Evidence from nitrogen-15 and solvent deuterium isotope effects on the chemical mechanism of adenosine deaminase.
Evidence from nitrogen-15 and solvent deuterium isotope effects on the chemical mechanism of adenosine deaminase.
复制标题
来自氮 15 和溶剂氘同位素对腺苷脱氨酶化学机制影响的证据。
DOI：
10.1021/bi00397a027
复制
发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Cleland,WW
中科院分区：
文献类型：
--
作者：
Weiss,PM;Cook,PF;Hermes,JD;Cleland,WW
材料和方法材料。小牛肠粘膜甘油中的腺苷脱氨酶、牛肝中甘油中的谷氨酸脱氢酶、-酮戊二酸、还原型/3-烟酰胺腺嘌呤二核苷酸(DPNH)和腺苷来自Sigma。NaOD和A-溴乙酰胺来自奥尔德里奇。D20(99.9原子%D)来自剑桥同位素实验室。氨的测定采用纳氏SigmaAmmonia显色剂。AG 50W-X8阳离子交换树脂为Bio-Rad.7，8-二氢-8-氧代腺苷(6-氨基-9-？-呋喃-9-核糖基-9H-嘌呤-8(7H)-酮)。三氧乙酰腺苷(II)是根据Bredereck和Martini(1947)制备的。将50g(187 Mmol)腺苷(I)溶于740毫升的干吡啶中。加入530毫升乙酸酐，在25℃下搅拌2.5小时，真空脱除溶剂，用300毫升乙醇洗涤残渣两次。淡黄色糖浆用150毫升冷乙醇在4℃下重结晶，收集白色晶体，在真空下干燥，得到47g II(I得率)。薄层层析法
Materials and MethodsMaterials. Adenosine deaminase from calf intestinal mucosa in glycerol, glutamate dehydrogenase from bovine liver in glycerol,-ketoglutarate, reduced/3-nicotinamide adenine dinucleotide (DPNH), and adenosine were from Sigma. NaOD and A-bromoacetamide were from Aldrich. D20 (99.9 atom% D) was from Cambridge Isotope Laboratories. Assay of ammonia was with the Nessler’s-based SigmaAmmonia Color Reagent. AG 50W-X8 cation-exchange resin was from Bio-Rad.7, 8-Dihydro-8-oxoadenosine (6-Amino-9-ß--ribo-furanosyl-9H-purin-8 (7H)-one). Tri-O-acetyladenosine (II) was prepared according to Bredereck and Martini (1947). A total of 50 g (187 mmol) of adenosine (I) was dissolved in 740 mL of dry pyridine. To this was added 530 mL of acetic anhydride, and this solution was allowed to stir at 25 C for 2.5 h. Solvent was removed in vacuo and the residue washed twice with 300 mL of ethanol. The yellowish syrup was re-crystallized from 150 mL of cold ethanol at 4 C. The white crystals were collected and dried under vacuum to yield 47 g of II (64% yield from I). Thin-layer chromatography of this