Nunnally M H, Blumenthal D K, Krebs E G, Stull J T
Department of Physiology, University of Texas Health Science Center at Dallas 75235-9040.
Biochemistry. 1987 Sep 8;26(18):5885-90. doi: 10.1021/bi00392a046.
A synthetic peptide representing the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase (K-R-R-W-K-K-N-F-I-A-V-S-A-A-N-R-F-K-K-I-S-S-S-G-A-L) was used as an antigen to produce a monoclonal antibody. The antibody (designated MAb RSkCBP1, of the IgM class) reacted with similar affinity (KD approximately 20 nM) by competitive enzyme-linked immunoassay (ELISA) with the antigen peptide and intact rabbit skeletal muscle myosin light chain kinase. MAb RSkCBP1 inhibited rabbit skeletal muscle myosin light chain kinase activity competitively with respect to calmodulin (Ki = 20 nM). The antibody also inhibited myosin light chain kinase activity in extracts of skeletal muscle from several mammalian species (rabbit, sheep, and bovine) and an avian species (chicken). The concentration of MAb RSKCBP1 required for 50% inhibition of enzyme activity was similar for the mammalian species (80 nM) but was significantly higher for the avian species (1.2 microM). A competitive ELISA protocol was used to analyze weak cross-reactivity to other calmodulin-binding peptides and proteins. This assay demonstrated no cross-reactivity with the venom peptides melittin or mastoparan; smooth muscle myosin light chain kinases from hog carotid, bovine trachea, or chicken gizzard; bovine brain calmodulin-dependent calcineurin; or rabbit skeletal muscle troponin I. These data support the contention that the synthetic peptide used as the antigen represents the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase and that the calmodulin-binding domains of different calmodulin-regulated proteins may have distinct primary and/or higher order structures.
一种代表兔骨骼肌肌球蛋白轻链激酶钙调蛋白结合结构域的合成肽（K-R-R-W-K-K-N-F-I-A-V-S-A-A-N-R-F-K-K-I-S-S-S-G-A-L）被用作抗原以产生单克隆抗体。该抗体（命名为MAb RSkCBP1，IgM类）通过竞争性酶联免疫吸附测定（ELISA）与抗原肽和完整的兔骨骼肌肌球蛋白轻链激酶以相似的亲和力（KD约为20 nM）发生反应。MAb RSkCBP1相对于钙调蛋白竞争性地抑制兔骨骼肌肌球蛋白轻链激酶活性（Ki = 20 nM）。该抗体还抑制了几种哺乳动物（兔、绵羊和牛）以及一种禽类（鸡）骨骼肌提取物中的肌球蛋白轻链激酶活性。对于哺乳动物，抑制酶活性50%所需的MAb RSKCBP1浓度相似（80 nM），但对于禽类则显著更高（1.2 microM）。采用竞争性ELISA方案分析与其他钙调蛋白结合肽和蛋白质的弱交叉反应性。该测定表明与蜂毒肽蜂毒明肽或mastoparan、猪颈动脉、牛气管或鸡砂囊的平滑肌肌球蛋白轻链激酶、牛脑钙调蛋白依赖性钙调神经磷酸酶或兔骨骼肌肌钙蛋白I无交叉反应。这些数据支持这样的观点，即用作抗原的合成肽代表兔骨骼肌肌球蛋白轻链激酶的钙调蛋白结合结构域，并且不同钙调蛋白调节蛋白的钙调蛋白结合结构域可能具有不同的一级和/或高级结构。
