Kinetics of Denaturation of Rabbit Skeletal Muscle Glycogen Phosphorylase b by Guanidine Hydrochloride

The kinetics of denaturation and aggregation of rabbit muscle glycogen phosphorylase b in the presence of guanidine hydrochloride (GuHCl) have been studied. The curve of inactivation of phosphorylase b in time includes a region of the fast decline in the enzymatic activity,an intermediate plateau,and a part with subsequent decrease in the enzymatic activity. The fact that the shape of the inactivation curves is dependent on the enzyme concentration testifies to the dissociative mechanism of inactivation. The dissociation of phosphorylase b dimers into monomers in the presence of GuHCl is supported by sedimentation data. The rate of phosphorylase b aggregation in the presence of GuHCl rises as the denaturant concentration increases to 1.12 M; at higher concentration of GuHCl, suppression of aggregation occurs. At rather low concentration of the protein (0.25 mg/ml), the terminal phase of aggregation follows the kinetics of a monomolecular reaction (the reaction rate constant is equal to 0.082 min^–1;1 M GuHCl, 25°C). At higher concentration of phosphorylase b (0.75 mg/ml), aggregation proceeds as a trimolecular reaction.     

肌糖原磷酸化酶a,b亚基的杂交

本文用标记在肌糖原磷酸化酶ａ（ＧＰＡ）亚基上的荧光探针ＩＡＥＤＡＮＳ与标记在肌糖原磷酸化酶ｂ（ＧＰＢ）亚基上的ＩＡＦ之间无辐射能量转移;高压力下亚基交换动力学和外源荧光探针荧光偏振等三种方法证实了肌糖原磷酸化酶ａ、ｂ的亚基可相互交换,形成磷酸化酶ａ、ｂ的杂交形式。     

Kinetic Mechanism of Allosteric Regulation of Muscle Glycogen Phosphorylase b by Adenosine 5"-Monophosphate

Kinetic analysis of the glycogen chain growth reaction catalyzed by glycogen phosphorylase b from rabbit skeletal muscle has been carried out over a wide range of AMP concentration under the saturation of the enzyme by glycogen. Applicability of some variants of the kinetic model involving the interaction of AMP- and glucose 1-phosphate-binding sites in the dimeric enzyme molecule is considered. A kinetic model of the enzymatic reaction describing adequately the activation of the enzyme by AMP and inhibition at sufficiently high concentrations of AMP is proposed.     

高强度间歇训练：调节骨骼肌质量及功能的新手段

近年来,高强度间歇训练（high-intensity interval training,HIIT）被认为是一种调节骨骼肌质量及功能的运动方式,但其具体作用和机制以及运动和检测中需要注意的问题尚不明确。因此,梳理HIIT与骨骼肌质量及功能的关系显得尤为重要。本文综述HIIT上调骨骼肌蛋白质合成速率和下调萎缩速率、引发肌肉重塑和调节肌纤维类型、促进血管生成和血流灌注、介导骨骼肌线粒体含量上调和功能改善、增加肌肉力量和与膳食补充的协同作用等影响骨骼肌质量及功能的研究进展,为HIIT预防和改善肌肉丢失和功能下降提供理论依据和应用策略。     

糖原磷酸化酶BB在心脏直视手术后心肌损伤中的判定价值

目的:探讨糖原磷酸化酶BB对心脏直视手术后心肌损伤的判定价值.方法:108例行心脏瓣膜置换手术患者,分别于围术期多个时点取桡动脉血,测定血清糖原磷酸化酶BB、心肌肌钙蛋白I(cTnI)、肌酸磷酸激酶同工酶(CKMB)的水平,并检测心肌细胞凋亡程度.结果:血清糖原磷酸化酶BB于体外循环主动脉开放后最早达峰值,其峰值与cT...     

Immunohistochemical Localization of Glycogen Phosphorylase Isozymes in the Rat Gastrointestinal Muscle Layers and Enteric Nervous System

Glycogen represents the major brain energy reserve though its precise functions are still under debate. Glycogen has also been found in different cell types of the enteric nervous system (ENS), the largest and most complex component of the peripheral nervous system. In the present work we have demonstrated, by application of isozyme-specific antibodies, the presence of isozymes of glycogen phosphorylase (GP), one of the major control sites in glycogen metabolism, in the rat ENS. Immunohistochemistry revealed that isoform BB (brain) is the predominant isozyme expressed in enteric glial cells (EGC) and rare neurons of the myenteric and submucosal plexuses. Isoform MM (muscle) appears in cells which are, according to their location and morphology, probably interstitial cells of Cajal (ICC). In addition, both GP isoforms are expressed in longitudinal and circular intestinal smooth muscle layers. As GP BB is mainly regulated by the cellular AMP level, a special function of glycogen in the energy supply of neural gut functions is suggested.     

Selective Increase of Tetrameric (G4) Acetylcholinesterase Activity in Rat Hindlimb Skeletal Muscle Term Denervation

Acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes in gracilis muscles from adult Sprague-Dawley rats were studied 24-96 h after obturator nerve transection. Results show a selective denervation-induced increase in the globular G4 isoform, which is predominantly associated with the plasmalemma. This enzymatic increase was (a) transient (occurring between 24 and 60 h) and accompanied by declines in all other identifiable AChE isoforms; (b) observed after concurrent denervation and inactivation of the enzyme with diisopropylfluorophosphate, but not following treatment with cycloheximide; and (c) more prominent in the extracellular compartment of muscle endplate regions. Aside from this transient change, G4 activity did not fall below control levels, indicating that at least the short-term maintenance of G4 AChE (i.e., at both normal and temporarily elevated levels) does not critically depend on the presence of the motor nerve. In addition, this isoform's activity increases in response to perturbations of the neuromuscular system that are known to produce elevated levels of acetylcholine (ACh), such as short-term denervation and exercise-induced enhancement of motor activity. The present study is consistent with the hypothesis that individual AChE isoforms in gracilis muscle are subject to distinct modes of neural regulation and suggests a role for ACh in modulating the activity of G4 AChE at the motor endplate.     

Influence of Osmolytes on Inactivation and Aggregation of Muscle Glycogen Phosphorylase <Emphasis Type="Italic">b</Emphasis> by Guanidine Hydrochloride. Stimulation of Protein Aggregation under Crowding Conditions

The effects of the osmolytes trimethylamine-N-oxide (TMAO), betaine, proline, and glycine on the kinetics of inactivation and aggregation of rabbit skeletal muscle glycogen phosphorylase b by guanidine hydrochloride (GuHCl) have been studied. It is shown that the osmolytes TMAO and betaine exhibit the highest protective efficacy against phosphorylase b inactivation. A test system for studying the effects of macromolecular crowding induced by osmolytes on aggregation of proteins is proposed. TMAO and glycine increase the rate of phosphorylase b aggregation induced by GuHCl.     

心肌肌钙蛋白I和糖原磷酸化酶同工酶BB金标记免疫层析联合检测法的建立

目的：建立人心肌肌钙蛋白I（cTnI）及糖原磷酸化酶同工酶BB（GPBB）的胶体金免疫层析联合检测法。方法：以纯化的人心肌cTnI和GPBB为免疫原免疫小鼠,制备抗cTnI和抗GPBB单克隆抗体,并用胶体金标记cTnI和GPBB抗体,采用免疫层析技术建立快速准确检测cTnI和GPBB的胶体金免疫层析法。结果：建立的检测方法灵敏度高,可检出血液样品中1ng／mL的cTnI和7ng／mL的GPBB;特异性强,与心肌肌钙蛋白T、心肌肌钙蛋白C、肌酸激酶同工酶均无交叉反应。结论：该方法特异性强,灵敏度高,快速、简便,弥补了传统心肌梗死诊断方法的不足,对急性心肌梗死的早期筛查有重要意义,具有较高的临床应用价值和广泛的应用前景。     

Control of Cell Volume in Skeletal Muscle

Regulation of cell volume is a fundamental property of all animal cells and is of particular importance in skeletal muscle where exercise is associated with a wide range of cellular changes that would be expected to influence cell volume. These complex electrical, metabolic and osmotic changes, however, make rigorous study of the consequences of individual factors on muscle volume difficult despite their likely importance during exercise. Recent charge-difference modelling of cell volume distinguishes three major aspects to processes underlying cell volume control: (i) determination by intracellular impermeant solute; (ii) maintenance by metabolically dependent processes directly balancing passive solute and water fluxes that would otherwise cause cell swelling under the influence of intracellular membrane-impermeant solutes; and (iii) volume regulation often involving reversible short-term transmembrane solute transport processes correcting cell volumes towards their normal baselines in response to imposed discrete perturbations. This review covers, in turn, the main predictions from such quantitative analysis and the experimental consequences of comparable alterations in extracellular pH, lactate concentration, membrane potential and extracellular tonicity. The effects of such alterations in the extracellular environment in resting amphibian muscles are then used to reproduce the intracellular changes that occur in each case in exercising muscle. The relative contributions of these various factors to the control of cell volume in resting and exercising skeletal muscle are thus described.     

人骨骼肌M蛋白的提取与纯化

依据Ｔｒｉｎｉｃｋ－Ｅｐｐｅｎｂｅｒｇｅｒ对鸡骨骼肌Ｍ蛋白的提取方法,由人骨骼肌中得到的Ｍ蛋白粗提物除含分子量为１６５０００的Ｍ蛋白外,还含有分子量为１８５０００和１４００００（Ｃ成分）的两组分。由于在粗提物中未发现分子量为９００００的磷酸化酶,我们将最终纯化步骤中的亲和层析改为制备电泳,同样获得了纯化的Ｍ蛋白。     

Mechanism of Ion Permeation in Skeletal Muscle Chloride Channels

Voltage-gated Cl⁻ channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl⁻ channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I⁻ and related anions. Extracellular and intracellular I⁻ exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br⁻) and polyatomic (SCN⁻, NO₃ ⁻, CH₃SO₃ ⁻) anions. In addition, I⁻ block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I⁻ causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I⁻ slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined K _D values for I⁻ block in three distinct kinetic states and found that binding of I⁻ to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I⁻ binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.     

Postexercise Muscle Triacylglycerol and Glycogen Metabolism in Obese Insulin‐Resistant Zucker Rats

Objective: To determine the impact of insulin resistance and obesity on muscle triacylglycerol (IMTG) and glycogen metabolism during and after prolonged exercise. Research Methods and Procedures: Female lean (fa/?; N = 40, ZL) and obese insulin-resistant (fa/fa; N = 40, ZO) Zucker rats performed an acute bout of swimming exercise (8 times for 30 minutes) followed by 6 hours of carbohydrate supplementation (CHO) or fasting (FAST). IMTG and glycogen were measured in the extensor digitorum longus (EDL) and red vastus lateralis (RVL) muscles. Results: Despite resting IMTG content being 4-fold higher in ZO compared with ZL rats, IMTG levels were unchanged in either EDL or RVL muscles immediately after exercise. Resting glycogen concentration in EDL and RVL muscles was similar between genotypes, with exercise resulting in glycogen use in both muscles from ZL rats (∼85%, p < 0.05). However, in ZO rats, there was a much smaller decrease in postexercise glycogen content in both EDL and RVL muscles (∼30%). During postexercise recovery, there was a decrease in EDL muscle levels of IMTG in ZL rats supplemented with CHO after 30 and 360 minutes (p < 0.05). In contrast, IMTG content was increased above resting levels in RVL muscles of ZO rats fasted for 360 minutes. Six hours of CHO refeeding restored glycogen content to resting levels in both muscles in ZL rats. However, after 6 hours of FAST in ZO animals, RVL muscle glycogen content was still lower than resting levels (p < 0.05). At this time, IMTG levels were elevated above basal (p < 0.05). Discussion: In both healthy and insulin-resistant skeletal muscle, there was negligible net IMTG degradation after a single bout of prolonged exercise. However, during postexercise recovery, there was differential metabolism of IMTG between phenotypes.     

Bcl-2 Expression Identifies an Early Stage of Myogenesis and Promotes Clonal Expansion of Muscle Cells

We show that Bcl-2 expression in skeletal muscle cells identifies an early stage of the myogenic pathway, inhibits apoptosis, and promotes clonal expansion. Bcl-2 expression was limited to a small proportion of the mononucleate cells in muscle cell cultures, ranging from ∼1–4% of neonatal and adult mouse muscle cells to ∼5–15% of the cells from the C2C12 muscle cell line. In rapidly growing cultures, some of the Bcl-2–positive cells coexpressed markers of early stages of myogenesis, including desmin, MyoD, and Myf-5. In contrast, Bcl-2 was not expressed in multinucleate myotubes or in those mononucleate myoblasts that expressed markers of middle or late stages of myogenesis, such as myogenin, muscle regulatory factor 4 (MRF4), and myosin. The small subset of Bcl-2–positive C2C12 cells appeared to resist staurosporine-induced apoptosis. Furthermore, though myogenic cells from genetically Bcl-2–null mice formed myotubes normally, the muscle colonies produced by cloned Bcl-2–null cells contained only about half as many cells as the colonies produced by cells from wild-type mice. This result suggests that, during clonal expansion from a muscle progenitor cell, the number of progeny obtained is greater when Bcl-2 is expressed.     

脂联素在骨骼肌中的新功能及其与运动的关系

既往研究认为脂联素（adiponectin,ADPN）是一种脂肪因子,可以促进骨骼肌的糖原合成来调节血糖。但是近期研究表明,骨骼肌同样是分泌ADPN的重要器官。此外,ADPN在骨骼肌中不仅可以调节糖代谢,还在改变肌肉类型、介导线粒体功能、改善胰岛素抵抗、提升肌肉收缩和钙调节、促进肌肉再生等均发挥重要的生物学作用。运动可以调节血清及骨骼肌中ADPN表达水平,但其结果还存在争议。因此,探索ADPN在骨骼肌中新的生物学功能以及运动对骨骼肌ADPN表达的确切效果可能为治疗骨骼肌相关疾病提供新思路。     

肥胖诱导的骨骼肌萎缩机制研究进展

骨骼肌是人体氨基酸和蛋白质的主要贮存、代谢库,其正常功能和代谢过程受到多种病理因素的影响。骨骼肌萎缩发生于骨骼肌稳态严重失衡状态下,对患者生活和社会医疗造成了沉重负担。近年来,由于世界肥胖人群数量激增,肥胖诱导的骨骼肌萎缩正日益成为公共卫生的严峻挑战之一。肥胖诱导的骨骼肌萎缩过程涉及多种信号分子或通路的改变,如泛素蛋白酶系统、自噬溶酶体系统、胰岛素/IGF1-PI3K-Akt、肌肉生长抑制素、白细胞介素-6、肿瘤坏死因子等;这些信号分子或通路在肥胖状态下被激活或抑制后,可共同影响蛋白质合成/分解平衡进而造成骨骼肌萎缩。基于上述信号分子或通路,系统总结并讨论了肥胖诱导的骨骼肌萎缩机制,以期为寻找缓解/治疗肥胖诱导的肌萎缩靶点和进一步开发利用天然植物化学物提供理论依据。     

A Role for Acetylcholine-Nicotinic Receptor Interactions in the Selective Increase of Rat Skeletal Muscle G4 Acetylcholinesterase Following Short-Term Denervation

The present work addresses the effects of short-term denervation on acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes in anterior gracilis muscles from adult male Sprague-Dawley rats. It examines possible relationships between AChE isoform changes and other denervation phenomena, and evaluates the importance of acetylcholine (ACh)-nicotinic receptor interactions in selectively modulating the activity of G4 AChE. Results confirm that denervation causes a specific, transient increase in G4 AChE and show that: most of the increment can be explained by the hydrophobic species of this isoenzyme; changes in AChE isoforms markedly precede the onset of spontaneous electromechanical activity (fibrillation), as well as acetylcholine receptor (AChR) proliferation; and the G4 AChE response is eliminated when AChRs are blocked by alpha-bungarotoxin treatment performed before but not after (24 h) denervation. These data point to the absence of direct causal relationships between the G4 AChE increment and fibrillation, AChR proliferation, or changes in the release of this isoform from denervated muscle. In turn, they suggest the participation of AChR activation in triggering the G4 AChE response and emphasize the possible role of ACh-AChR interactions in modulating the production of this isoenzyme in not only denervated but also innervated fast-twitch muscles.     

小鼠肌母细胞的增殖与分化

通过对小鼠肌母细胞C2C12的培养,研究C2C12细胞的增殖与分化的关系以及胰岛素在细胞分化过程中的作用。在对照组中,C2C12细胞增殖占了明显的优势,细胞形态几乎没有发生变化;而在实验组中,C2C12细胞在换为分化培养基24小时后,就出现了部分细胞衰亡和死亡的现象,尤其是在48小时细胞的死亡率达到最高,存活细胞开始从增殖期进入分化期,72小时出现了少量肌管,在96小时细胞分化效果达到最好。而在添加了胰岛素的分化培养基中的细胞分化效果明显好于没有添加胰岛素的分化培养基中的细胞,结果表明,胰岛素促进C2C12细胞的分化。     

The JAK2/STAT3 Signal Pathway Regulates the Expression of Genes Related to Skeletal Muscle Development and Energy Metabolism in Mice and Mouse Skeletal Muscle Cells

The gene encoding a β-galactosidase from Entevobacter cloacae GAO was cloned and expressed in Escherichia coli. The nucleotide sequence of the insert of a positive clone had an open reading frame of 3084 bp that encoded a polypeptide of 1028 amino acid residues with a calculated molecular mass of 116,677 daltons. The amino acid sequence of β-galactosidase deduced from the nucleotide sequence, especially the sequence around the putative active site and of the fourteen regions, showed significant homology to β-galactosidases of other microorganisms, E. coli, Klebsiella pneumoniae, Lactobacillus bulgaricus, and Clostridium acetobutylicum.