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Title: Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

Abstract

The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-({sup 3}H)ethylcarboxamidoadenosine (({sup 3}H)NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the ({sup 3}H)NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.

Authors:
; ; ;  [1]
  1. (University Hospital, Ann Arbor, MI (USA))
Publication Date:
OSTI Identifier:
6828305
Resource Type:
Journal Article
Resource Relation:
Journal Name: Molecular Pharmacology; (USA); Journal Volume: 37:4
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ADENOSINE; RECEPTORS; FRACTIONATION; BIOCHEMICAL REACTION KINETICS; BLOOD PLATELETS; CELL MEMBRANES; MAN; PLACENTA; RADIOASSAY; TRACER TECHNIQUES; TRITIUM COMPOUNDS; ULTRACENTRIFUGATION; VASODILATORS; ANIMALS; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY FLUIDS; CARDIOVASCULAR AGENTS; CELL CONSTITUENTS; CENTRIFUGATION; DRUGS; FETAL MEMBRANES; HYDROGEN COMPOUNDS; ISOTOPE APPLICATIONS; KINETICS; MAMMALS; MATERIALS; MEMBRANE PROTEINS; MEMBRANES; NUCLEOSIDES; NUCLEOTIDES; ORGANIC COMPOUNDS; PRIMATES; PROTEINS; REACTION KINETICS; RIBOSIDES; SEPARATION PROCESSES; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Zolnierowicz, S., Work, C., Hutchison, K., and Fox, I.H. Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein. United States: N. p., 1990. Web.
Zolnierowicz, S., Work, C., Hutchison, K., & Fox, I.H. Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein. United States.
Zolnierowicz, S., Work, C., Hutchison, K., and Fox, I.H. 1990. "Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein". United States. doi:.
@article{osti_6828305,
title = {Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein},
author = {Zolnierowicz, S. and Work, C. and Hutchison, K. and Fox, I.H.},
abstractNote = {The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-({sup 3}H)ethylcarboxamidoadenosine (({sup 3}H)NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the ({sup 3}H)NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.},
doi = {},
journal = {Molecular Pharmacology; (USA)},
number = ,
volume = 37:4,
place = {United States},
year = 1990,
month = 4
}
  • Considerable discrepancies exist in the literature concerning the size and activity of bovine placental lactogen. Our bovine placental lactogen purification preparations were 20% as active as bovine PRL (bPRL) on a per weight basis when compared to bPRL in a lactogenic radioreceptor assay. To identify the active component in these preparations, the proteins were radioiodinated and bound to membrane receptors in the presence and absence of competing hormones, bPRL, and bovine GH (bGH). After centrifugation, membrane pellets were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gels were autoradiographed. Only one radioiodinated protein band was present. This protein wasmore » displaced in the presence of competing hormone and comigrated with a major component of the purification preparations. In radioreceptor assays the active component was as active as bPRL and bGH. From the migration of protein standards included with the radioiodinated purification preparations in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we estimate that the protein is 32,000 mol wt--about 10,000 mol wt larger than placental lactogens isolated in other species. The possibility that the active molecule was a precursor protein was investigated by examining proteins secreted by bovine placenta tissue cultures. The binding activity in these secretions, as well as in the purification preparations, eluted between ovalbumin (43,000 mol wt) and bPRL (22,000 mol wt) under nondenaturing conditions using high performance gel filtration chromatography. Analysis of this secreted protein, also by binding to membrane receptors, showed that the protein had the same molecular weight as that isolated from the purification preparations and was specifically displaced by the same hormones.« less
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