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.

We have used a fluorescence recovery after photobleaching (FRAP) technique to measure radial diffusion of myoglobin and other proteins in single skeletal and cardiac muscle cells. We compare the radial diffusivities, Dr (i.e., diffusion perpendicular to the long fiber axis), with longitudinal ones, Dl (i.e., parallel to the long fiber axis), both measured by the same technique, for myoglobin (17 kDa), lactalbumin (14 kDa), and ovalbumin (45 kDa). At 22°C, D l for myoglobin is 1.2 ؋ 10 ؊7 cm 2 ͞s in soleus fibers and 1.1 ؋ 10 ؊7 cm 2 ͞s in cardiomyocytes. Dl for lactalbumin is similar in both cell types. Dr for myoglobin is 1.2 ؋ 10 ؊7 cm 2 ͞s in soleus fibers and 1.1 ؋ 10 ؊7 cm 2 ͞s in cardiomyocytes and, again, similar for lactalbumin. Dl and Dr for ovalbumin are 0.5 ؋ 10 ؊7 cm 2 ͞s. In the case of myoglobin, both Dl and Dr at 37°C are about 80% higher than at 22°C. We conclude that intracellular diffusivity of myoglobin and other proteins (i) is very low in striated muscle cells, Ϸ1͞10 of the value in dilute protein solution, (ii) is not markedly different in longitudinal and radial direction, and (iii) is identical in heart and skeletal muscle. A Krogh cylinder model calculation holding for steady-state tissue oxygenation predicts that, based on these myoglobin diffusivities, myoglobin-facilitated oxygen diffusion contributes 4% to the overall intracellular oxygen transport of maximally exercising skeletal muscle and less than 2% to that of heart under conditions of high work load. Since Wittenberg (1, 2) demonstrated that myoglobin (Mb) diffusion facilitates O 2 transport in aqueous solutions, it has been postulated that facilitated O 2 diffusion may play an important role in respiring muscle. The extent of Mb-facilitated sarcoplasmic O 2 transport will depend mainly on the intracellular concentration of Mb, which is fairly well known for various muscle fiber types, and on the magnitude of its sarcoplasmic diffusion coefficient (D), which still is a matter of discussion. The direct measurement of translational Mb diffusivity in living skeletal muscle fibers (3-5) revealed an unexpectedly small value for D. However, in these studies, Mb diffusivity-for technical reasons-has been measured only along the longitudinal axis of muscle fibers and over long distances (tens or hundreds of sarcomere lengths). In contrast, facilitated O 2 transport in vivo mainly requires radial diffusion of Mb, from the sarcolemma to the mitochondria, i.e., in the direction perpendicular to the longitudinal fiber axis. It is conceivable that the highly ordered sarcoplasmic structures, e.g., the myofilaments, might give rise to significant differences between longitudinal (D l ) and radial (D r ) diffusion coefficients. Such an anisotropy has been reported for small molecules such as H 2 O (6), Ca 2ϩ (7), and O 2 (8), but it was not clear whether this also would be true for protein diffusion. Thus, the magnitude of D r in living heart and skeletal muscle remained a matter of speculation. For example, Baylor and Pape (3) argued that the...

1. In skeletal muscle an extracellular sarcolemmal carbonic anhydrase (CA) has been demonstrated. We speculate that this CA accelerates the interstitial CO 2 /HCO 3 _ buffer system so that H + ions can be rapidly delivered or buffered in the interstitial fluid. Because > 80 % of the lactate which crosses the sarcolemmal membrane is transported by the H + -lactate cotransporter, we examined the contributions of extracellular and intracellular CA to lactic acid transport, using ion-selective microelectrodes for measurements of intracellular pH (pH i ) and fibre surface pH (pH s ) in rat extensor digitorum longus (EDL) and soleus fibres.2. Muscle fibres were exposed to 20 mM sodium lactate in the absence and presence of the CA inhibitors benzolamide (BZ), acetazolamide (AZ), chlorzolamide (CZ) and ethoxzolamide (EZ The membrane-permeable CA inhibitors CZ (0.5 mM) and EZ (0.1 mM), which inhibit the extracellular as well as the intracellular CAs, exerted no greater effects than the poorly permeable inhibitors BZ and AZ did.6. In soleus, 10 mM cinnamate inhibited the lactate influx by 47 %. Addition of 0.01 mM BZ led to a further inhibition by only 10 %. BZ alone reduced the influx by 37 %.7. BZ (0.01 mM) had no influence on the K m value of the lactate transport, but led to a decrease in maximal transport rate (V max ). In EDL, BZ reduced V max by 50 % and in soleus by about 25 %.8. We conclude that the extracellular sarcolemmal CA plays an important role in lactic acid transport, while internal CA has no effect, a difference most likely attributable to the high internal vs. low extracellular BF non-HCO 3 . The fact that the effects of cinnamate and BZ are not additive indicates that the two inhibitors act at distinct sites on the same transport pathway for lactic acid.