deBelle I, Mak A S
Biochim Biophys Acta. 1987 Jul 16;925(1):17-26. doi: 10.1016/0304-4165(87)90143-7.
Tropomyosin kinase is partially purified from 14-day-old chicken embryos using DEAE-cellulose, cellulose phosphate and gel filtration chromatography. The purest enzyme preparation consists of two major bands of Mr = 76,000 and 43,000 on SDS-polyacrylamide gel electrophoresis. The molecular weight of the enzyme is 250,000 determined by gel filtration chromatography. It phosphorylates casein and skeletal tropomyosin equally well but histone and phosvitin at a much slower rate. Smooth muscle myosin light chain, tropomyosin from platelet, erythrocyte and smooth muscle are not phosphorylated. The apparent Km for skeletal alpha-tropomyosin and ATP is 50 microM and 200 microM, respectively. Vmax varies between 100-300 nmol/min per mg depending on the purity of the preparation. Mg2+ and dithiothreitol are essential for activity but Ca+, calmodulin and cAMP are not required. The optimum temperature is 37 degrees C and optimum pH is about 7.5. Heparin, a potent inhibitor of casein kinase II, has no inhibitory effect on the enzyme. Similar tropomyosin kinase activity is not detected in skeletal muscle in adult rabbit and chicken. The tropomyosin kinase described here represents a hitherto uncharacterized kinase responsible for phosphorylation of tropomyosin in the chicken embryo.
利用二乙氨基乙基纤维素、磷酸纤维素和凝胶过滤色谱法从14日龄鸡胚中部分纯化原肌球蛋白激酶。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，最纯的酶制剂由两条主要条带组成，其相对分子质量分别为76,000和43,000。通过凝胶过滤色谱法测定该酶的分子量为250,000。它对酪蛋白和骨骼肌原肌球蛋白的磷酸化效果同样良好，但对组蛋白和卵黄高磷蛋白的磷酸化速率要慢得多。平滑肌肌球蛋白轻链、来自血小板、红细胞和平滑肌的原肌球蛋白均未被磷酸化。骨骼肌α-原肌球蛋白和ATP的表观米氏常数分别为50微摩尔和200微摩尔。最大反应速度根据制剂的纯度在每毫克100 - 300纳摩尔/分钟之间变化。镁离子和二硫苏糖醇对酶活性至关重要，但钙离子、钙调蛋白和环磷酸腺苷并非必需。最适温度为37℃，最适pH约为7.5。肝素是酪蛋白激酶II的强效抑制剂，对该酶无抑制作用。在成年兔和鸡的骨骼肌中未检测到类似的原肌球蛋白激酶活性。这里描述的原肌球蛋白激酶代表了一种迄今未被表征的激酶，负责鸡胚中原肌球蛋白的磷酸化。
