Kosk-Kosicka D
University of Maryland, School of Medicine, Department of Biological Chemistry, Baltimore 21201.
Mol Cell Biochem. 1990 Dec 20;99(2):75-81. doi: 10.1007/BF00230336.
We have compared properties of the red blood cell Ca2(+)-ATPase in two types of preparations: red cell membrane ghosts (enzyme in unfractionated membranes) and after purification (detergent-soluble enzyme). The Ca2(+)-ATPase activity was studied with respect to its requirement for: calmodulin, calcium, magnesium, monovalent cations, ionic strength, pH, and temperature. Sensitivity of the Ca2(+)-ATPase activity in the two preparations to anticalmodulin drugs and to engineered calmodulins with amino acid substitutions was determined. Finally, stoichiometry of the formation of phosphorylated enzyme intermediate (EP) and titrations of the ATP binding region with fluorescein 5'-isothiocyanate (FITC) were characterized. For the first time a high phosphorylation level of 2.0-2.4 mmol EP/mg of purified enzyme is reported. The two enzyme preparations have been found to be very similar with respect to the dependency of all the regulating factors described here. These results complement findings reported from various laboratories on the similarity of other kinetic properties as well as the similarity of modulation of the Ca2(+)-ATPase activity by phospholipids and proteolysis in the membraneous and purified enzyme. Thus, the purified detergent-soluble enzyme is very well suited for kinetic characterization of the red cell Ca2(+)-ATPase.
我们比较了两种制剂中红细胞Ca2(+)-ATP酶的特性：红细胞膜空壳（未分级膜中的酶）和纯化后（去污剂可溶酶）的制剂。针对其对钙调蛋白、钙、镁、单价阳离子、离子强度、pH值和温度的需求，研究了Ca2(+)-ATP酶活性。测定了两种制剂中Ca2(+)-ATP酶活性对抗钙调蛋白药物和具有氨基酸取代的工程钙调蛋白的敏感性。最后，对磷酸化酶中间体（EP）形成的化学计量关系以及用异硫氰酸荧光素5'-异硫氰酸盐（FITC）对ATP结合区域的滴定进行了表征。首次报道了纯化酶的高磷酸化水平为2.0 - 2.4 mmol EP/mg。已发现这两种酶制剂在本文所述的所有调节因子的依赖性方面非常相似。这些结果补充了各个实验室报道的关于其他动力学特性的相似性以及膜结合酶和纯化酶中磷脂和蛋白水解对Ca2(+)-ATP酶活性调节的相似性的研究结果。因此，纯化的去污剂可溶酶非常适合用于红细胞Ca2(+)-ATP酶的动力学表征。
