Nicotinamide adenine dinucleotide binding and promotion of enzyme activity: model based on affinity labeling of 3 alpha, 20 beta-hydroxysteroid dehydrogenase with a nucleoside.
Nicotinamide adenine dinucleotide binding and promotion of enzyme activity: model based on affinity labeling of 3 alpha, 20 beta-hydroxysteroid dehydrogenase with a nucleoside.
复制标题
烟酰胺腺嘌呤二核苷酸结合和酶活性促进：基于 3α、20β-羟基类固醇脱氢酶与核苷的亲和标记的模型。
DOI：
10.1021/bi00521a010
复制
发表时间：
1981
期刊：
影响因子：
2.9
通讯作者：
Samant,BR
中科院分区：
文献类型：
--
作者：
Sweet,F;Samant,BR
MethodsEnzyme化验(200年活动)进行匹配1 X 1厘米比色皿25摄氏度以下解决方案添加到最后一卷3.0 mL: 2.6毫升的0.05米磷酸钾缓冲,pH值6.5,0.100毫升整除的酶解(3 225µg, 2 o0-hsd 5.0毫升的0.05米磷酸钾缓冲,pH值7.0),0.200毫升的孕酮(0.1µ)乙醇,和0.100毫升的NADH(0.35µ)0.05米磷酸钾缓冲,pH值7.0。在340 nm处吸光度的初始线性下降（由于NADH的氧化）随时间的斜率被用来计算酶的活性。在贝克曼25型记录分光光度计中，在25±1℃下进行三次测定。除用0.200 mL（0.5µ）乙醇中的5a-二氢睾酮代替黄体酮外，按上述程序对酶孵育液中0.300 mL的等分进行3aase活性测定。亲合标记实验。FSA （12.25 mg; 25µ）溶解于乙二醇单甲醚（1.0 mL）中1使用缩写：FSA， 5'-[对-（氟磺酰）苯甲酰]腺苷；3a, 200 - hsd, 3a， 200 -羟基类固醇脱氢酶；NADH，烟酰胺腺嘌呤二核苷酸（还原形式）；NAD，烟酰胺腺嘌呤二核苷酸（氧化形式）；腺苷S'-二磷酸核糖；腺苷S'-二磷酸；腺苷S'-单磷酸；NMN，烟酰胺单核苷酸（氧化形式）；尼古丁酰胺单核苷酸（还原形式）。
MethodsEnzyme assays (for 200 activity) were conducted in matched 1 X 1 cm cuvettes at 25 C with the following solutions added to a final volume of 3.0 mL: 2.6 mL of 0.05 M potassium phosphate buffer, pH 6.5, 0.100 mL aliquot of enzyme solution (225 µg of 3a, 2O0-HSD in 5.0 mL of 0.05 M potassium phosphate buffer, pH 7.0), 0.200 mL of progesterone (0.1 µ) in ethanol, and 0.100 mL of NADH (0.35 µ) in 0.05 M potassium phosphate buffer, pH 7.0. The slope of the initial linear decrease in absorbance at 340 nm (due to oxidation of NADH) as a function of time was used to calculate enzyme activity. Assays were performed in triplicate at 25±1 C in a Beckman Model 25 recording spectrophotometer. Assays for 3aenzyme activity were performed on 0.300-mL aliquots from the enzyme incubation solutions according to the above procedure except that 0.200 mL of 5a-dihydrotestosterone (0.5 µ) in ethanol was used in place of progesterone. Affinity-Labeling Experiments. FSA (12.25 mg; 25 µ) dissolved in ethyleneglycol monomethyl ether (1.0 mL) served1 Abbreviations used: FSA, 5'-[p-(fluorosulfonyl) benzoyl] adenosine; 3a, 2O0-HSD, 3a, 2O0-hydroxysteroid dehydrogenase; NADH, nicotin-amide adenine dinucleotide (reduced form); NAD, nicotinamide adenine dinucleotide (oxidized form); ADPR, adenosine S'-diphosphate-ribose; ADP, adenosine S'-diphosphate; AMP, adenosine S'-monophosphate; NMN, nicotinamide mononucleotide (oxidized form); NMNH, nicotin-amide mononucleotide (reduced form).