Lees Simon J, Williams Jay H
Muscular Function Laboratory, Department of Human Nutrition, Foods, & Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
Am J Physiol Cell Physiol. 2004 Jan;286(1):C97-104. doi: 10.1152/ajpcell.00188.2003. Epub 2003 Sep 10.
The purpose of this investigation was to determine whether there is a link between sarcoplasmic reticulum (SR) glycogen status and SR Ca2+ handling. In this investigation, skeletal muscle SR was purified from female Sprague-Dawley rats (200-250 g). Glycogen was extracted from the SR purified from one hindlimb, whereas the SR purified from the contralateral limb served as control. Before removal of the tissue, the animals were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Both alpha-amylase treatment (AM) and removal of EDTA from the homogenization and storage buffers reduced the amount of glycogen associated with the SR (P < 0.05). AM treatment reduced the glycogen phosphorylase content of SR (P < 0.05). In contrast, creatine kinase (CK) and pyruvate kinase (PK) contents were increased after both glycogen extraction protocols (P < 0.05). Under exogenous ATP conditions, both AM and EDTA-free (EF) treatments resulted in an increase in Ca2+-stimulated ATPase activity when normalized to sarco(endo)plasmic reticulum calcium-ATPase (SERCA) content (P < 0.05). CK and PK-supported SR Ca2+ uptake was decreased (P < 0.05) in the AM group when normalized to SERCA and CK or SERCA and PK content, respectively. AM was more effective than the EF for extracting glycogen associated with purified SR. Glycogen extraction alters the yield of purified SR proteins and must be taken into account when investigating SR calcium handling. Removal of glycogen from purified SR causes a change in Ca2+-handling properties as measured by ATPase and uptake activities.
本研究的目的是确定肌浆网（SR）糖原状态与SR钙处理之间是否存在联系。在本研究中，从雌性Sprague-Dawley大鼠（200-250 g）中纯化骨骼肌SR。从一侧后肢纯化的SR中提取糖原，而从对侧肢体纯化的SR用作对照。在取出组织之前，通过腹腔注射氯胺酮（80 mg/kg）和赛拉嗪（10 mg/kg）对动物进行麻醉。α-淀粉酶处理（AM）以及从匀浆和储存缓冲液中去除EDTA均降低了与SR相关的糖原量（P < 0.05）。AM处理降低了SR的糖原磷酸化酶含量（P < 0.05）。相反，在两种糖原提取方案后，肌酸激酶（CK）和丙酮酸激酶（PK）含量均增加（P < 0.05）。在外源ATP条件下，当以肌浆（内质）网钙ATP酶（SERCA）含量进行标准化时，AM和无EDTA（EF）处理均导致钙刺激的ATP酶活性增加（P < 0.05）。当分别以SERCA和CK或SERCA和PK含量进行标准化时，AM组中CK和PK支持的SR钙摄取减少（P < 0.05）。AM在提取与纯化SR相关的糖原方面比EF更有效。糖原提取会改变纯化SR蛋白的产量，在研究SR钙处理时必须予以考虑。从纯化的SR中去除糖原会导致通过ATP酶和摄取活性测量的钙处理特性发生变化。
