Stout J G, Kirley T L
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, OH 45267-0575.
J Biochem Biophys Methods. 1994 Jul;29(1):61-75. doi: 10.1016/0165-022x(94)90057-4.
The ecto-Mg-ATPase isolated from chicken gizzard smooth muscle was solubilized, purified and characterized. The purification did not require the use of expensive or specialized apparatus. The chromatographic and electrophoretic characteristics of the ecto-Mg-ATPase from chicken are similar to those reported earlier for the ecto-Mg-ATPase isolated from rabbit skeletal muscle transverse tubule membranes [1992, J. Biol. Chem. 267, 11777-11782]. One obvious difference found was that the solubilized chicken ecto-Mg-ATPase can be stimulated approximately 1900% by the lectin Concanavalin A (Con A) under the same conditions that the rabbit enzyme is inhibited by approximately 50%. This stimulatory effect of Con A is useful for following the purification, and also increases the specific activity of the chicken enzyme to a very high level similar to that observed for the rabbit enzyme. After purification of the solubilized chicken ecto-Mg-ATPase by three steps of anion exchange chromatography, as well as Con A and erythroagglutinating Phaseolus vulgaris (PHA-E) lectin affinity chromatographies, a single diffuse glycoprotein band at approximately 66 kDa is observed after SDS-PAGE. This protein could be deglycosylated to a core protein of 53 kDa. Thus, the chicken gizzard protein is very similar in molecular size to the rabbit skeletal muscle ecto-Mg-ATPase both before and after deglycosylation [1992, J. Biol. Chem. 267, 11777-11782]. The N-terminal sequence of the 66 kDa chicken gizzard protein was found to be: Ala-Arg-Arg-Ala-Ala-Ala-Val-Leu-Leu-Leu-Leu-Ala. This is a unique sequence which, while very different from the rabbit ecto-Mg-ATPase N-terminus, exhibits some of the same characteristics, since it contains basic residues as the second and third amino acids, with the remainder of the N-terminus being very hydrophobic in nature. Furthermore, the chicken gizzard ecto-Mg-ATPase can be separated from the adhesion molecule, truncated cadherin (T-cadherin) by anion exchange chromatography, and is therefore not identical to that protein, as had been recently proposed [1993, Arch. Biochem. Biophys. 303, 32-43].
从鸡砂囊平滑肌中分离出的胞外镁 - 三磷酸腺苷酶（ecto - Mg - ATPase）被增溶、纯化并进行了特性鉴定。纯化过程无需使用昂贵或专门的仪器。鸡的胞外镁 - 三磷酸腺苷酶的色谱和电泳特性与先前报道的从兔骨骼肌横管膜中分离出的胞外镁 - 三磷酸腺苷酶相似[1992年，《生物化学杂志》267卷，11777 - 11782页]。发现一个明显的差异是，在相同条件下，兔酶被抑制约50%，而增溶的鸡胞外镁 - 三磷酸腺苷酶可被凝集素伴刀豆球蛋白A（Con A）刺激约1900%。Con A的这种刺激作用有助于跟踪纯化过程，还能将鸡酶的比活性提高到与兔酶相似的非常高的水平。通过三步阴离子交换色谱以及Con A和红细胞凝集菜豆（PHA - E）凝集素亲和色谱对增溶的鸡胞外镁 - 三磷酸腺苷酶进行纯化后，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）后观察到一条约66 kDa的单一弥散糖蛋白带。该蛋白可去糖基化成为53 kDa的核心蛋白。因此，鸡砂囊蛋白在去糖基化前后的分子大小与兔骨骼肌胞外镁 - 三磷酸腺苷酶非常相似[1992年，《生物化学杂志》267卷，11777 - 11782页]。发现66 kDa鸡砂囊蛋白的N端序列为：丙氨酸 - 精氨酸 - 精氨酸 - 丙氨酸 - 丙氨酸 - 丙氨酸 - 缬氨酸 - 亮氨酸 - 亮氨酸 - 亮氨酸 - 亮氨酸 - 丙氨酸。这是一个独特的序列，虽然与兔胞外镁 - 三磷酸腺苷酶的N端非常不同，但表现出一些相同的特征，因为它的第二个和第三个氨基酸是碱性残基，N端的其余部分本质上非常疏水。此外，鸡砂囊胞外镁 - 三磷酸腺苷酶可通过阴离子交换色谱与黏附分子截短钙黏蛋白（T - cadherin）分离，因此与最近提出的该蛋白并不相同[1993年，《生物化学与生物物理学报》303卷，32 - 43页]。
