Purification of the human blood platelet thromboxane A2/prostaglandin H2 receptor protein.
The human platelet thromboxane A2/prostaglandin H2 receptor has been purified 6100-fold to apparent homogeneity by a three-step chromatographic procedure with an overall yield of 6%. A 6-fold purification of the receptor was first achieved by chromatography of 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS)-solubilized membrane proteins from human platelets on a diethylaminoethyl (DEAE)-Sepharose column. The DEAE eluate fractions containing receptor activity were then applied to a newly developed affinity column using the cyclohexyl derivative of SQ30,741 (SQ31,491) as the immobilized ligand. Elution of the receptor from the affinity column with BM13.177 yielded a further purification of 1700-fold. An additional 4-fold receptor purification from the affinity column eluate was achieved by HPLC using GPC 500 and GPC 100 columns connected in tandem. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the HPLC eluate containing purified receptor revealed a single, distinct band with a molecular weight of 55,000. The receptor binding activity was detected with [3H]SQ29,548 using a newly developed binding assay which involved immobilization of the receptor on polyethyleneimine-treated glass fiber filters. The binding of [3H]SQ29,548 to the purified receptor was time dependent, saturable, reversible and highly specific. Unlabeled SQ29,548, BM13.505, and U46619 (but not thromboxane B2 or 6-keto prostaglandin F1 alpha) competed for [3H]SQ29,548 binding to the purified receptor in a concentration-dependent manner. Scatchard analysis of [3H]SQ29,548 binding to the purified receptor revealed the presence of a single class of high-affinity binding sites, with a Kd of 4 nM and a Bmax of 17 nmol/mg protein.
Kim
S O
SO
Department of Medicinal Chemistry, University of Illinois, Chicago 60612.
Lim
C T
CT
Lam
S C
SC
Hall
S E
SE
Komiotis
D
D
Venton
D L
DL
Le Breton
G C
GC
D001792:Blood Platelets / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005231:Fatty Acids, Unsaturated; D006801:Humans; D006834:Hydrazines; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D015539:Platelet Activation; D011982:Receptors, Prostaglandin / Q000302:isolation & purification*; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism*
D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; D006834:Hydrazines; D008565:Membrane Proteins; D011982:Receptors, Prostaglandin; D044244:Receptors, Thromboxane A2, Prostaglandin H2; C054215:SQ 30741; D013928:Thromboxane A2; C045749:SQ 29548