Purification and characterization of the adenosine A2-like binding site from human placental membrane.
We have purified and characterized the adenosine A2-like binding site from human placental membranes. 5'-N-Ethylcarboxamido[2,8-3H]adenosine ([3H]NECA) binds to this site, with a Kd of 240 nM and a Bmax of 13.0 pmol/mg in human placental membranes. The adenosine A2-like binding site was purified after extraction from placental membranes with 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The purification included ammonium sulfate precipitation and concanavalin A, DEAE-Sephadex, and Sepharose 6B gel filtration chromatographies. The protein was purified 127-fold to homogeneity, with a final specific activity of 1.5-1.9 nmol/mg of protein and a 5.5-8.1% yield of binding activity from the membranes. The purified protein had similar binding properties and an identical potency order for displacement of [3H] NECA by adenosine analogs as the initial membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa which coeluted with [3H]NECA binding activity during Sepharose 6B gel filtration chromatography. In 0.1% Triton X-100, the binding complex has a Stokes radius of 70 A, a sedimentation coefficient of 6.9 S, and a partial specific volume of 0.698 ml/g. The detergent-protein complex has a calculated molecular mass of 230 kDa. The estimated frictional ratio is 1.5. The native binding complex appears to consist of a dimer of identical subunits. The function of this ubiquitous protein remains unclear.
Hutchison
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Department of Internal Medicine, University of Michigan, Ann Arbor 48109-0108.
Fox
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IH
D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005260:Female; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011487:Protein Conformation; D011983:Receptors, Purinergic / Q000302:isolation & purification* / Q000378:metabolism
D011983:Receptors, Purinergic