Parvalbumin concentration and diffusion coefficient in frog myoplasm

Abstract

The concentrations and diffusivity of two isoforms of parvalbumin, IVa and IVb, were measured using quantitative SDS PAGE in single fibers from semitendinosus muscles of the frog Rana temporaria. The concentrations of IVa and IVb were 2.9 ± 0.3 (SEM) and 4.5 ± 0.5 gl−1 total fiber volume, respectively. The total concentration of parvalbumin (7.4 ± 0.8 gl−1 total fiber) corresponds to a cytosolic concentration of 0.9 ± 0.1 mmoll−1 myoplasmic water. Estimates for the transverse and longitudinal diffusion coefficients for parvalbumin at 4°C were obtained in two ways: (1) by diffusion of parvalbumin out of skinned fibers into droplets of relaxing solution, and (2) by diffusion of parvalbumin between two juxtaposed skinned fibers under oil. The transverse diffusion coefficient obtained using the droplet method was significantly lower than that obtained using juxtaposed fibers, but the longitudinal diffusion coefficients obtained from both methods were similar. The juxtaposed fiber method more accurately approximates parvalbumin diffusion in undisturbed myoplasm because no artificial solutions were used and, upon fiber-to-fiber contact, a potentially confounding oil barrier at the interface rapidly disperses. The juxtaposed fiber method yielded values for transverse (4.27 ± 0.87 × 10−7 cm2 s−1) and longitudinal (3.20 ± 0.74 × 10−7 cm2 s−1) diffusion coefficients that were not significantly different, suggesting that diffusion of parvalbumin in myoplasm is essentially isotropic. The average diffusion coefficient of frog parvalbumin in myoplasm (3.74 ± 0.81 × 10−7 cm2 s−1; 4°C) is approximately a third of that estimated for frog parvalbumin diffusing in bulk water into and out of 3% agarose cylinders (10.6 × 10−7 cm2 s−1; 4°C). The reduced translational mobility of parvalbumin in myoplasm reflects an elevated effective viscosity due to tortuosity and viscous drag imposed by the fixed proteins of the cytomatrix and the numerous diffusible particles of the cytosol.