Papadopoulos S, Jürgens K D, Gros G
Department of Physiology, Medizinische Hochschule Hannover, 30623 Hannover, Germany.
Biophys J. 2000 Oct;79(4):2084-94. doi: 10.1016/S0006-3495(00)76456-3.
Sarcoplasmic protein diffusion was studied under different conditions, using microinjection in combination with microspectrophotometry. Six globular proteins with molecular masses between 12 and 3700 kDa, with diameters from 3 to 30 nm, were used for the experiments. Proteins were injected into single, intact skeletal muscle fibers taken from either soleus or extensor digitorum longus (edl) muscle of adult rats. No correlation was found between sarcomere spacing and the sarcoplasmic diffusion coefficient (D) for all proteins studied. D of the smaller proteins cytochrome c (diameter 3.1 nm), myoglobin (diameter 3.5 nm), and hemoglobin (diameter 5.5 nm) amounted to only approximately 1/10 of their value in water and was not increased by auxotonic fiber contractions. D for cytochrome c and myoglobin was significantly higher in fibers from edl (mainly type II fibers) compared to fibers from soleus (mainly type I fibers). Measurements of D for myoglobin at 37 degrees C in addition to 22 degrees C led to a Q(10) of 1.46 for this temperature range. For the larger proteins catalase (diameter 10.5 nm) and ferritin (diameter 12.2 nm), a decrease in D to approximately 1/20 and approximately 1/50 of that in water was observed, whereas no diffusive flux at all of earthworm hemoglobin (diameter 30 nm) along the fiber axis could be detected. We conclude that 1) sarcoplasmic protein diffusion is strongly impaired by the presence of the myofilamental lattice, which also gives rise to differences in diffusivity between different fiber types; 2) contractions do not cause significant convection in sarcoplasm and do not lead to increased diffusional transport; and 3) in addition to the steric hindrance that slows down the diffusion of smaller proteins, diffusion of large proteins is further hindered when their dimensions approach the interfilament distances. This molecular sieve property progressively reduces intracellular diffusion of proteins when the molecular diameter increases to more than approximately 10 nm.
利用显微注射结合显微分光光度法，在不同条件下研究了肌浆蛋白扩散。实验使用了六种分子量在12至3700 kDa之间、直径为3至30 nm的球状蛋白。将蛋白质注射到取自成年大鼠比目鱼肌或趾长伸肌（edl）的单个完整骨骼肌纤维中。在所研究的所有蛋白质中，肌节间距与肌浆扩散系数（D）之间未发现相关性。较小的蛋白质细胞色素c（直径3.1 nm）、肌红蛋白（直径3.5 nm）和血红蛋白（直径5.5 nm）的D值仅约为其在水中值的1/10，且等张纤维收缩并未使其增加。与比目鱼肌纤维（主要是I型纤维）相比，edl纤维（主要是II型纤维）中细胞色素c和肌红蛋白的D值显著更高。除了在22℃下，还在37℃下测量了肌红蛋白的D值，在此温度范围内得到的Q(10)为1.46。对于较大的蛋白质过氧化氢酶（直径10.5 nm）和铁蛋白（直径12.2 nm），观察到D值降至约为其在水中值的1/20和约1/50，而沿纤维轴根本检测不到蚯蚓血红蛋白（直径30 nm）的扩散通量。我们得出结论：1）肌丝晶格的存在严重阻碍了肌浆蛋白扩散，这也导致了不同纤维类型之间扩散率的差异；2）收缩不会在肌浆中引起显著对流，也不会导致扩散运输增加；3）除了空间位阻减缓较小蛋白质的扩散外，当大蛋白质的尺寸接近丝间距离时，其扩散会进一步受阻。当分子直径增加到超过约10 nm时，这种分子筛特性会逐渐降低蛋白质在细胞内的扩散。
