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

Abstract

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.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Univ. of Illinois College of Medicine, Chicago
OSTI Identifier:
5509362
Report Number(s):
CONF-8604222-
Journal ID: CODEN: FEPRA; TRN: 86-026534
Resource Type:
Conference
Journal Name:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
Additional Journal Information:
Journal Volume: 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
Subject:
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

Citation Formats

Lim, C T, Kattelman, E J, Arora, S K, Venton, D L, and Le Breton, G C. Partial purification and identification of the thrombozane A/sub 2//prostaglandin H/sub 2/ receptor protein in human platelets. United States: N. p., 1986. Web.
Lim, C T, Kattelman, E J, Arora, S K, Venton, D L, & Le Breton, G C. Partial purification and identification of the thrombozane A/sub 2//prostaglandin H/sub 2/ receptor protein in human platelets. United States.
Lim, C T, Kattelman, E J, Arora, S K, Venton, D L, and Le Breton, G C. 1986. "Partial purification and identification of the thrombozane A/sub 2//prostaglandin H/sub 2/ receptor protein in human platelets". United States.
@article{osti_5509362,
title = {Partial purification and identification of the thrombozane A/sub 2//prostaglandin H/sub 2/ receptor protein in human platelets},
author = {Lim, C T and Kattelman, E J and Arora, S K and Venton, D L and Le Breton, G C},
abstractNote = {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.},
doi = {},
url = {https://www.osti.gov/biblio/5509362}, journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 45:3,
place = {United States},
year = {Sat Mar 01 00:00:00 EST 1986},
month = {Sat Mar 01 00:00:00 EST 1986}
}

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