Evidence for myosin associated with bovine photoreceptor cell outer segments [proceedings].
Evidence for myosin associated with bovine photoreceptor cell outer segments [proceedings].
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肌球蛋白与牛感光细胞外节相关的证据[会议记录]。
DOI：
10.1042/bst0061271
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发表时间：
1978
影响因子：
3.9
通讯作者：
D. Aunis
中科院分区：
文献类型：
--
作者：
J. Hesketh;N. Virmaux;D. Aunis
大量的证据表明，肌动蛋白和肌球蛋白都是非肌肉细胞的正常成分(Pollard&魏兴，1974；Clarke&Spudich，1977)。酶分析(Hesketh et al.，1977)和凝胶电泳法(Blitz&Fine，1974；Philps&Slate，1975)都被用来研究这些蛋白质在亚细胞组分中的分布。我们最近从牛视网膜中提纯了肌球蛋白(Hesketh et a/.，1978)，在目前的交流中，我们提出了证据表明，这种肌球蛋白部分地与光感受器细胞外节相关。用Virmaux等人(1971)的方法制备牛视网膜光感受器细胞外段，用37%蔗糖在10OrnM-Tris/HCI中匀浆，pH=7。取上清液于25000g离心20min，在蔗糖/Tris缓冲液中匀浆。然后将该悬浮液分层到Tris缓冲液下面，并在45000g的条件下离心20min。感光细胞外节在界面上呈条带状，通过反复浮选步骤进行纯化。在乳化剂存在下对依赖于(K+-EDTA)的三磷酸腺苷酶活性的测量(方法见Hesketh et a/1978)显示，光感受器细胞外段的比活性与起始匀浆相比翻了一番，尽管这种增加大多发生在去核时(表1)。与光感受器细胞外段定位的鸟氨酸环化酶相比，增加的幅度很小(Virmaux等人，1976)。然而，值得注意的是，尽管光感受器细胞外节只回收了1.3%的匀浆蛋白，但这些结构中发现了4.6%的依赖于(K+-EDTA)的三磷酸腺苷酶活性。这种3.5的相对比活性表明肌球蛋白三磷酸腺苷酶活性与光感受器细胞外节有关，尽管显然这种活性并不局限于这些结构。光感受器细胞外节进一步用十二烷基硫酸钠/聚丙烯酰胺凝胶电泳法分析。样品制成10%的2-巯基乙醇(或二硫苏糖醇)，1%的十二烷基硫酸钠，并在100℃加热2分钟。最初，样品在7.5%的聚丙烯酰胺凝胶上使用Tris/甘氨酸缓冲体系在0.1%的十二烷基硫酸钠存在下进行分析(Waehnheldt&Mandel，1970)。结果(图1)显示，在光感受器细胞外节中，存在一条与纯化的视网膜肌球蛋白重链非常接近或相同的迁移率带。此外，使用平板梯度(4-30%聚丙烯酰胺)凝胶的分析也表明，在光感受器细胞外段制备的视网膜肌球蛋白重链中有一条强条带的共同迁移。酶的结果是不明确的；光感受器细胞外节制备过程中比活力的增加很小，但4.6%的活力恢复了
Much evidence has accrued suggesting that both actin and myosin are normal constituents of non-muscle cells (Pollard & Weihing, 1974; Clarke & Spudich, 1977). Both enzymic (Hesketh et al., 1977) and gel-electrophoresis analysis (Blitz & Fine, 1974; Philips & Slater, 1975) have been used to study the distribution of these proteins in subcellular fractions. We have recently purified myosin from bovine retinae (Hesketh et a/., 1978) and in the present communication we present evidence that, in part, this myosin is associated with photoreceptor cell outer segments. Bovine photoreceptor cell outer segments were prepared by the method of Virmaux et al.(1971); retinae were homogenized in 37% sucrose in lOOrnM-Tris/HCI, pH7. 0, centrifuged at 2000g for 15min to remove nuclei, and then the supernatant was centrifuged at 25000g for 20min and the pellet was homogenized in the sucrose/Tris buffer. This suspension was then layered below Tris buffer and centrifuged at 45000g for 20min. Photoreceptor cell outer segments appeared as a band at the interface and were purified by repeated flotation steps. Measurement of (K+-EDTA)-dependent adenosine triphosphatase activity (for the method, see Hesketh et a/., 1978) in the presence of emulphogen showed a doubling of specific activity in photoreceptor cell outer segments compared with the starting homogenate, although most of this increase occurred on removal of nuclei (Table 1). The increase is small compared with the photoreceptor cell outer segments-localized enzyme guanylate cyclase (Virmaux et al., 1976). It is noteworthy, however, that although only 1.3% of homogenate protein was recovered in photoreceptor cell outer segments, 4.6% of the (K+-EDTA)-dependent adenosine triphosphatase activity was found in these structures. Such a relative specific activity of 3.5 suggests an association of the myosin adenosine triphosphatase activity with photoreceptor cell outer segments, although obviously such activity is not restricted to these structures. Photoreceptor cell outer segments were further analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Samples were made 10% with respect to 2-mercaptoethanol (or dithiothreitol), 1% with respect to sodium dodecyl sulphate and heated for 2min at 100 C. Initially samples were analysed on 7.5% polyacrylamide gels by using a Tris/glycine buffer system in the presence of 0.1% sodium dodecyl sulphate (Waehnheldt & Mandel, 1970). The results (Fig. 1) showed the presence, in photoreceptor cell outer segments, of a band of mobility very close or identical to that of purified retinal myosin heavy chains. Furthermore, analysis by using slab gradient (4-30% polyacrylamide) gels also showed co-migration of a strong band in photoreceptor cell outer segments preparations with retinal myosin heavy chains. The enzymic results were equivocal; the increase in specific activity during photoreceptor cell outer segments preparation was small, but the 4.6% activity recovered