Properties of immunoaffinity purified 106-kDa Ca2+ release channels from the skeletal sarcoplasmic reticulum.
Properties of immunoaffinity purified 106-kDa Ca2+ release channels from the skeletal sarcoplasmic reticulum.
复制标题
骨骼肌浆网免疫亲和纯化 106-kDa Ca2 释放通道的特性。
DOI：
10.1016/0003-9861(92)90043-v
复制
发表时间：
1992
影响因子：
3.9
通讯作者：
Salama,G
中科院分区：
文献类型：
--
作者：
Hilkert,R;Zaidi,N;Shome,K;Nigam,M;Lagenaur,C;Salama,G
用生物素-亲和素柱层析从骨骼肌浆网(SR)小泡中纯化了巯基门控的106 kDa钙释放通道(SG-106)，并将其作为抗原制备多克隆抗体。免疫印迹显示，该抗血清可与抗原性SG-106交叉反应，而不与肌浆网钙、镁-三磷酸腺苷酶或足部结合蛋白(JFPS)交叉反应(Zaidial，1989，J.Biol)。化学264(36)，21,725-21,736；21,737-21,747)。多克隆抗体亲和柱被用来选择性地纯化SG-106-kDa蛋白，当被掺入平面双层时，发现存在一种阳离子通道，其性质类似于通过SR囊泡与平面双层融合而获得的“天然”钙释放通道。2，2‘-二硫代联吡啶和二硫苏糖醇分别增加和减少了106 kDa钙释放通道的开放概率，这与SR小泡钙释放的测量结果一致。与JFPS的报道相比，0.5-1nminRyanodine增加了开放时间的概率，在2-10nmlock的106 kDa钙释放通道处于关闭状态而不是开放的亚电导状态。毫摩尔的三磷酸腺苷激活SG-106，毫摩尔的镁离子抑制SG-106，微摩尔的Ru红抑制SG-106。阿霉素(2-10μm)引起SG-106Ca~(2+)释放通道的一过性激活，然后在约5min内关闭，并间歇性激活到亚电导状态。用于纯化SG-106的多克隆抗体在加入到双层的侧壁但不能激活通道时也会激活通道。因此，SG-106通道具有类似于“天然”SR钙释放通道的特征，在免疫学上有别于JFPS，并且在平面双层中与纳米分子ryanodine相互作用几秒钟。
The sulfhydryl-gated 106-kDa Ca2+-release channel (SG-106) was purified by biotin-avidin chromatography from skeletal sarcoplasmic reticulum (SR) vesicles and used as an antigen to raise polyclonal anti-bodies. Western blots showed that the antisera crossreacted with the antigenic SG-106 and not with SR Ca2+, Mg2+-ATPase or with junctional foot proteins (JFPs) (Zaidiet al., 1989,J. Biol. Chem.264(36), 21,725–21,736; 21,737–21,747). Polyclonal anti-body-affinity columns were used to selectively purify SG-106-kDa proteins which, upon incorporation in planar bilayers, revealed the presence of a cationic channels with properties similar to “native” Ca2+-release channels obtained through the fusion of SR vesicles with planar bilayers. In agreement with measurements of Ca2+release from SR vesicles, sulfhydryl oxidizing and reducing agents (i.e., 2,2′-dithiodipyridine and dithiothreitol) respectively increased and decreased the open-time probability of 106-kDa Ca2+-release channels. In contrast with reports on JFPs, ryanodine at 0.5–1 nmincreased the open-time probability and at 2–10 nmlocked 106-kDa Ca2+-release channels in a closed state rather than an open subconductance state. The SG-106 was activated by millimolar ATP, inhibited by millimolar Mg2+, and blocked by micromolar ruthenium red. Adriamycin (2–10 μm) caused a transient activation of SG-106 Ca2+-release channels, followed by closure in about 5 min, and intermittent activation to a subconductance state. Polyclonal antibodies used to purify the SG-106 also activated the channel when added to thecisside but not thetransside of the bilayer. Thus, SG-106 channels possess features that are similar to “native” SR Ca2+-release channels, are immunologically distinct from JFPs, and interact in seconds with nanomolar ryanodine in planar bilayers.