ARTERIAL ACTOMYOSIN - EFFECTS OF PH AND TEMPERATURE ON SOLUBILITY AND ATPASE ACTIVITY
ARTERIAL ACTOMYOSIN - EFFECTS OF PH AND TEMPERATURE ON SOLUBILITY AND ATPASE ACTIVITY
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DOI：
10.1152/ajplegacy.1971.220.5.1494
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发表时间：
1971-01-01
影响因子：
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通讯作者：
MURPHY, RA
中科院分区：
文献类型：
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作者：
MURPHY, RA
方法肌动球蛋白。表 1 说明了离子强度、pH 和 ATP 浓度变化在动脉肌动球蛋白分离中的应用。收集猪颈动脉（250-400 条动脉，去除疏松外膜组织后每条重约 1 g），并按先前所述提取收缩蛋白 (16)。坚韧纤维组织的充分均质化对于高产量至关重要，并且最好在具有剪刀般切割作用的搅拌机（例如 Polytron（Brinkman Instruments））中实现。每克匀浆动脉使用 5-10 ml 指定溶液进行两次提取，第一次提取过夜，第二次提取 5 小时。缓冲液是 22 mM 吗啉代丙磺酸 (MOPS)，在 pH 7.0 和 0°C 时缓冲值为 -10mM Hf/pH 单位。通过用每毫升蛋白质溶液 15 mg Dowex 21 阴离子交换树脂去除 ATP，然后通过用 4.6 mM 组氨酸（0°C 时 pH 6.0，缓冲值）针对所示 KC1 浓度进行透析来进行沉淀。 -2 mM H+/pH 单位）。通过将KCl浓度提高至0.6M、通过添加固体NaHCO 3 将pH调节至7.0并添加1nlM ATP来溶解沉淀的蛋白质沉淀物。纯化的肌动球蛋白的最终产物在0.1 M KCl中洗涤两次，溶解在0.6 M KCl中，并将等分试样在液氮中冷冻保存。在 196°C 下储存期间，酶活性没有发生变化。该程序比在 20°C 下的 50% 甘油中储存有优势，因为蛋白质在使用前不必清洗，也无需重新测定蛋白质浓度。然而，液氮储存确实保留了污染性微粒体或线粒体 ATP 酶活性，这些活性通常在 50% 甘油中储存几周后就会消失。如前所述，从混合兔骨骼肌中分离出肌动球蛋白（肌球蛋白 B）(16)。在本报告中，从动脉平滑肌和骨骼肌分离的蛋白质用术语“动脉肌动球蛋白”和“骨骼肌肌动球蛋白”表示。假设 16% 的蛋白质是氮，通过微量凯氏定氮法测量蛋白质浓度。
METHODSActomyosin. Application of changes in ionic strength, pH, and ATP concentration to the isolation of arterial actomyosin is illustrated in Table 1. Hog carotid arteries (250-400 arteries weighing approximately 1 g each after removal of the loose adventitial tissue) were collected and the contractile proteins extracted as previously described(16). Adequate homogenization of the tough fibrous tissue is critical for high yields and was best achieved in a blender which has a scissors-like cutting action such as the Polytron (Brinkman Instruments). Two extractions using 5-10 ml of the indicated solution per gram of homogenized arteries were carried out, the first overnight and the second for 5 hr. The buffer was 22 mM morpholinopropane sulfonic acid (MOPS) with a buffering value of-1OmM Hf/pH unit at pH 7.0 and 0 C. Precipitations were carried out by removing ATP with 15 mg Dowex 21 anion exchange resin per milliliter protein solution, then by dialysis against the indicated KC1 concentrations with 4.6 mM histidine (pH 6.0 at 0 C, buffering value of-2 mM H+/pH unit). Sedimented protein precipitates were dissolved by raising the KC1 concentration to 0.6 M, adjusting the pH to 7.0 by additions of solid NaHC03, and adding 1 nlM ATP. The final product of purified actomyosin was washed twice in 0.1 M KCl, dissolved in 0.6 M KCl, and aliquots were frozen in liquid nitrogen for storage. No alterations in enzymatic activity occurred during storage at-196 C. This procedure has an advantage over storage at-20 C in 50% glycerol because the protein does not have to be washed before use and the protein concentration redetermined. However, liquid nitrogen storage does preserve contaminating microsomal or mitochondrial ATPase activity which was usually lost after a few weeks storage in 50% glycerol.Actomyosin(myosin B) from mixed rabbit skeletal muscles was isolated as previously described (16). The proteins isolated from arterial smooth and skeletal muscle are indicated by the terms arterial actomyosin and skeletal actomyosin in this report. Protein concentrations were measured by micro-Kjeldahl methods assuming that 16% of the protein was nitrogen.