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.

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.

Presence of enolase in the M-band of skeletal muscle and possible indirect interaction with the cytosolic muscle isoform of creatine kinase.

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.

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.

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.

The effect of pH on the covalent and metabolic control of C4 phosphoenolpyruvate carboxylase from Sorghum leaf.

The influence of pH on the in vitro activity and regulatory properties of Sorghum leaf C4 phosphoenolpyruvate carboxylase (PEPC) was investigated with respect to the phosphorylation status of the enzyme. In vitro protein phosphorylation was achieved using the catalytic subunit of a cAMP-dependent protein kinase (PKA) and a recombinant, immunopurified PEPC (0.9 mol of covalent Pi/mol PEPC subunit).

Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase (A Cardinal Event Influencing the Photosynthesis Rate in Sorghum and Maize).

C4 leaf phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.

A model system of coupled activity of co-immobilized creatine kinase and myosin.

Myosin and creatine kinase were co-immobilized onto Immunodyne films to mimic the behaviour of creatine kinase bound to the M-line of myofilaments. The Mg-ATPase activity of bound myosin was studied by a coupled enzymatic assay, which detects Mg-ADP in the bulk solution by means of pyruvate kinase and lactate dehydrogenase. The competition for Mg-ADP between pyruvate kinase and creatine kinase either free in solution or co-immobilized with myosin was studied at various creatine phosphate concentrations.

Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase. Identification of the protein-serine kinase and some elements of the signal-transduction cascade.

The phosphoenolpyruvate (PPrv) carboxylase isozyme involved in C4 photosynthesis undergoes a day/night reversible phosphorylation process in leaves of the C4 plant, Sorghum. Ser8 of the target enzyme oscillates between a high (light) and a low (dark) phosphorylation status. Both in vivo and in vitro, phosphorylation of dark-form carboxylase was accompanied by an increase in the apparent Ki of the feedback inhibitor L-malate and an increase in Vmax.

Diffusion-limited kinetics of immobilized myosin ATPase.

To test the possibility that ATP diffusion limits the kinetics of myosin ATPase (EC. 3.6.

An example of substrate channeling between co-immobilized enzymes. Coupled activity of myosin ATPase and creatine kinase bound to frog heart myofilaments.

In myofilaments obtained by Triton X-100 lysis of frog heart cells in high ionic strength medium, the activity of bound creatine kinase cannot be detected by a coupled enzymatic assay. ATP is channelized toward myosin ATPase, through the unstirred layer near myofilaments and cannot diffuse into the bulk solution. Model systems based upon the coupled kinetics of enzymes co-immobilized on the same surface may explain this behaviour.

High-energy phosphates in quiescent, beating and contracted cardiac cells.

Isolated myocytes from ventricles are quiescent in the presence of 0.9 mM calcium. However, it is possible to induce beating by adding 0.

Compartmentation of high-energy phosphates in resting and beating heart cells.

The subcellular distribution of ATP, ADP, creatine phosphate and creatine has been analyzed by fast detergent fractionation of isolated frog heart cells. Digitonin fractionation (0.5 mg/ml, 10 s at 2 degrees C in 20 mM 4-morpholinepropanesulfonic acid/3 mM EDTA/230 mM mannitol medium) was used to separate mitochondria and myofilaments from cytosol.

Coupled reaction of immobilized aspartate aminotransferase and malate dehydrogenase. A plausible model for the cellular behaviour of these enzymes.

To study the effect of facilitated diffusion of the intermediate metabolite, oxaloacetate, on the coupled reaction of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.

Binding of substrates to aspartate aminotransferase. Evidence for a dissymmetrical binding.

The binding of substrates L-glutamate and 2-oxoglutarate to aspartate aminotransferase was studied by spectrophotometric titration according to Jenkins and D'Ari [J. Biol. Chem.

Interactions between apoaspartate aminotransferase and pyridoxal 5'-phosphate. A stopped-flow study.

The fast kinetics and mechanism of the reconstitution reaction of holoaspartate aminotransferase from apoenzyme and pyridoxal 5'-phosphate were investigated by the stopped-flow method. When the absorbance change was monitored at 362 nm, the process was shown to involve three steps. The dependence of the three relaxation times on pyridoxal 5'-phosphate concentration and the analysis of the amplitudes enabled us to propose a mechanism in which the initial reversible binding step was followed by two irreversible isomerization steps.

Fluorescence of aromatic amino acids in a pyridoxal phosphate enzyme: aspartate aminotransferase.

At pH 8.3, the fluorescence spectrum of apoaspartate aminotransferase is characteristic of buried tryptophans (maximum at 330 nm and width at half-height equal to 51 nm). Its quantum yield is 1.

Dissociation of aspartate aminotransferase into subunits. Effect of ligands upon this dissociation.

Frontal and zonal analysis of the chromatography of aspartate aminotransferase (EC2.61.1), pig heart cytosolic enzyme, on Bio-Gel P150 shows that holo- and apoenzyme can dissociate at pH 8.