Purification and characterization of an adenotin-like adenosine binding protein from human platelets.
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 0.3% 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Further purification included Sepharose CL6B gel filtration, DEAE-Sepharose CL6B, 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-ethylcarboxamidoadenosine ([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-methylcarboxamidoadenosine (CIMECA) and 2-chloro-5'-N-ethylcarboxamidoadenosine (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-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine (CIA) > 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21,680) > R-N6-phenylisopropyl-adenosine (R-PIA). Amino-terminal sequence analysis revealed homologies to endoplasmin, glucose regulated protein (GRP94), tumor rejection antigen precursor (GP96), and some stress related proteins.
Fein
T
T
Pharmakologisches Institut, Universität Heidelberg, Germany.
Schulze
E
E
Bär
J
J
Schwabe
U
U
D000241:Adenosine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011869:Radioligand Assay
D002352:Carrier Proteins; D003902:Detergents; C064982:adenotin; D000241:Adenosine