Mobility of creatine phosphokinase and beta-enolase in cultured muscle cells

Abstract

The diffusion of beta-enolase and creatine phosphokinase in muscle cells has been studied by modulated fringe pattern photobleaching. Beta-enolase is mobile in the sarcoplasm. At 20 degrees C, the diffusion coefficient is 13.5 +/- 2.5 microm2 s(-1) in the cytosol and 56 microm2 s(-1) in aqueous media. As in the case of dextrans of the same hydrodynamic radius, its mobility is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments. A fraction of creatine phosphokinase is mobile in the sarcoplasm. Its diffusion coefficient in the cytosol, 4.5 +/- 1 microm2 s(-1), is lower than that of the dextran of equivalent size. The other fraction (20 to 50%) is very slightly mobile, with an apparent diffusion coefficient varying from 0.0035 to 0.043 microm2 s(-1). This low mobility might be attributed to exchange between free and bound creatine phosphokinase. The bound fraction of the endogenous enzyme was localized by immunocytofluorescence on the cultured muscle cells. Our results favor a localization of bound cytosolic creatine phosphokinase on the M-line and a diffuse distribution in all myotubes.

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

“…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. Analyses of the influence of hydrodynamic radius on the D values of macromolecules demonstrated that for both proteins , 1996) the difference between D in the sarcoplasm and D in water increased with molecule size, until in the largest proteins D was essentially negligible.…”

From fixed to FRAP: measuring protein mobility and activity in living cells

“…It is beyond the scope of this review to discuss all mathematical models in detail, but we shall mention some biologically important parameters determining diffusion of soluble and membrane-bound molecules. Unrestricted diffusion of a particle in a free-volume model is described by the Stokes-Einstein formula 6 : D = kT/6πηR h which correlates the hydrodynamic behaviour of a sphere with the absolute temperature T, the viscosity of the solution η, the Boltzmann constant k and the hydrodynamic radius of the particle R h . Because the local absolute temperature is hardly affected by bleaching 7 , and the viscosity of water and cytosol are relatively constant under experimental conditions, D is determined mainly by R h .…”

Diffusion-limited encounter rate in a three-dimensional lattice of connected compartments studied by Brownian-dynamics simulations

“…For instance, (5-enolase has a hydrodynamic radius of about 4 nm and diffusion coefficient in buffer Do = 56 pm2 s ~1. The diffusion coefficient in the cytosol of muscle cells is reduced to D = 13.5 pm 2 s _l [31]. Assume that the muscle cell has a characteristic radius of L = 25 nm (the typical values for the intracellular environment are L = 10-50nm) [10,[32][33][34][35][36][37], In Fig.…”