Cavadore J C, Molla A, Harricane M C, Gabrion J, Benyamin Y, Demaille J G
Proc Natl Acad Sci U S A. 1982 Jun;79(11):3475-9. doi: 10.1073/pnas.79.11.3475.
Antibodies were elicited against turkey gizzard myosin light chain kinase (MLCK), purified by affinity chromatography on the enzyme bound to Sepharose, and used to localize myosin kinase--in rabbit fast skeletal, slow skeletal, cardiac, and smooth muscles--by indirect immunofluorescence. When studied on nitrocellulose replicas of NaDodSO4/polyacrylamide gel electrophoretograms, antibodies were specific for the Mr 140,000 MLCK of gizzard smooth muscle. By using the same technique, they were shown to recognize the Mr 140,000 MLCK and a Mr 75,000 polypeptide--presumably derived from the former by proteolysis--in rat arterial and stomach smooth muscle as well as in rat thyroid cells. The same antibodies reacted only with a Mr approximately equal to 75,000 protein from rat cardiac and skeletal muscle. Antibodies inhibited the activity of smooth and skeletal myosin kinases in an in vitro assay with approximately equal to 11 mole of antibody needed for 50% inhibition of 1 mole of gizzard enzyme. The antibodies stain vascular and gizzard smooth muscle cells with no apparent segregation of the enzyme in a specific part of the cell. In contrast, sarcomeric muscles exhibit a striated staining pattern, superimposable to the staining by antiactin antibodies. This shows that (i) antibodies are not species- or tissue-specific, (ii) they recognize kinases that differ in their molecular weight and ability to be phosphorylated, probably at the level of their common catalytic and calmodulin-binding domains, and (iii) sarcomeric muscle kinases are at least in part bound to the contractile apparatus and their distribution is restricted to a specific part of the sarcomere. This raises the possibility that myosin phosphorylation may be controlled not only by the Ca2+ concentration but also by actin-myosin interaction.
制备了针对火鸡砂囊肌球蛋白轻链激酶（MLCK）的抗体，该酶通过与琼脂糖结合的酶进行亲和层析纯化，并用其通过间接免疫荧光法在兔的快收缩骨骼肌、慢收缩骨骼肌、心肌和平滑肌中定位肌球蛋白激酶。当在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳图谱的硝酸纤维素复制品上进行研究时，抗体对砂囊平滑肌中分子量为140,000的MLCK具有特异性。通过使用相同技术，显示它们在大鼠动脉和胃平滑肌以及大鼠甲状腺细胞中识别分子量为140,000的MLCK和一种分子量为75,000的多肽（推测是前者经蛋白水解产生）。相同的抗体仅与大鼠心肌和骨骼肌中分子量约为75,000的蛋白质发生反应。在体外测定中，抗体抑制平滑肌和骨骼肌肌球蛋白激酶的活性，抑制1摩尔砂囊酶的50%活性大约需要11摩尔抗体。抗体对血管和平滑肌细胞进行染色，酶在细胞的特定部位没有明显的分隔。相反，肌节肌呈现出条纹状染色模式，与抗肌动蛋白抗体的染色重叠。这表明：（i）抗体不是物种或组织特异性的；（ii）它们识别分子量和磷酸化能力不同的激酶，可能在其共同的催化和钙调蛋白结合结构域水平上；（iii）肌节肌激酶至少部分与收缩装置结合，并且它们的分布限于肌节的特定部分。这增加了肌球蛋白磷酸化不仅可能受钙离子浓度控制，还可能受肌动蛋白-肌球蛋白相互作用控制的可能性。
