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Purification and characterization of an adenotin-like adenosine binding protein from human platelets

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Abstract

A low-affinity adenosine binding protein (adenotin) was purified from human platelet membranes by a four-step procedure. Purification was achieved after extraction from human platelet membranes with 0.30% 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Further purification included Sepharose CL 6 B gel filtration, DEAE-Sepharose CL 6 B, and hydroxylapatite chromatography. The protein was purified 884-fold to homogeneity with a 25% yield of binding activity from the membranes. 5′-[8(n)-3H]-N-ethyl-carboxamidoadenosine ([3H]NECA) binds to the purified protein with a KD of 155 (144–167) nmol/l and a Bmax of 1.85±0.10 nmol/mg of protein. Sodium dodecylsulfate polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa. The 2-chloro-substituted adenosine analogs 2-chloro-5′-N-methylcarb-oxamidoadenosine (CIMECA) and 2-chloro-5′-N-ethyl-carboxamidoadenosine (CINECA) were identified as new high affinity ligands of the purified protein showing Ki values of 18 nmol/l and 28 nmol/l, respectively. The low-affinity adenosine binding protein showed a pharmacological profile as follows: CIMECA > 5′-N-ethylcarbox-amidoadenosine (NECA) > 2-chloroadenosine (CIA) > 2-[4-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarbox amidoadenosine (CGS 21680) > R-N6-phenylisopropyl-adenosine (R-PIA).

Amino-terminal sequence analysis revealed homologies to endoplasmin, glucose regulated protein (GRP 94), tumor rejection antigen precursor (GP96), and some stress related proteins.

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Abbreviations

CGS 21,680:

2-[4-(2-carboxyethyl)phenethylamino]-5′N-ethylcarboxamidoadenosine

CIA:

2-chloroadenosine

CIMECA:

2-chloro-5′-N-methylcarboxamidoadenosine

CINECA:

2-chloro-5′-Nethylcarboxamidoadenosine

CHAPS:

3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate

NECA:

5′-N-ethylcarboxamidoaden-osine

DPCPX:

1,3-dipropyl-8-cyclopentylxanthine

MECA:

5′-N-meth-ylcarboxamidoadenosine

R-PIA:

R-N6-phenylisopropyladenosine

SDS-PAGE:

sodium dodecylsulfate polyacrylamide gel electrophoresis

XAC:

xanthine amine congener, 8-{4-8[([{(2-aminoethyl)amino}carbonyl]-methyl)oxy]phenyl}-1,3-dipropyl-xanthine

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Authors and Affiliations

  1. Pharmakologisches Institut, Universität Heidelberg, Im Neuenheimer Feld 366, D-69120, Heidelberg, Germany

    T. Fein, E. Schulze & U. Schwabe

  2. Institut für Biochemie, Bereich Medizin, Universität Leipzig, Liebigstrasse 16, D-04103, Leipzig, Germany

    J. Bär

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  1. T. Fein
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  2. E. Schulze
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  3. J. Bär
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  4. U. Schwabe
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Correspondence to: T. Fein at the above adress

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Fein, T., Schulze, E., Bär, J. et al. Purification and characterization of an adenotin-like adenosine binding protein from human platelets. Naunyn-Schmiedeberg’s Arch Pharmacol 349, 374–380 (1994). https://doi.org/10.1007/BF00170883

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  • DOI: https://doi.org/10.1007/BF00170883

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