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 ac hieved after extraction from human platelet membranes with 0.3% holami dopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Further purifica tion included Sepharose CL6B gel filtration, DEAE-Sepharose CL6B, and hydroxylapatite chromatography. The protein was purified 884-fold to h omogeneity with a 25% yield of binding activity from the membranes. 5' -[8(n)-H-3]-N-ethylcarboxamidoadenosine ([H-3]NECA) binds to the purif ied protein with a K-D of 155 (144-167) nmol/l and a B-max of 1.85 +/- 0.10 nmol/mg of protein. Sodium dodecylsulfate polyacrylamide gel ele ctrophoresis of purified protein revealed a single band at 98 kDa. The 2-chlorosubstituted adenosine analogs 2-chloro-5'-N-methylcarboxamido adenosine (C1MECA) and 2-chloro-5'-N-ethylcarboxamidoadenosine (C1NECA ) were identified as new high affinity ligands of the purified protein showing K-i values of 18 nmol/l and 28 nmol/l, respectively. The low- affinity adenosine binding protein showed a pharmacological profile as follows: C1MECA > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroade nosine (C1A) > yl)phenethylamino]-5'-N-ethylcarbox-amidoadenosine (CGS 21 680) > R-N-6-phenylisopropyladenosine (R-PIA). Amino-terminal sequ ence analysis revealed homologies to endoplasmin, glucose regulated pr otein (GRP 94), tumor rejection antigen precursor (GP 96), and some st ress related proteins.