Protein Diffusion in Living Skeletal Muscle Fibers: Dependence on Protein Size, Fiber Type, and Contraction

Abstract

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.

Diffusion tensor imaging of the human calf muscle: distinct changes in fractional anisotropy and mean diffusion due to passive muscle shortening and stretching

“…In the literature, several factors that influence the diffusion properties of a muscle tissue have been discussed: fiber type and composition (7), myofilament lattices (22), temperature (23), and cell volume (24). It seems obvious that the diffusional anisotropy of musculature is the result of physical barriers due to the muscular architecture.…”

Molecules in motion: influences of diffusion on metabolic structure and function in skeletal muscle

“…The diffusion of Mb in skeletal muscle has been of particular interest because of its role in reversibly binding O 2 and therefore serving as a temporary O 2 store and facilitating O 2 diffusion (reviewed in Wittenberg and Wittenberg, 2003). Several studies have found that D of Mb in muscle fibers is about 1/6 to 1/10 of that in aqueous solution (Baylor and Pape, 1988;Jurgens et al, 1994;Papadopoulos et al, 1995;Papadopoulos et al, 2000;Papadopoulos et al, 2001). Measurements of protein diffusion in skeletal muscle from frog (Maughan and Lord, 1988;Maughan and Godt, 1999) and cultured mammalian fibers (Arrio-Dupont et al, 1997;Arrio-Dupont et al, 2000) yielded D values that were minimally 1/3 lower than that in water, and in many cases much more dramatically reduced.…”

Protein self‐association in crowded protein solutions: A time‐resolved fluorescence polarization study

“…If the dye is effectively bound to the protein the ANS-fluorescein separation should be less than the diameter of apoMb (∼3.5 nm) (Papadopoulos et al 2000). Because the Förster critical distance, R 0 , for the ANS-fluorescein pair is 5 nm (Sassaroli et al 1984), the ANS → fluorescein energy transfer efficiency should be higher than 90%, and virtually all the ANS fluorescence would be quenched.…”