Single fiber analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle
Single fiber analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle
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DOI：
10.1152/ajpcell.00252.2012
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发表时间：
2012-12-01
影响因子：
5.5
通讯作者：
Stapleton, David I.
中科院分区：
文献类型：
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作者：
Murphy, Robyn M.;Xu, Hongyang;Stapleton, David I.
墨菲 RM、徐 H、拉奇曼 H、拉金斯 NT、古利 PR、斯台普顿 DI。糖原相关蛋白的单纤维分析揭示了它们与大鼠骨骼肌中糖原的差异关联。 Am J Physiol Cell Physiol 303：C1146-C1155，2012。首次发表于 2012 年 9 月 26 日； doi:10.1152/ajpcell.00252.2012.-为了了解糖原如何影响骨骼肌生理学，我们使用来自静止和刺激的大鼠骨骼肌的单纤维检查了肌肉糖原合成和降解所必需的酶。呈现范式的转变，我们表明这些蛋白质与糖原颗粒有不同的相关性。在从大鼠快肌伸指长肌 (EDL) 和慢肌比目鱼肌 (SOL) 分离的纤维中检查了糖原蛋白、糖原分支酶 (GBE)、脱支酶 (GDE)、磷酸化酶 (GP) 和合酶 (GS) 的蛋白质扩散性和/或丰度。与 SOL 肌肉相比，EDL 肌肉纤维中的 GDE 和 GP 蛋白更丰富（接近 10 至 100 倍）。肌肉之间的 GS 和糖原蛋白相似，而 SOL 肌肉中的 GBE 丰度大约是其四倍。暴露于生理缓冲液 10 分钟的机械剥皮纤维显示，GBE 和 GP 总量的 70% 是可扩散的（非结合），而 GDE 和 GS 的扩散性要低得多。强烈的体外刺激，足以引起细胞内糖原减少约 50%，GDE、GP 和 GS 的扩散性增加（约 15-60%），GBE 扩散性降低（约 20%）。淀粉酶处理会破坏糖原的 α-1,4 连接，表明扩散性不同，因此 GDE 和 GS 的糖原关联也不同。膜溶解（1% Triton-X-100）允许少量额外的 GDE 和 GS 从纤维中扩散，表明大多数非糖原相关的 GDE/GS 与肌肉的肌原纤维/收缩网络而不是膜相关。鉴于糖原代谢所需的酶存在差异，目前的研究结果表明糖原颗粒具有纤维类型依赖性结构。快肌纤维中糖原分解的更大分解代谢潜力可能解释了不同的收缩诱导的糖原利用率。
Murphy RM, Xu H, Latchman H, Larkins NT, Gooley PR, Stapleton DI. Single fiber analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle. Am J Physiol Cell Physiol 303: C1146-C1155, 2012. First published September 26, 2012; doi:10.1152/ajpcell.00252.2012.-To understand how glycogen affects skeletal muscle physiology, we examined enzymes essential for muscle glycogen synthesis and degradation using single fibers from quiescent and stimulated rat skeletal muscle. Presenting a shift in paradigm, we show these proteins are differentially associated with glycogen granules. Protein diffusibility and/or abundance of glycogenin, glycogen branching enzyme (GBE), debranching enzyme (GDE), phosphorylase (GP), and synthase (GS) were examined in fibers isolated from rat fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscle. GDE and GP proteins were more abundant (similar to 10-to 100-fold) in fibers from EDL compared with SOL muscle. GS and glycogenin proteins were similar between muscles while GBE had an approximately fourfold greater abundance in SOL muscle. Mechanically skinned fibers exposed to physiological buffer for 10 min showed similar to 70% total pools of GBE and GP were diffusible (nonbound), whereas GDE and GS were considerably less diffusible. Intense in vitro stimulation, sufficient to elicit a similar to 50% decrease in intracellular glycogen, increased diffusibility of GDE, GP, and GS (similar to 15-60%) and decreased GBE diffusibility (similar to 20%). Amylase treatment, which breaks alpha-1,4 linkages of glycogen, indicated differential diffusibilities and hence glycogen associations of GDE and GS. Membrane solubilization (1% Triton-X-100) allowed a small additional amount of GDE and GS to diffuse from fibers, suggesting the majority of nonglycogen-associated GDE/GS is associated with myofibrillar/contractile network of muscle rather than membranes. Given differences in enzymes required for glycogen metabolism, the current findings suggest glycogen particles have fiber-type-dependent structures. The greater catabolic potential of glycogen breakdown in fast-twitch fibers may account for different contraction induced rates of glycogen utilization.