2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins.
2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins.
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2-和8-叠氮基光亲和探针。
DOI：
10.1021/bi00424a023
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Haley,BE
中科院分区：
文献类型：
--
作者：
Suhadolnik,RJ;Karikó,K;SobolJr,RW;Li,SW;Reichenbach,NL;Haley,BE
材料和方法材料。2/，5/-腺苷酸三聚体和四聚体5/-三磷酸、2/，5/-和3/，5/-腺苷酸三聚体核心和聚（U）获自Pharmacia;[a-32 P] ATP（410 Ci/mmol），p3A4 [32P] pCp（3000 Ci/mmol）和[32 P] pCp（3000 Ci/mmol）来自阿默舍姆;细胞培养用培养基和血清来自GIBCO; T4 RNA连接酶来自Bethesda Research Laboratories;兔网织红细胞裂解物来自绿色海葵。2-5核心纤维素是R.西尔弗曼小鼠干扰素（1.5 × 107单位/mg蛋白质）由D.穆拉斯科细胞培养。L929细胞在补充有5%牛血清的Dulbecco改良Eagle培养基中维持单层培养。如有需要，用200单位/mL小鼠干扰素处理单层细胞20小时。细胞提取物。通过胰蛋白酶消化收集L929细胞，用PBS洗涤一次，并如所述在甘油缓冲液中裂解（Kariko & Ludwig，1985）。2-叠氮基ATP和8-叠氮基ATP的化学合成由2-氯腺苷（Sigma）合成2-N3 ATP（Czarnecki等人，1982年）。8-N3 ATP是通过Michelson（1964）的方法从8-N3 AMP合成的。如前所述由AMP合成8-N3 AMP（Czarnecki等人，1979年）。如所报道的制备和纯化[γ-32 P] 2-N3 ATP和[α-32 P] 8-N3 ATP（Haley，1977; Potter & Haley，1982）。通过HPLC和TLC分析测定2-和8-N3 ATP的纯度（Czarnecki等人，1982年）。经DEAE-纤维素柱层析和HPLC纯化后，2-和8-N3 ATP显示不含任何污染的ATP、8-溴ATP、8-氨基ATP或核苷酸3，（2 ′）-磷酸。
Materials and Methods Materials. 2/, 5/-Adenylate trimer and tetramer 5'-tri-phosphates, 2', 5'-and 3', 5'-adenylate trimer cores, and poly (U) were obtained from Pharmacia;[a-32P] ATP (410 Ci/mmol), p3A4 [32P] pCp (3000 Ci/mmol), and [32P] pCp (3000 Ci/mmol) were from Amersham; media and sera for cell culture were from GIBCO; T4 RNA ligase was from Bethesda Research Laboratories; rabbit reticulocyte lysates were from Green Hectares. 2-5 A core-cellulose was a generous gift from Dr. R. Silverman. Mouse interferon (1.5 X 107 units/mg of protein) was generously supplied by Dr. D. Murasko. Cell Culture. L929 cells were maintained in monolayer culture in Dulbecco’s modified Eagle medium supplemented with 5% bovine serum. Monolayers were treated where indicated with 200 units/mL mouse interferon for 20 h. Cell Extracts. L929 cells were collected by trypsinization, washed once with PBS, and lysed in glycerol buffer as de-scribed (Kariko & Ludwig, 1985). Chemical Synthesis of 2-AzidoATP and 8-AzidoATP. 2-N3ATP was synthesized from 2-chloroadenosine (Sigma)(Czarnecki et al., 1982). 8-N3ATP was synthesized from 8-N3AMP by the method of Michelson (1964). 8-N3AMP was synthesized from AMP as previously described (Czarnecki et al., 1979).[y-32P] 2-N3ATP and [a-32P] 8-N3ATP were prepared and purified as reported (Haley, 1977; Potter & Haley, 1982). Purity of the 2-and 8-N3ATP was determined by HPLC and TLC analysis (Czarnecki et al., 1982). Following purification by DEAE-cellulose column chromatography and HPLC, the 2-and 8-N3ATP were shown to be free of any contaminating ATP, 8-bromoATP, 8-aminoATP, or nucleotide 3,(2')-phosphates.