Martine Arrio-Dupont

Translational Diffusion of Globular Proteins in the Cytoplasm of Cultured Muscle Cells

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.

Diffusion of fluorescently labeled macromolecules in cultured muscle cells.

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 effect of solvent on the fluorescence of Schiff bases of pyridoxal 5′ phosphate

Summary In non aqueous solvents, the Schiff base form of PLP absorbing at 330 nm fluoresces either at 525 nm or at 430 nm. The former emission, observed in CCl4 or CHCl3, is due to H displacement in the intramolecular H bond between phenolic group and imine nitrogen; the latter, observed in H accepting solvents, is due to the ionized form in the excited state. The spectral properties of PLP in glycogen phosphorylase and in aspartate aminotransferase are ascribed to the possibility of H-bonding interaction between the coenzyme and its microenvironment.

Interactions between beta-enolase and creatine kinase in the cytosol of skeletal muscle cells.

We studied interactions in vivo between the cytosolic muscle isoform of creatine kinase (M-CK) and the muscle isoform of 2-phospho-D-glycerate hydrolyase (beta-enolase) in muscle sarcoplasm by incubating glycerol-skinned fibres with FITC-labelled beta-enolase in the presence or absence of free CK. A small amount of bound beta-enolase was observed in the presence of large concentrations of CK. The mobility of enolase was measured in cultured satellite cells by modulated-fringe-pattern photobleaching. FITC-labelled beta-enolase was totally mobile in both the presence and the absence of CK but its diffusion coefficient was slightly lower in the presence of CK. This suggests a weak interaction in vivo between enolase and CK.

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

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.

The thiol groups of aspartate aminotransferase. Reactions of specific reagents with aldimine and aminic forms of enzyme

Summary The reaction of thiol groups of aspartate aminotransferase with DTNB and p-MB has been studied with the aldimine and the aminic forms of the enzyme. The result indicate that two cysteine residues by dimeric enzyme are exposed to the solvent after the transformation of the aldimine enzyme into the aminic form.

Functional interactions between subunits of aspartate aminotransferase: Formation of monoliganded dimers during titration of the apoenzyme by pyridoxal 5′-phosphate☆☆☆

Abstract The interaction between apoaspartate aminotransferase and pyridoxal 5′-phosphate at either pH 8.3 (active form of holoenzyme) or pH 5.0 (inactive form) corresponds to a strong quenching of tryptophan fluorescence. The hybrid molecule containing one pyridoxal 5′-phosphate bound per dimer has been prepared both by electrofocusing and by ion exchange chromatography. At both pH values, the fluorescence of the hybrid is 80 to 85% of the arithmetic mean between the fluorescence of the symmetrical holoenzyme and apoenzyme. This is direct evidence of energy transfer from tryptophan residues of the subunit of apoenzyme to the coenzyme of the other subunit. Fluorescence intensity was used to determine the quantity of hybrid holoapoenzyme formed during titration of the apoenzyme by pyridoxal 5′-phosphate. At pH 8.3 a non-linear decrease in the fluorescence is observed, corresponding to 60% of hybrid for the point of half reactivation; this value corresponds to the percentage obtained by electrofocusing (Schlegel & Christen, 1974). At pH 5.0, the decrease in fluorescence is linear during pyridoxal binding; this indicates that at this pH the hybrid is never obtained at detectable concentrations. These results indicate strong interactions between subunits of aspartate aminotransferase corresponding to a weakly negative co-operativity at alkaline pH and a positive cooperativity at acidic pH for the binding of the coenzyme.

Mechanism of aspartate aminotransferase inhibition by 5-5'-dithiobis-2-nitrobenzoate

Summary By DTNB reaction on aspartate aminotransferase 50 p. cent inhibition of the enzyme activity is observed. Of the 2 sites of the dimeric enzyme only one remains active. The other site is able to bind the substrate aspartate giving an abortive complex with the coenzyme pyridoxal-5-phosphate. The 2 subunits of the DTNB modified enzyme are not identical.

Presence of β‐enolase on the m‐line of skeletal muscle fibers. Possible interaction with MM‐CK

Glycerol skinned skeletal muscle fibers washed with 0.1 M KCI, 5mM EDTA, 2 mM EGTA pH 7.0 in the presence of protease inhibitors retained the defined sarcomeric structure of the myofibrlls.These myofibrils which represented a functional compartment for muscle contraction retained few percent of total MM-CK and I?-enolase activity. These two enzymes were co-localized on the M-line of the sarcomere as observed by indirect immunofluorescence and confocal microscopy. Cross-linking experiments were performed on skinned fibers with two bifunctional succinimidyl esters (EGS : 1.6 nm long, DSP : 1.2 nm long). Immunoblotting analysis showed a protein complex which reacted with antibodies directed against either MM-CK or &enolase. The cross-linking efficiency was greater for the longer reagent. Immunolocalization and cross-linking indicated a possible interaction of the two enzymes on the M-line.