Reedy M K, Lucaveche C
Adv Exp Med Biol. 1984;170:29-45. doi: 10.1007/978-1-4684-4703-3_4.
We have measured filament lattice spacing in fibrils using X-ray diffraction, and find that STEM-determined mass/length values reported for myofilaments should give 13% w/v as the filament protein concentration in the lattice of the AO-band (filament overlap zone) of both insect flight muscle (IFM) and vertebrate skeletal muscle ( VSkM ). This is well below the actual mass concentration of AO-bands as measured by immersion refractometry of detergent-washed rigor myofibrils under the phase microscope. This technique identifies an immersion fluid of suitable refractive index (RI) for matching out all image contrast between background and the selected cross-band. We used RI fluids in which the effective RI matching component is a large-particle solute (Percoll or hemocyanin) excluded by the filament lattice. The measured RI indicates that protein concentration in AO-bands is 16-17% in Lethocerus and other IFM fibrils including CAF-digested fibrils, and is 18-19% in rabbit VSkM fibrils. On IFM fibrils we also measured absolute buoyant density in Percoll as greater than or equal to 1.042; this supports the value for mass concentration as determined by RI. The mass discrepancy between fibrils and filaments does not seem to arise from faults in the methods used. We therefore accept the STEM-determined mass/length values for thick filaments which indicate 4 myosins/crown in IFM and 3 in VSkM , and we believe there is considerable extra nonfilament material (concentration: 30-50 mg/ml) between the filaments in fibrils. In stretched VSkM , it is the H-bands, not the I-bands, which have an excess over filament mass content. The extra mass has not been identified.
我们使用X射线衍射测量了肌原纤维中细丝晶格间距，发现针对肌丝报告的扫描透射电子显微镜（STEM）测定的质量/长度值，应得出13%（w/v）作为昆虫飞行肌（IFM）和脊椎动物骨骼肌（VSkM）的AO带（细丝重叠区）晶格中的细丝蛋白浓度。这远低于通过相显微镜下对去污剂洗涤的僵直肌原纤维进行浸没折射测量所测得的AO带实际质量浓度。该技术可确定一种具有合适折射率（RI）的浸没液，以消除背景与选定交叉带之间的所有图像对比度。我们使用的RI液体中，有效的RI匹配成分是一种被细丝晶格排除的大颗粒溶质（聚蔗糖或血蓝蛋白）。测量的RI表明，在田鳖及其他IFM肌原纤维（包括经钙激活因子消化的肌原纤维）中，AO带中的蛋白质浓度为16 - 17%，在兔VSkM肌原纤维中为18 - 19%。在IFM肌原纤维上，我们还测量了其在聚蔗糖中的绝对浮力密度大于或等于1.042；这支持了通过RI确定的质量浓度值。肌原纤维和肌丝之间的质量差异似乎并非源于所用方法的缺陷。因此，我们接受STEM测定的粗肌丝质量/长度值，该值表明IFM中每条冠有4个肌球蛋白，VSkM中有3个，并且我们认为在肌原纤维的肌丝之间存在大量额外的非细丝物质（浓度：30 - 50 mg/ml）。在拉伸的VSkM中，具有超过细丝质量含量的是H带，而非I带。尚未确定额外的质量物质。
