Nguyen L T, Stephenson D G, Stephenson G M
Department of Chemical Sciences, Victoria University of Technology, Melbourne, Australia.
J Muscle Res Cell Motil. 1998 Aug;19(6):631-8. doi: 10.1023/a:1005377030193.
The main objective of this study was to analyse glycogen in single muscle fibres, using a recently developed microfluorometric method which detects subpicomol amounts of NADPH, glucose and glycogen (as glucosyl units) (detection limit 0.16-0.17 pmol in a 25 nl sample) without fluorochrome amplification. The fibres were freshly dissected from the twitch region of the iliofibularis muscle of the cane toad (Bufo marinus), and were mechanically skinned under paraffin oil to gain access to the intracellular compartments. The results show that (1) glycogen concentrations in toad skeletal muscle fibres range between 25.8 and 369 mmol glucosyl units/litre fibre volume; (2) there is a large variation in glycogen content between individual fibres from the iliofibularis muscle of one animal; (3) there are seasonal differences in the glycogen content of toad single muscle fibres; (4) the total amount of glycogen in single muscle fibres of the toad does not decrease significantly when storing the tissue, under paraffin oil, at 20-25 degree C for up to 6 h or at 4 degree C for up to 24 h; and (5) 15-26% of fibre glycogen can be washed in an aqueous solution at pH 5-7, within 5 min, while 74-85% of fibre glycogen remains associated with the washed skinned fibre, even after 40 min exposure of the skinned fibre preparation to the aqueous environment. The retention of most glycogen in the fibre preparation after mechanical removal of the plasma membrane and extensive washing indicates that in toad skeletal muscle fibres the largest proportion of glycogen is tightly bound to intracellular structures. The results also show that the skinned muscle fibre preparation is well suited for microfluorometric glycogen determination, since low molecular weight non-glycogen contributors to the fluorescence signal can be removed from the myoplasmic space prior to the glycogen hydrolysis step.
本研究的主要目的是利用一种最近开发的显微荧光测定法分析单根肌纤维中的糖原，该方法可检测亚皮摩尔量的烟酰胺腺嘌呤二核苷酸磷酸（NADPH）、葡萄糖和糖原（以葡糖基单位计）（在25 nl样品中的检测限为0.16 - 0.17 pmol），无需荧光染料放大。纤维取自蔗蟾（Bufo marinus）髂腓肌的抽动区域，新鲜解剖后在石蜡油下进行机械去膜，以进入细胞内区室。结果表明：（1）蟾蜍骨骼肌纤维中的糖原浓度在25.8至369 mmol葡糖基单位/升纤维体积之间；（2）同一动物髂腓肌中各单根纤维的糖原含量存在很大差异；（3）蟾蜍单根肌纤维的糖原含量存在季节性差异；（4）将蟾蜍单根肌纤维组织在石蜡油中于20 - 25℃保存长达6小时或在4℃保存长达24小时时，单根肌纤维中糖原的总量没有显著减少；（5）在pH 5 - 7的水溶液中，15 - 26%的纤维糖原可在5分钟内被洗去，而即使在去膜纤维制剂暴露于水环境40分钟后，仍有74 - 85%的纤维糖原与洗后的去膜纤维结合。在机械去除质膜并进行大量洗涤后，大部分糖原保留在纤维制剂中，这表明在蟾蜍骨骼肌纤维中，大部分糖原与细胞内结构紧密结合。结果还表明，去膜肌纤维制剂非常适合用于显微荧光法测定糖原，因为在糖原水解步骤之前，可以从肌浆空间中去除荧光信号的低分子量非糖原成分。
