Brandt N R, Caswell A H, Carl S A, Ferguson D G, Brandt T, Brunschwig J P, Bassett A L
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida.
J Membr Biol. 1993 Feb;131(3):219-28. doi: 10.1007/BF02260110.
Dyads (transverse tubule--junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the M(r) 102K Ca2+ ATPase were associated with a diffuse protein band (22-30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same M(r) as triadin. Western blots of muscle microsomes from preparations which had been treated with 100 mM iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of M(r) 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.
通过连续蔗糖梯度从大鼠心室微粒体中富集二联体（横小管 - 连接肌浆网复合体）。在36%蔗糖处的主要囊泡峰含有高达90%具有二氢吡啶（DHP）结合位点的膜（横小管的标志物）以及所有具有兰尼碱受体和假定连接足蛋白的膜（连接肌浆网的标志物）。此外，36%蔗糖峰含有一半带有毒蕈碱受体的囊泡。每毒蕈碱受体中二氢吡啶结合位点含量低所定义的非连接质膜来源的囊泡以及由M(r) 102K Ca2+ ATP酶所定义的游离肌浆网来源的囊泡与梯度较轻区域的一条弥散蛋白带（22 - 30%蔗糖）相关。这些细胞器回收率低。假定的二联体在8000磅力的法国压榨处理下未被破坏，在14000磅力时仅部分被破坏。针对骨骼肌三联蛋白的单克隆抗体GE4.90，一种在骨骼肌三联体连接中将DHP受体与连接足蛋白相连的蛋白质，与大鼠二联体中与三联蛋白相同M(r)的一种蛋白质发生交叉反应。在整个分离过程中用100 mM碘乙酰胺处理的制剂的肌肉微粒体的蛋白质免疫印迹显示，心脏三联蛋白主要由一条M(r) 95 kD的条带组成。与大鼠腰大肌微粒体中的寡聚体形式阶梯相比，可检测到较高分子量聚合物但含量低。心脏三联蛋白不会因高渗盐或 Triton X - 100从微粒体中溶解，表明它以及骨骼肌三联蛋白是连接肌浆网的整合蛋白。通过比较其在总微粒体和法国压榨破坏的二联体制剂中与细胞器标志物的分布，将心脏表位定位到连接肌浆网。使用单克隆抗体GE4.90对三联蛋白进行免疫荧光定位显示，完整的大鼠心室肌组织在每个肌节都按照明确的条带模式染色。这些条带的间距与三联蛋白存在于二联体连接区域的解释一致。
