Oligomeric state of the muscle glycogen phosphorylase b in the system of reversed micelles of aerosol OT in octane

The oligomeric state and formation of supramolecular structures of glycogen phosphorylase b from rabbit skeletal muscle was studied in the system of aerosol OT (AOT) reversed micelles in octane. The sedimentation experiments have shown that the enzyme oligomeric state depends on the degree of micelle hydration. The enzyme monomer, dimer, trimer, tetramer, hexamer, and octamer were observed, depending on the degree of hydration.     

Activity of lipoxygenase incorporated into reversed micelles of aerosol OT in octane

Soybean lipoxygenase (EC 1.13.11.12) incorporated into the reversed micelles of aerosol OT in octane has been studied for its catalytic properties. The enzyme is shown to preserve up to 10% activity as compared with the activity in the aqueous solution. In this case Km of lipoxygenase for linoleic acid increases from 10(-5) M to 5 X 10(-4) M. The activity of lipoxygenase is maximal, the aerosol OT concentration being 0.03 M and a degree of reversed micelle hydratation 40. Cationic detergents of the cetyltrimethyl ammonium bromide type are not good to form reversed micelles of lipoxygenase, since they inhibit the latter with IC50 = (4 divided by 6) x 10(-4) M. The lipoxygenase preparations in reversed micelles of aerosol OT in octane may be used to synthesize natural metabolites of polyunsaturated fatty acids, for instance of eicosanoids.     

Solubilization of rat kidney lysosomes in reversed micelles of aerosol OT in octane.

Intact lysosomes from rat kidneys were solubilized in the ternary system: surfactant (Aerosol OT)-buffer-organic solvent. According to data of laser light-scattering analysis and kinetic experiments with the lysosomal marker enzyme, N-acetyl-beta-D-hexosaminidase (EC 3.2.1.30), the solubilization of lysosomes in this system resulted in the destruction of the lysosomes and the entrapping of their components in reversed micelles.     

Catalytic properties and stability of catalase in reversed micelles of aerosol OT in octane]

The catalytic function of catalase and its peroxidatic activity during tetramethylbenzidine (TMB) oxidation by cumene hydroperoxide were studied in reversed micelles of Aerosol OT (AOT) in octane relative to the [H2O]/[AOT] ratio and the initial catalase concentration. The optimum conditions permitting to retain the catalytic activity of the enzyme and its ability to induce peroxidation of TMB, were found. The catalytic function of the enzyme was shown to be dependent on its concentration in AOT micelles. The catalase stability monitored by the catalytic reaction and the decrease of the Soret band were analyzed. Both processes have two phases differing by the rate constants of the pseudo-first order. The catalase inserted into AOT micelles is characterized by the high stability as compared to other hemoproteins (cytochrome P-450, myoglobin, hemoglobin, peroxidase) under identical conditions.     

Thermobarostability of alpha-chymotrypsin in reversed micelles of aerosol OT in octane solvated by water-glycerol mixtures

Thermostability of alpha-chymotrypsin at normal pressure in reversed micelles depends on both an effective surfactant solvation degree and glycerol content in the system. The difference in alpha-chymotrypsin stability in reversed micelles at various glycerol concentrations [up to 60% (v/v)] was more pronounced at high surfactant degrees of solvation, R >/= 16. After a 1-h incubation at 40 degrees C in "aqueous" reversed micelles (in the absence of glycerol), alpha-chymotrypsin retained only 1% of initial catalytic activity and 10, 22, 59, and 48% residual activity in glycerol-solvated micelles with 20, 30, 50, and 60% (v/v) glycerol, respectively. The explanation of the observed effects is given in the frames of micellar matrix structural order increasing in the presence of glycerol as a water-miscible cosolvent that leads to the decreasing mobility of the alpha-chymotrypsin molecule and, thus the increase of its stability. It was found that glycerol or hydrostatic pressure could be used to stabilize alpha-chymotrypsin in reversed micelles; a lower pressure is necessary to reach a given level of enzyme stability in the presence of glycerol.     

Comparative study of the kinetics of the reduction of cytochrome c and the nitroxide radical in reversed aerosol OT micelles in octane

The comparison of the reduction kinetics of cytochrome c and nitroxide radical by ascorbate in reversed micelles of aerosol OT in octane was studied. The plot of the dependence of the reduction rate constant on the micelle hydratation is bell-shaped in the case of protein but shows the plateau form for the radical. The reaction rates decreases at high micelle concentrations. The equations have been drawn that connect the experimental rate constants with intramicellar biomolecular rate constant (km) for reagents unsolved in organic phase. In the case of strong hydratated micelles km for the radical reduction is practically equal to the rate constant in aqueous solution. For cytochrome c the ratio of these constants is less than 0.22, that may be explained by protein conformational changes detected by optical methods. For small micelle hydratation the dependence of the cytochrome reduction rate on ascorbate concentration is characterized by plateau. Under these conditions the limited stage of reaction is apparently the transition of the protein to the active conformation.     

Effectiveness of acylation of protein amino groups with fatty acid chlorides in a system of reversed micelles of aerosol OT in octane

The characteristics of water-soluble enzyme (alpha-chymotrypsin) modification with [3H] palmitoyl chloride in the reversed Aerosol OT micelles in octane were determined. The degree of enzyme modification depends on the molar ratio [palmitoyl chloride]/[protein]. The modification reaction is characterized by the wide pH-optimum range and proceeds with high speed.     

The interaction of muscle glycogen phosphorylase b with glycogen

Interaction of muscle glycogen phosphorylase b (EC 2.4.1.1) with glycogen was studied by sedimentation, stopped-flow and temperature-jump methods. The equilibrium enzyme concentration was determined by sedimentation in an analytical ultracentrifuge equipped with absorption optics and a photoelectric scanning system. The maximum adsorption capacity of pig liver glycogen is 3.64 mumol dimeric glycogen phosphorylase b per g glycogen, which corresponds to 20 dimeric enzyme molecules per average glycogen molecule of Mr 5.5 X 10(6). Microscopic dissociation constants were determined for the enzyme-glycogen complex within the temperature range from 12.7 to 30.0 degrees C. Enzyme-glycogen complexing is accompanied by increasing light scattering and its increment depends linearly on the concentration of the binding sites on a glycogen particle that are occupied by the enzyme. Complex formation and relaxation kinetics are in accordance with the proposed bimolecular reaction scheme. The monomolecular dissociation rate constant of the complex increases as the temperature increases from 12.7 to 30.0 degrees C, whereas the bimolecular rate constant changes slightly and is about 10(8) M-1 X S-1. These data point to the possibility of diffusional control of the complex formation.     

Enzyme kinetics of muscle glycogen phosphorylase b

Interest in the kinetics of glycogen phosphorylase has recently been renewed by the hypothesis of a glycogen shunt and by the potential of altering phosphorylase to treat type II diabetes. The wealth of data from studies of this enzyme in vitro and the need for a mathematical representation for use in the study of metabolic control systems make this enzyme an ideal subject for a mathematical model. We applied a two-part approach to the analysis of the kinetics of glycogen phosphorylase b (GPb). First, a continuous state model of enzyme-ligand interactions supported the view that two phosphates and four ATP or AMP molecules can bind to the enzyme, a result that agrees with spectroscopic and crystallographic studies. Second, using minimum error estimates from continuous state model fits to published data (that agreed well with reported error), we used a discrete state model of internal molecular events to show that GPb exists in three discrete states (two of which are inactive) and that state transitions are concerted. The results also show that under certain concentrations of substrate and effector, ATP can activate the enzyme, while under other conditions, it can competetively inhibit or noncompetitively inhibit the enzyme. This result is unexpected but is consistent with spectroscopic, crystallographic, and kinetic experiments and can explain several previously unexplained phenomena regarding GPb activity in vivo and in vitro.     

Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles

The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307deg;C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.     

Phosphorescent analysis of the intramolecular dynamics of the muscle glycogen phosphorylase b

Large-scale functionally significant changes in the intramolecular dynamics of muscle glycogen phosphorylase b (EC 2.4.1.1) in solution upon ligand binding, transition from dimeric to tetrameric form, temperature denaturation and aggregation were registered at room temperature using the tryptophan phosphorescence technique. It was shown that binding of glucose-1-phosphate (substrate, 0.25-4 mM) and glucose (competitive inhibitor, 0.5-8 mM) to the active site and temperature-induced protein aggregation decrease large-scale structural fluctuations of the protein matrix at the level of domains and subunits; whereas the transition of glycogen phosphorylase b to tetrameric form (R-conformation) leads to a dramatic increase in the structural flexibility of the peripheral parts of the protein globule.     

Hydrophobic formate dehydrogenase from Pseudomonas sp. 101 in the system of reverse micelles of aerosol OT in octane

NAD(+)-dependent formate dehydrogenase (FDH) was hydrophobized with palmitoyl chloride to give the samples with various modification degrees (2-10). The native and modified FDHs were comparatively studied in the system of reverse micelles of Aerosol OT in octane. Like the native, the modified enzyme displayed three maxima in the curve of dependence of its catalytic activity on the degree of surfactant hydration (the micelle size), which reflect the enzyme functioning in the form of a monomer, dimer, or octamer. The peak corresponding to the functioning of the FDH dimer was found to decrease along with an increase in the modification degree. Thus, the modified enzyme mainly functions in the form of monomer and octamer. The modified FDH displayed membranotropy and revealed the dependence of catalytic activity on surfactant concentration.     

Catalytic properties of the membrane enzyme prostaglandin H synthetase in a system of inverted micelles of aerosol OT in octane

Prostaglandin H synthetase solubilized in octane with the aid of hydrated reversed micelles of Aerosol OT (AOT) exhibits a catalytic activity dependent on hydration of the surface-active substance and its concentration. The maximum rate of the reaction is attained at the H2O/AOT molar ratio equal to 20 and amounts to a value close to the one observed in an aqueous solution. The inactivation rate of the enzyme in the course of the reaction does not depend on the water content in the system and is described by a Kin value commensurable with the Kin in an aqueous solution.     

Kinetics of soybean lipoxygenase reaction in hydrated reversed micelles

The rate of linoleic acid peroxidation catalysed by soybean lipoxygenase I was studied as a function of the hydration degree of aerosol OT (bis(2-ethylhexyl) sulfosuccinate sodium salt) reversed micelles in octane. Lipoxygenase reaction parameters for the micelle-bound substrate were spectrophotometrically determined. The linoleic acid distribution between the micelles and octane was detected by the sedimentation method, with the concentration of linoleic acid in supernatant after settling of micelles (i.e. the concentration of free linoleic acid) being estimated by the enzymatic method. The apparent constant of linoleic acid distribution (the ratio of the bound and free substrate concentrations) was enhanced with increasing hydration of reversed micelles. The dependence of the enzymatic reaction rate on the bound substrate concentration obeyed the empiric Hill equation. The Hill coefficient remained practically constant (h = 1.34) as the hydration degree changed. Parameters of the lipoxygenase reaction, enzyme reaction limiting rate V and semi-saturation substrate concentration [S]0.5 increased with increasing degree of hydration and reached the optimum at [H2O]/[AOT] approximately 30, where dimensions of the micellar internal cavity coincided with those of the enzyme molecule. Some aspects of kinetic behavior of membrane-bound enzymes participating in chemical transformation of non-polar compounds dispersed in lipid phase are discussed.     

Catalysis by enzymes entrapped into reversed micelles of surfactants in organic solvents. Peroxidase in the aerosol OT-water-octane system

Spectral and catalytic parameters of peroxidase solubilized in the aerosol OT-water-octane system have been studied. The spectrum of peroxidase solubilized in octane with AOT reversed micelles, a degree of surfactant hydration being above 12, is actually identical to that of the enzyme aqueous solution. On the other hand, significant spectral changes have been detected when transferring the enzyme from water to the reversed micelle medium at low degrees of surfactant hydration, precisely [H2O]/[AOT] less than 12. The reversed micelle-entrapped peroxidase catalyses the oxidation of pyrogallol with hydrogen peroxide much more actively (at [H2O]/[surfactant] approximately 13) than that in aqueous solution. The entrapment of peroxidase into surfactant reversed micelles increases precisely the catalytic constant of the reaction, i.e. the virtual reactivity of the enzyme increases ten and hundred times depending on degrees of surfactant hydration and concentration. The systems of reversed micelles may be considered as models of biomembranes. Our findings hence show that enzymes in vivo can be much more catalytically active then it appears possible to reveal in conventional experiments in vitro in aqueous solutions.     

Binding of oxalyl derivatives of beta-d-glucopyranosylamine to muscle glycogen phosphorylase b

Five oxalyl derivatives of beta-d-glucopyranosylamine were synthesized as potential inhibitors of glycogen phosphorylase (GP). The compounds 1-4 were competitive inhibitors of rabbit muscle GPb (with respect to alpha-d-glucose-1-phosphate) with K(i) values of 0.2-1.4 mM, while compound 5 was not effective up to a concentration of 10 mM. In order to elucidate the structural basis of their inhibition, we analysed the structures of compounds 1-4 in complex with GPb at 1.93-1.96 Angstrom resolution. The complex structures reveal that the inhibitors can be accommodated at the catalytic site at approximately the same position as alpha-d-glucose and stabilize the T-state conformation of the 280 s loop by making several favourable contacts to Asp283 and Asn284 of this loop. Comparison with the lead compound N-acetyl-beta-d-glucopyranosylamine (6) shows that the hydrogen bonding interaction of the amide nitrogen with the main-chain carbonyl oxygen of His377 is not present in these complexes. The differences observed in the K(i) values of the four analogues can be interpreted in terms of subtle conformational changes of protein residues and shifts of water molecules in the vicinity of the catalytic site, variations in van der Waals interactions, conformational entropy and desolvation effects.     

Mechanism of extraction of chymotrypsin into isooctane at very low concentrations of aerosol OT in the absence of reversed micelles

Chymotrypsin is easily extracted from an aqueous solution into isooctane containing the anionic surfactant aerosol OT (AOT). The concentration of AOT needed to efficiently extract 0.5 mg/mL CMT is as low as 1 mM and as low as 0.2 mM AOT was sufficient to extract the protein into isooctane. The extraction process was unaffected by 10% (v/v) ethyl acetate in the isooctane phase. Moreover, spectroscopic analysis by electron paramagnetic resonance indicated that CMT did not exist inside a discreet water pool of a reversed micelle. Calculations of the number of AOT molecules associated per extracted CMT molecule indicate that only ca. 30 surfactant molecules interact with the protein, a value too low for reversed micellar incorporation of the protein in isooctane. These studies suggested that reversed micelles do not need to be involved in the actual transfer of the protein from the aqueous to the organic phase and protein solubilization in the organic phase is possible in the absence of reversed micelles. Based on these findings, a new mechanism has been proposed herein for protein extraction via the phase transfer method involving ionic surfactants. The central theme of this mechanism is the formation of an electrostatic complex between CMT and AOT at the aqueous/organic interface between AOT and CMT, thereby leading to the formation of a hydrophobic species that partitions into the organic phase. Consistent with this mechanism, the efficiency of extraction is dependent on the interfacial mass transfer, the concentrations of CMT and AOT in the aqueous and organic phases, respectively; the ionic strength of the aqueous phase; and the presence of various cosolvents. (c) 1994 John Wiley & Sons, Inc.     

Inhibition of muscle glycogen phosphorylase b by heterocyclic vitamins and coenzymes

Inhibition of rabbit skeletal muscle glycogen phosphorylase b by biotin, pyridoxine, lipoic acid, as well as by thiamine and cobalamine vitamins and coenzymes has been found. The values of "half-saturation" concentration and Hill coefficients are determined for biotin (27 mM, 1.3), pyridoxine (19 mM, 1.7), 5'-deoxyadenosyl-cobalamine (2.5 mM, 1.5), lipoic acid (3.4 mM, 1.1), thiamine (11 mM, 1.3), thiamine diphosphate (11 mM, 1.0). Effectiveness of the enzyme inhibition by vitamins and coenzymes containing different heterocyclic groups is analysed; riboflavin and its coenzymic forms are suggested to be the most effective inhibitors.     

Segmental mobility in glycogen phosphorylase b

The dynamics and structuredness of the pyridoxal 5'-phosphate-binding region in glycogen phosphorylase b (EC 2.4.1.1) has been investigated with different techniques of fluorescence spectroscopy. Fluorescence polarization data of the thermal Perrin plot indicate some mobility in the cofactor binding site, while the isothermic measurements (at 20 degrees C, in high-viscosity solvents) demonstrate that the mobile unit carrying the emission oscillator is practically insensitive to the external viscosity. Characteristics of the thermal Perrin plots obtained for both native and reduced phosphorylase b can be interpreted either as a freely moving cofactor in a medium of high viscosity (0.3 P) or as the motion of a unit larger than a lysine-bonded pyridoxal 5'-phosphate in a medium with the viscosity of water. Data for acrylamide quenching and time-resolved fluorescence measurements suggest that the latter interpretation should valid. These data also suggest a tightly packed microenvironment around the pyridoxal moiety.