Pfeuffer E, Dreher R M, Metzger H, Pfeuffer T
Proc Natl Acad Sci U S A. 1985 May;82(10):3086-90. doi: 10.1073/pnas.82.10.3086.
The guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG)-activated adenylate cyclase from rabbit myocardial membranes was purified approximately equal to 60,000-fold to a specific activity of 15 mumol X mg-1 X min-1 by Lubrol PX extraction, affinity chromatography, and gel permeation HPLC. The major purification (greater than 2000-fold) was achieved by affinity chromatography on forskolin-Sepharose, a method previously developed in this laboratory. The final product appeared as two major peptides of Mr 150,000 and 42,000 and one minor peptide of Mr 45,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis. It is suggested that the Mr 42,000 and 150,000 components represent the alpha-subunit of the stimulatory guanine nucleotide binding regulatory protein (GS) and the catalytic unit, respectively, because upon crosslinking of a reconstituted adenylate cyclase containing the [32P]ADP-ribosylated alpha-subunit of GS (Mr, 42,000), a single radiolabeled product of Mr 190,000 appeared on NaDodSO4/polyacrylamide gels. Further identification is based on the correlation of the Mr 150,000/42,000 bands with enzymatic activity when the purified enzyme was analyzed by various chromatographic procedures.
通过Lubrol PX抽提、亲和层析和凝胶渗透高效液相色谱法，从兔心肌细胞膜中纯化出鸟苷5'-[β,γ-亚氨基]三磷酸（p[NH]ppG）激活的腺苷酸环化酶，纯化倍数约为60000倍，比活性达到15 μmol·mg⁻¹·min⁻¹。主要纯化步骤（超过2000倍）是通过在毛喉素-琼脂糖上进行亲和层析实现的，这是本实验室先前开发的一种方法。当通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳分析时，最终产物呈现为两条主要肽段，分子量分别为150000和42000，以及一条次要肽段，分子量为45000。有人提出，分子量为42000和150000的组分分别代表刺激性鸟嘌呤核苷酸结合调节蛋白（GS）的α亚基和催化单位，因为在对含有经[³²P]ADP-核糖基化的GSα亚基（分子量42000）的重组腺苷酸环化酶进行交联后，在十二烷基硫酸钠/聚丙烯酰胺凝胶上出现了一条分子量为190000的单一放射性标记产物。当通过各种色谱方法分析纯化的酶时，基于分子量为150000/42000的条带与酶活性的相关性进行了进一步鉴定。
