Levitsky D O, Syrbu S I, Cherepakhin V V, Rokhlin O V
Eur J Biochem. 1987 Apr 15;164(2):477-84. doi: 10.1111/j.1432-1033.1987.tb11081.x.
Purified sarcoplasmic reticulum (SR) vesicles from dog heart were used as an antigen to produce monoclonal antibodies (mAbs) to the Ca2+-ATPase. Nine of twelve clones of hybridoma cells produce mAbs which cross-react with seven SR preparation isolated from cardiac and skeletal muscles of various species. Three mAbs of IgM type interact with the 45-kDa tryptic fragment of rabbit skeletal muscle Ca2+-ATPase and markedly inhibit Ca2+ uptake (by 95%) and ATPase activity (by 80%) and decrease (by 30-50%) the steady-state level of the Ca2+-ATPase phosphoenzyme. The ATPase activity could be completely blocked by one of these mAbs if the incubation medium was supplemented with 2 microM orthovanadate. On the other hand, when SR vesicles were treated with increasing concentrations of a nonionic detergent C12E8, the inhibiting effect of mAb 4B4 is diminished. It is concluded that the mAbs inhibit the Ca2+-ATPase only if the enzyme exists in an oligomeric form. The inhibition of the SR activities is due to an effect of the mAbs on the whole active center of the enzyme, rather than on a single partial reaction.
用从犬心脏中纯化得到的肌浆网（SR）囊泡作为抗原，制备针对Ca2 + -ATP酶的单克隆抗体（mAb）。杂交瘤细胞的12个克隆中有9个产生的mAb与从不同物种的心脏和骨骼肌中分离得到的7种SR制剂发生交叉反应。3种IgM型mAb与兔骨骼肌Ca2 + -ATP酶的45 kDa胰蛋白酶片段相互作用，并显著抑制Ca2 +摄取（95%）和ATP酶活性（80%），并使Ca2 + -ATP酶磷酸酶的稳态水平降低（30 - 50%）。如果在孵育培养基中添加2 microM原钒酸盐，其中一种mAb可完全阻断ATP酶活性。另一方面，当用浓度不断增加的非离子去污剂C12E8处理SR囊泡时，mAb 4B4的抑制作用减弱。得出的结论是，只有当酶以寡聚体形式存在时，mAb才会抑制Ca2 + -ATP酶。SR活性的抑制是由于mAb对酶的整个活性中心产生影响，而不是对单个部分反应产生影响。
