Ab. This findi ng indicates sequence homology between the 55- and 52-kDa proteins. In dependent identification of brain TXA(2)/PGH(2) receptors was provided by P(2)Ab/TxAb immunohistochemistry, which demonstrated specific labe ling of discrete myelin-containing fiber tracts. P(2)Ab/TxAb purificat ion of TXA(2)/PGH(2) receptors from aorta also revealed a major protei n band at 55 kDa and a minor band at 52 kDa. These results represent t he first purification of TXA(2)/PGH(2) receptors from either brain or aorta.