Bourguignon L Y, Jin H, Iida N, Brandt N R, Zhang S H
Department of Cell Biology and Anatomy, School of Medicine, University of Miami, Florida 33101.
J Biol Chem. 1993 Apr 5;268(10):7290-7.
Mouse T-lymphoma cells contain a unique type of internal vesicle which bands at the relatively light density of 1.07 g/cc. These vesicles do not contain any detectable Golgi, endoplasmic reticulum, plasma membrane, or lysosomal marker protein activities. Binding of [3H]inositol 1,4,5-trisphosphate (IP3) to these internal vesicles reveals the presence of a single, high affinity class of IP3 receptor with a dissociation constant (Kd) of 1.6 +/- 0.3 nM. Using a panel of monoclonal and polyclonal antibodies against IP3 receptor, we have established that the IP3 receptor (approximately 260 kDa) displays immunological cross-reactivity with the rat brain IP3 receptor. Polymerase chain reaction analysis of first-strand cDNAs from both mouse T-lymphoma cells and rat brain tissues reveals that the IP3 receptor transcript in mouse T-lymphoma cells belongs to the short form (non-neuronal form) and not the long form (neuronal form) detected in rat brain tissue. Scatchard plot analysis shows that high affinity binding occurs between ankyrin and the IP3 receptor with a Kd of 0.2 nM. Most importantly, the binding of ankyrin to the light density vesicles significantly inhibits IP3 binding and IP3-induced internal Ca2+ release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of IP3 receptor-mediated internal Ca2+ release during lymphocyte activation.
小鼠T淋巴瘤细胞含有一种独特的内部囊泡，其在相对较轻的密度1.07 g/cc处形成条带。这些囊泡不含有任何可检测到的高尔基体、内质网、质膜或溶酶体标记蛋白活性。[3H]肌醇1,4,5-三磷酸（IP3）与这些内部囊泡的结合揭示了存在一类单一的高亲和力IP3受体，其解离常数（Kd）为1.6±0.3 nM。使用一组针对IP3受体的单克隆和多克隆抗体，我们确定IP3受体（约260 kDa）与大鼠脑IP3受体表现出免疫交叉反应性。对来自小鼠T淋巴瘤细胞和大鼠脑组织的第一链cDNA进行聚合酶链反应分析表明，小鼠T淋巴瘤细胞中的IP3受体转录本属于短形式（非神经元形式），而非在大鼠脑组织中检测到的长形式（神经元形式）。Scatchard图分析表明，锚蛋白与IP3受体之间发生高亲和力结合，Kd为0.2 nM。最重要的是，锚蛋白与低密度囊泡的结合显著抑制IP3结合和IP3诱导的细胞内Ca2+释放。这些发现表明，细胞骨架在淋巴细胞激活过程中IP3受体介导的细胞内Ca2+释放的调节中起关键作用。
