Modulated fringe pattern photobleaching (MFPP) was used to measure the translational diffusion of microinjected fluorescein isothiocyanate (FITC)-labeled proteins of different sizes in the cytoplasm of cultured muscle cells. This technique, which is an extension of the classical fluorescence recovery after photobleaching (FRAP) technique, allows the measurement of the translational diffusion of macromolecules over several microns. Proteins used had molecular masses between 21 and 540 kDa. The results clearly indicated that the diffusivity of the various proteins is a decreasing function of their hydrodynamic radius. This decrease is more rapid with globular proteins than with FITC-labeled dextrans (, Biophys. J. 70:2327-2332), most likely because, unlike globular proteins, dextrans are randomly coiled macromolecules with a flexible structure. These data do not exclude the possibility of a rapid diffusion over a short distance, unobservable with our experimental set-up, which would take place within the first milliseconds after bleaching and would correspond to the diffusion in restricted domains followed by impeded diffusion provoked by the network of microtubules, microfilaments, and intermediate filaments. Thus our results may complement rather than contradict those of Verkman and collaborators (, J. Cell Biol. 138:1-12). The biological consequence of the size-dependent restriction of the mobility of proteins in the cell cytoplasm is that the formation of intracellular complexes with other proteins considerably reduces their mobility.

Myotubes were obtained from culture of satellite cells. They had a sarcomeric organization similar to that of muscle. The diffusion in the direction perpendicular to the fibers of microinjected fluorescein isothiocyanate-dextrans of molecular weight ranging from 9500 to 150,000 was examined by modulated fringe pattern photobleaching. On the time scale of the observation, 10-30 S, all of the dextrans were completely mobile in the cytoplasm. The diffusion coefficients were compared to the values obtained in water. The ratio D(cytoplasm)/D(w) decreased with the hydrodynamic radius R(h) of the macromolecules. The mobility of inert molecules in muscle cells is hindered by both the crowding of the fluid phase of the cytoplasm and the screening effect due to myofilaments: D(cytoplasm)/D(w) = (D/D(w)) protein crowding x (D/D(w))(filament screening). The equation (D/D(w))filament screening = exp(-K(L)RCh) was used for the contribution of the filaments to the restriction of diffusion. A free protein concentration of 135 mg/ml, a solvent viscosity of cytoplasm near that of bulk water, and a calculated K(L) of 0.066 nm(-1), which takes into account the sarcomeric organization of filaments, accurately represent our data.

The kinetics of ester substrate hydrolysis catalysed by the a, j, and $ forms of trypsin have been compared. An affinity chromatography method for +-trypsin purification is described. At optimum pH, with esters as substrates, there is no significant difference between the activity of the a and j forms; j-trypsin, however, is about 40 % more active than a-trypsin on benzoyl-L-arginine amide.At acidic pH, (= 3.6), j3-trypsin is more reactive toward p-nitrophenyl-p'-guanidino benzoate HCl than a-trypsin, and differential active-site titrations of the a and fi forms can be carried out in mixtures of both of them. Hydrolysis of specific esters by $-trypsin is very slow compared to native trypsin but it is not completely aspecific. Carbobenzoxy-L-lysine benzyl ester is a relatively good substrate of this enzyme form; k,,, values for $-trypsin are very sensitive to the substrate leaving group, in contrast to that which is observed with native trypsin. Furthermore, experiments involving nucleophilic competition indicate that the acylation step becomes rate-limiting for the hydrolysis of cationic ester substrates by $-trypsin.It is concluded that in tryptic catalysis enzyme acylation is a more specific step than substrate binding or enzyme deacylation. The acylation of the enzyme is greatly perturbed both by substrate modifications and by structural alterations of the native enzyme molecule. The role of the essential active-site carboxylic group (Asp-177) is discussed. It is proposed that its function in tryptic catalysis is to decrease the activation free energy for enzyme acylation instead of stabilizing the initial enzymesubstrate complex.The kinetic and structural properties of trypsin, a proteolytic enzyme, have been studied extensively [l -31. Tryptic catalysis is specific for peptide, amide and ester bonds formed with the carboxylic function of the basic amino acids lysine and arginine [4,5]. Tryptic specificity is not absolute, however, and some non-basic amino acid ester derivatives are also substrates of this enzyme [6,7].The kinetics of tryptic hydrolysis of synthetic ester substrates can be described by a three-step mechanism involving an acyl-enzyme intermediate (ES') in addition to the classical Michaelis complex (ES), as given in Eqn (1) :+ PI where PI and P, are the reaction products (alcohol and acid, respectively, in the case of an ester substrateAbbreviations. Amino-acid derivatives are abbreviated according to the general rules of IUPAC-IUB, Commission on Biochemical Nomenclature, see Eur. J. Biochern. 27, 9 . 201-207 (1972).Enzyme. Bovine trypsin (EC 3.4.4.4).In a previous paper [8] we analysed and discussed tryptic specificity in terms of the formalism of Eqn (1). The following conclusions were reported.a) The deacylation step (k,) is rate-limiting for specific ester substrates (lysine or arginine derivatives).b) The acylation step (k,) is at least partly ratelimiting for nonspecific ester substrates (tyrosine, phenylalanine and glycine derivatives). c) Structural modifications of a specific substra...

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.

Glycerol-skinned skeletal muscle fibres retain the defined sarcomeric structure of the myofibrils. We show here that a small fraction of two enzymes important for energy metabolism, the cytosolic muscle isoform of creatine kinase (EC 2.7.3.2), MM-creatine kinase (MM-CK), and enolase (EC 4.2.1.11), remains bound to skinned fibres. CK is slowly exchangeable, whereas enolase is firmly bound. Two-dimensional gel electrophoresis followed by Western blot analyses demonstrates that both alpha (ubiquitous) and beta (muscle-specific) subunits of enolase are present in these preparations. Enolase and CK were co-localized at the M-band of the sarcomeres, as observed by indirect immunofluorescence and confocal microscopy. Cross-linking experiments were performed on skinned fibres with three bifunctional succinimidyl esters of different lengths and yielded a protein complex of 150 kDa that reacted with antibodies directed against either M-CK or beta-enolase. The cross-linking efficiency was greatest for the longest reagent and zero for the shortest one. The length of the cross-linker giving a covalent complex between the two enzymes does not support the notion of a direct interaction between M-CK and enolase. This is the first demonstration of the presence of an enzyme of energy metabolism other than CK at the M-band of myofibres.