Kinetics of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle: mechanism of protective action of alpha-crystallin

The kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle have been studied by dynamic light scattering (0.08M Hepes, pH 6.8, containing 0.1M NaCl; 48 degrees C). The hydrodynamic radius of the start aggregates determined from the initial linear parts of the dependences of the hydrodynamic radius (R(h)) on time was found to be 16.7 +/- 1.0 nm. At rather high values of time, the R(h) value for the protein aggregates becomes proportional to t(1/1.8) = t(0.56) suggesting that the aggregation process proceeds in the regime of diffusion-limited cluster-cluster aggregation. In the presence of alpha-crystallin, a protein possessing the chaperone-like activity, the process of protein aggregation switches to the regime of reaction-limited cluster-cluster aggregation as indicated by the exponential dependence of the R(h) value on time. It was shown that the addition of alpha-crystallin raises the rate of thermal inactivation of Phb. These data in combination with the results of the study of interaction of Phb with alpha-crystallin by analytical ultracentrifugation suggest that alpha-crystallin interacts with the intermediates of unfolding of the Phb molecule.     

Structural changes and aggregation process of Cu/containing amine oxidase in the presence of 2,2,2'-trifluoroethanol

Conformational and structural changes of lentil seedlings amine oxidase (LSAO) were studied in the presence of trifluoroethanol (TFE) by spectroscopic and analytical techniques. At TFE concentrations up to 5%, the induction of a structural transition from beta-sheet to alpha-helix and up to 10% TFE a structural transition from alpha-helix to beta-sheet as well as inactivation of the enzyme are observed. At TFE concentrations between 10-35%, LSAO proves to be prone to aggregation and beyond 35% TFE leads to a non-native protein structure with a high alpha-helix content. The obtained results revealed that the aggregation of LSAO is strongly linked to the nature of secondary structures.     

Cooperative self-association of phosphorylase kinase from rabbit skeletal muscle

Ca(2+)- and Mg(2+)-induced association of phosphorylase kinase (PhK) from rabbit skeletal muscle has been studied at the magnitudes of the ionic strength close to the physiological values (40 mM Hepes, pH 6.8, containing 0.1 M NaCl, 0.1 mM Ca(2+), 10 mM Mg(2+); 25 degrees C) and under the molecular crowding conditions produced by high concentrations (1 M) of the natural osmolyte, trimethylamine N-oxide (TMAO). In the presence of 0.1 M NaCl two forms of PhK were registered, namely the "basic form" and "highly associated form", suggesting that PhK association may be treated as an example of cooperative association. According to the data on dynamic light scattering the average hydrodynamic radii of these forms were 16 and 144 nm. The addition of 1 M TMAO produces the time dependent increase in the light scattering intensity caused by the conversion of the basic form into the highly associated form. According to the data of the sedimentation analysis the basic form of PhK comprises a hexadecamer (M(r)=1320 kDa) and its small associates. The removal of Ca(2+) by addition of EGTA results in the reverse conversion of the highly associated form into the basic form suggesting reversibility of self-association of PhK. FAD, the ligand that is specifically bound to PhK, blocks the conversion of the basic form of PhK into the highly associated form.     

Kinetics of Denaturation of Rabbit Skeletal Muscle Glycogen Phosphorylase b by Guanidine Hydrochloride

The kinetics of denaturation and aggregation of rabbit muscle glycogen phosphorylase b in the presence of guanidine hydrochloride (GuHCl) have been studied. The curve of inactivation of phosphorylase b in time includes a region of the fast decline in the enzymatic activity,an intermediate plateau,and a part with subsequent decrease in the enzymatic activity. The fact that the shape of the inactivation curves is dependent on the enzyme concentration testifies to the dissociative mechanism of inactivation. The dissociation of phosphorylase b dimers into monomers in the presence of GuHCl is supported by sedimentation data. The rate of phosphorylase b aggregation in the presence of GuHCl rises as the denaturant concentration increases to 1.12 M; at higher concentration of GuHCl, suppression of aggregation occurs. At rather low concentration of the protein (0.25 mg/ml), the terminal phase of aggregation follows the kinetics of a monomolecular reaction (the reaction rate constant is equal to 0.082 min^–1;1 M GuHCl, 25°C). At higher concentration of phosphorylase b (0.75 mg/ml), aggregation proceeds as a trimolecular reaction.     

Combined kinetic mechanism describing activation and inhibition of muscle glycogen phosphorylase b by adenosine 5'-monophosphate

The kinetic analysis of the glycogen chain growth reaction catalyzed by glycogen phosphorylase b from rabbit skeletal muscle has been carried out over a wide range of concentrations of AMP under the saturation of the enzyme by glycogen. The applicability of 23 different variants of the kinetic model involving the interaction of AMP and glucose 1-phosphate binding sites in the dimeric enzyme molecule is considered. A kinetic model has been proposed which assumes: (i) the independent binding of one molecule of glucose 1-phosphate in the catalytic site on the one hand, and AMP in both allosteric effector sites and both nucleoside inhibitor sites of the dimeric enzyme molecule bound by glycogen on the other hand; (ii) the binding of AMP in one of the allosteric effector sites results in an increase in the affinity of other allosteric effector site to AMP; (iii) the independent binding of AMP to the nucleoside inhibitor sites of the dimeric enzyme molecule; (iv) the exclusive binding of the second molecule of glucose 1-phosphate in the catalytic site of glycogen phosphorylase b containing two molecules of AMP occupying both allosteric effector sites; and (v) the catalytic act occurs exclusively in the complex of the enzyme with glycogen, two molecules of AMP occupying both allosteric effector sites, and two molecules of glucose 1-phosphate occupying both catalytic sites.     

Sedimentation analysis of muscle glycogen phosphorylase b entrapped in hydrated reversed micelles of aerosol OT in octane

The oligomeric state and formation of supramolecular structures of glycogen phosphorylase b from rabbit skeletal muscles have been studied in the system of hydrated reversed micelles of aerosol OT (AOT) in octane. Sedimentation studies show that the oligomeric state of the enzyme is controlled by the degree of hydration of micelles. Monomeric, dimeric, trimeric, tetrameric, hexameric, or octameric forms of the enzyme were observed depending on the degree of micelle hydration.     

Supramolecular complexes of polyethylene glycol-alpha-Chymotrypsin covalent and noncovalent adducts with polyoxyethylene-modified cyclo dextrins

Complexes of covalent and noncovalent adducts of polyethylene glycol (PEG) and alpha-chymotrypsin (ChT), PEG-ChT, were generated in the presence of beta-cyclodextrin derivatives of polyoxyethylene (beta CD-PEO), and their thermal stability was studied. The covalent [PEG-ChT]c conjugates were obtained by chemical modification of the protein amino groups with the monoaldehyde derivatives of monomethoxypolyethylene glycol. The noncovalent [PEG-ChT]n complexes were obtained by the treatment of ChT-PEG mixtures with increasing pressure (1.1-400 MPa). Supramolecular structures resulting from complex formation between PEG chains of the PEG-ChT adducts (PEGad) and beta CD-PEO were studied. The decrease in the rate constant of the slow stage of ChT thermal inactivation in PEG-ChT adducts (k2) can serve as confirmation of complex formation between beta CD-PEO and PEGad. The stoichiometric composition of our supramolecular structures was determined from the k2 dependence on the molar ratio of beta CD-PEO to PEGad. It was shown that each polymeric chain in the [PEG-ChT]c conjugates forms an inclusion complex with beta CD-PEO, whereas only half of the PEGad polymeric chains participate in the formation of supramolecular structures in the case of [PEG-ChT]n complexes. Although covalent and noncovalent PEG-ChT adducts of the same composition significantly differ in their thermal stability, the maximal values of the k2 rate constants for [PEG-ChT]c and [PEG-ChT]n adducts in the triple system attainable at the (beta CD-PEO) to (PEGad) ratio corresponding to the stoichiometry of the resulting ternary systems are practically the same (k2 = 0.007 c-1 at 45 degrees C in 0.02 M Tris-HCl buffer solution, pH 8.0). Structures for the supramolecular dendrite-like ensembles formed upon the interaction of covalent and noncovalent PEG-ChT adducts with beta CD-PEO were suggested.     

A tentative mechanism of the ternary complex formation between phosphorylase kinase, glycogen phosphorylase b and glycogen

The kinetics of rabbit skeletal muscle phosphorylase kinase interaction with glycogen has been studied. At pH 6.8 the binding of phosphorylase kinase to glycogen proceeds only in the presence of Mg2+, whereas at pH 8.2 formation of the complex occurs even in the absence of Mg2+. On the other hand, the interaction of phosphorylase kinase with glycogen requires Ca2+ at both pH values. The initial rate of the complex formation is proportional to the enzyme and glycogen concentrations, suggesting the formation of the complex with stoichiometry 1:1 at the initial step of phosphorylase kinase binding by glycogen. According to the kinetic and sedimentation data, the substrate of the phosphorylase kinase reaction, glycogen phosphorylase b, favors the binding of phosphorylase kinase with glycogen. We suggest a model for the ordered binding of phosphorylase b and phosphorylase kinase to the glycogen particle that explains the increase in the tightness of phosphorylase kinase binding with glycogen in the presence of phosphorylase b.     

The study of amorphous aggregation of tobacco mosaic virus coat protein by dynamic light scattering

The kinetics of heat-induced and cetyltrimethylammonium bromide induced amorphous aggregation of tobacco mosaic virus coat protein in Na(+)/Na(+) phosphate buffer, pH 8.0, have been studied using dynamic light scattering. In the case of thermal aggregation (52 degrees C) the character of the dependence of the hydrodynamic radius (R(h)) on time indicates that at certain instant the population of aggregates is split into two components. The size of the aggregates of one kind remains practically constant in time, whereas the size of aggregates of other kind increases monotonously in time reaching the values characteristic of aggregates prone to precipitation (R(h)=900-1500 nm). The construction of the light scattering intensity versus R(h) plots shows that the large aggregates (the start aggregates) exist in the system at the instant the initial increase in the light scattering intensity is observed. For thermal aggregation the R(h) value for the start aggregates is independent of the protein concentration and equal to 21.6 nm. In the case of the surfactant-induced aggregation (at 25 degrees C) no splitting of the aggregates into two components is observed and the size of the start aggregates turns out to be much larger (107 nm) than on the thermal aggregation. The dependence of R(h) on time for both heat-induced aggregation and surfactant-induced aggregation after a lapse of time follows the power law indicating that the aggregation process proceeds in the kinetic regime of diffusion-limited cluster-cluster aggregation. Fractal dimension is close to 1.8. The molecular chaperone alpha-crystallin does not affect the kinetics of tobacco mosaic virus coat protein thermal aggregation.