PURIFICATION AND CHARACTERIZATION OF AN ADENOTIN-LIKE ADENOSINE BINDING-PROTEIN FROM HUMAN PLATELETS

被引:16
作者
FEIN, T [1 ]
SCHULZE, E [1 ]
BAR, J [1 ]
SCHWABE, U [1 ]
机构
[1] UNIV LEIPZIG, INST BIOCHEM, BEREICH MED, D-04103 LEIPZIG, GERMANY
关键词
ADENOTIN; HEAT SHOCK PROTEINS; RADIO-LIGAND BINDING; PURIFICATION; H-3] NECA;
D O I
10.1007/BF00170883
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
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)-H-3]-N-ethylcarboxamidoadenosine ([H-3]NECA) binds to the purified 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 electrophoresis of purified protein revealed a single band at 98 kDa. The 2-chlorosubstituted adenosine analogs 2-chloro-5'-N-methylcarboxamidoadenosine (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-chloroadenosine (C1A) > 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarbox-amidoadenosine (CGS 21 680) > R-N-6-phenylisopropyladenosine (R-PIA). Amino-terminal sequence analysis revealed homologies to endoplasmin, glucose regulated protein (GRP 94), tumor rejection antigen precursor (GP 96), and some stress related proteins.
引用
收藏
页码:374 / 380
页数:7
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