Partial purification and identification of the thrombozane A/sub 2//prostaglandin H/sub 2/ receptor protein in human platelets

Conference · Sat Mar 01 00:00:00 EST 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5509362

Lim, C T; Kattelman, E J; Arora, S K; Venton, D L; Le Breton, G C

The thromboxane A/sub 2//prostaglandin H/sub 2/ (TXA/sub 2//PGH/sub 2/) receptor antagonist (/sup 3/H)-13-azaprostanoic acid (13-APA) was used to identify and purify the platelet TXA/sub 2//PGH/sub 2/ receptor protein. Optimal solubilization of the 13-APA binding protein was achieved by extraction with 3-((3-cholamidopropyl)dimethyl-ammonio)-1-propanesulfonate (CHAPS) detergent. Preliminary purification of the crude solubilized membrane fraction was performed by gel filtration chromatography using a Sepharose 4B column. Further purification was accomplished by high performance liquid chromatography (HPLC) using a Synchropak GPC-500 column. The HPLC protein profile revealed two protein peaks, only one of which was enriched in (/sup 3/H)-13-APA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of this peak revealed two bands with molecular weights of 65,000 and 60,000 daltons. In binding studies using the 60,000 dalton-enriched subfraction, unlabelled 13-APA, the TXA/sub 2//PGH/sub 2/ mimetic U46619 and the TXA/sub 2//PGH/sub 2/ antagonist SQ 29,548 all competed for (/sup 3/H)-13-APA binding whereas TXB/sub 2/ did not compete for binding. Heat denaturation of this subfraction resulted in a complete loss of binding activity. These findings indicate that a protein of approximately 60,000 daltons represents the human platelet TXA/sub 2//PGH/sub 2/ receptor.

• Research Organization: Univ. of Illinois College of Medicine, Chicago
• OSTI ID: 5509362
• Report Number(s): CONF-8604222-; TRN: 86-026534
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:3; Conference: 70. annual meeting of the Federation of American Society for Experimental Biology, St. Louis, MO, USA, 13 Apr 1986
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PROSTAGLANDINS
RECEPTORS
PROTEINS
PURIFICATION
BIOCHEMICAL REACTION KINETICS
BLOOD PLATELETS
CHROMATOGRAPHY
ELECTROPHORESIS
LIQUID COLUMN CHROMATOGRAPHY
MAN
MOLECULAR WEIGHT
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ANIMALS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MAMMALS
MATERIALS
MEMBRANE PROTEINS
ORGANIC COMPOUNDS
PRIMATES
REACTION KINETICS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques