Purification of rat brain, rabbit aorta, and human platelet thromboxane A2/prostaglandin H2 receptors by immunoaffinity chromatography employing anti-peptide and anti-receptor antibodies.
In the present study, a new polyclonal antibody (TxAb) was raised against native thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor protein. Previously developed anti-peptide antibodies (P1Ab, P2Ab) and TxAb were then used to prepare immunoaffinity columns to purify TXA2/PGH2 receptors from platelets, brain, and aorta. In platelets, SDS-polyacrylamide gel electrophoresis revealed the purification of a 55-kDa protein by each affinity column. Identification of this protein as the TXA2/PGH2 receptor was based on: 1) an identical electrophoretic mobility to authentic receptor; 2) immunoblotting of TxAb against P1Ab and P2Ab-purified protein; 3) immunoblotting of P1Ab/P2Ab against TxAb-purified protein; and 4) specific [3H]SQ29,548 binding to TxAb-purified protein. P1Ab/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 [3H]SQ29,548 binding. In addition to the 55-kDa protein, P1Ab/TxAb also purified a minor protein in brain at 52 kDa, which when concentrated, cross-blotted with TxAb and P1Ab. This finding indicates sequence homology between the 55- and 52-kDa proteins. Independent identification of brain TXA2/PGH2 receptors was provided by P2Ab/TxAb immunohistochemistry, which demonstrated specific labeling of discrete myelin-containing fiber tracts. P2Ab/TxAb purification of TXA2/PGH2 receptors from aorta also revealed a major protein band at 55 kDa and a minor band at 52 kDa. These results represent the first purification of TXA2/PGH2 receptors from either brain or aorta.
Borg
C
C
Department of Pharmacology, University of Illinois, Chicago 60612.
Lim
C T
CT
Yeomans
D C
DC
Dieter
J P
JP
Komiotis
D
D
Anderson
E G
EG
Le Breton
G C
GC
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001011:Aorta / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011463:Prostaglandins H / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011982:Receptors, Prostaglandin / Q000276:immunology / Q000302:isolation & purification*; D017482:Receptors, Thromboxane / Q000276:immunology / Q000302:isolation & purification*; D044244:Receptors, Thromboxane A2, Prostaglandin H2
D000906:Antibodies; D011463:Prostaglandins H; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; D044244:Receptors, Thromboxane A2, Prostaglandin H2