The supramolecular organization of ovomucin. Biophysical and morphological studies.

Ovomucin participates in the ovomucin-gel-forming properties because of its shape and its ability to interact in a specific spatial organization. Purified from chicken egg-white by exclusion chromatography with Sephacryl S-300 and Sepharose CL-2B and analysed by light-scattering, it exhibited an Mr of about 40 x 10(6). This large Mr can be explained by the aggregation of polymers that can be degraded into 3 x 10(6)-Mr fragments by reduction with dithiothreitol. The values for hydrodynamic parameters such as Mr, radius of gyration, hydrodynamic radius, mass per unit length and combinations of them suggested that ovomucin is a linear and highly flexible molecule conferring upon it a random-coil-like structure in 0.2 M-NaCl solution. Analysis of the ovomucin molecules by electron microscopy revealed its linear character but also indicated a lower Mr than that obtained in the light-scattering experiments. By temperature-induced non-specific aggregation of an ovomucin solution containing other globular egg proteins, an attempt was made to find out what conditions are required for gel formation and to examine the quality of aggregation that is obtained under these conditions. Results show that the viscosity of the solution did not increase after heat treatment. Apparently, in the ovomucin gel, specific spatial organization of the ovomucin molecules is required for hydrogel formation.     

Kinetic and structural aspects of reconstitution of phosphatidylcholine vesicles by dilution of phosphatidylcholine-sodium cholate mixed micelles

Dilution of mixed micellar dispersions of egg phosphatidylcholine (PC) and sodium cholate beyond a critical value results in formation of cholate-containing PC vesicles. The structure of the resultant vesicles and some mechanistic aspects of this process have been investigated by the use of light scattering and nuclear magnetic resonance techniques. The main findings and conclusions are the following: Both the state of aggregation (micellar or vesicular) and the apparent equilibrium size distribution of micelles or vesicles obtained by dilution of the PC-cholate mixed micellar dispersions are a function of the cholate to PC molar ratio in the mixed aggregates (micelles or vesicles). When this effective ratio (Re) is higher than 0.4, the dispersion is micellar, and the size of the mixed micelles increases with decreasing Re; when Re less than 0.3, the dispersion is essentially vesicular, and the mean hydrodynamic radius of the vesicles is an increasing function of Re; in dispersions with 0.3 less than Re less than 0.4, mixed micelles and vesicles coexist. Addition of cholate to vesicular dispersions, to Re values below 0.3, results in vesicle size growth through a concentration-independent lipid-exchange mechanism. Addition of cholate to higher Re values results in micellization (solubilization) of the vesicles. On the other hand, dilution of vesicular dispersions does not affect the size of the vesicles. Apparent equilibration of a mixed micellar dispersion following dilution to Re values below 0.3 is slow (many hours). The overall process involves a series of three subsequent categories of steps: (i) a rapid (approximately 1-2 min) prevesiculation equilibration of micellar sizes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography

Size exclusion chromatography is an established technique for the determination of hydrodynamic volumes of proteins or protein complexes. When applied to membrane proteins, the contribution of the detergent micelle, which is required to keep the protein soluble in the aqueous phase, needs to be determined to obtain accurate measurements for the protein. In a detergent series, in which the detergents differ only by the length of the alkyl chain, the contribution of the detergent micelle to the hydrodynamic volume is variable, whereas the contribution of the protein is constant. By using this approach, several parameters of membrane proteins can be estimated by extrapolation, such as the radius at the midpoint of the membrane, the average radius, the Stokes radius, and the excluded volume. The molecular mass of the protein can be determined by two independent measurements that arise from the behaviour of the free detergent micelle and protein-detergent micelle during size exclusion chromatography and the determination of the detergent-protein ratio. Determining the dimensions of protein-detergent micelles may facilitate membrane protein purification and crystallization by defining the accessibility of the protein surface.     

Impact of Phenolic Antioxidants on Structural Properties of Micellar Solutions

Antioxidants solubilized in micellar solutions can change micellar properties like the size and shape of micelles, critical micellar concentration (cmc) and viscosity. Interactions arising between antioxidants and the surfactant determine the locations of antioxidants and vice versa. The location and interaction are dependent on the type of both the antioxidant and surfactant. Influences of various antioxidants on the physical and structural properties were tested in micellar systems of cationic CTAB, non-ionic Brij 58 and anionic SDS. The antioxidants used to investigate the effects of gradually increasing lipophilicity were gallic acid (GA) and the gallate esters from methyl to octyl gallate (MG-OG). Hydroxy cinnamic acids (HCAs) like -coumaric acid (pC), caffeic acid (CA), ferulic acid (FA) and sinapic acid (SA) were employed to observe effects of functional groups like hydroxyl and methoxy groups. Micellar size and shape determined by small angle neutron scattering (SANS), viscosity and cmc were chosen to characterize the antioxidant influence. In Brij 58 systems propyl gallate (PG) did not affect the cmc or aggregation number but decreased micellar size slightly due to an intercalation of PG into the region of the polyoxyethylene chain and the first adjacent alkyl chain methylene groups. In SDS systems the micellar size and cmc decreased in the presence of PG. This was attributed to PG residing in the Stern layer. However, in CTAB systems micelles swelled at low PG concentration and in the presence of GA, while higher PG concentrations and more lipophilic antioxidants led to a sphere-to-rod transition with a simultaneous increase in viscosity and decrease in cmc. This revealed the intercalation of antioxidants in the palisade layer of CTAB micelles entering into strong interactions of electrostatic and hydrophobic origin. It could be demonstrated that the interactions became stronger the more lipophil an antioxidant is and the more hydroxyl groups are attached to the aromatic ring. Differences in the location and interaction of antioxidant and micelles are proposed as being responsible for the effectiveness of antioxidants.     

Micellar properties of glycosphingolipids in aqueous media

The aggregation properties of neutral glycosphingolipids and gangliosides were investigated by ultracentrifugal sedimentation and gel permeation chromatography. Initially, glycosphingolipids in buffer formed high molecular aggregates. On standing, the glycosphingolipids produced smaller and stable micelles in equilibrium with their monomers at the critical micellar concentration (cmc). The cmc's of monohexaosyl- to tetrahexaosylceramides were on the order of 10(-8) to 10(-7)M. In contrast, values of 10(-8)M for monosialogangliosides and 10(-6)-10(-5)M for di- and trisialogangliosides were found. From estimations of hydrodynamic radii, Svedberg coefficients, and partial specific volumes, relative masses of glycosphingolipid micelles were calculated to be in the range of 300 to 1000 for the neutral glycosphingolipids and 100 to 350 for the gangliosides.     

Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin

Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548-1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27-3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin-Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of approximately 16 nm, a sedimentation coefficient of 17-20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions.     

Determination of the B820 subunit size of a bacterial core light-harvesting complex by small-angle neutron scattering

The B820 subunit is an integral pigment-membrane protein complex and can be obtained by both dissociation of the core light-harvesting complex (LH1) in photosynthetic bacteria and reconstitution from its component parts in the presence of n-octyl beta-D-glucopyranoside (OG). Intrinsic size of the B820 subunit from Rhodospirillum rubrum LH1 complex was measured by small-angle neutron scattering in perdeuterated OG solution and evaluated by Guinier analysis. Both the B820 subunits prepared by dissociation of LH1 and reconstitution from apopolypeptides and pigments were shown to have a molecular weight of 11,400 +/- 500 and radius of gyration of 11.0 +/- 1.0 A, corresponding to a heterodimer consisting of one pair of alphabeta-polypeptides and two bacteriochlorophyll a molecules. Molecular weights of micelles formed by OG alone in solutions were determined in a range from 30,000 to 50,000 over concentrations of 1-5% (w/v), and thus are much larger than that of the B820 subunit. Similar measurement on the pigment-depleted apopolypeptides revealed highly heterogeneous behavior in the OG solutions, indicating that aggregates with various sizes were formed. The result provides evidence that bacteriochlorophyll a molecules play a crucial role in stabilizing and maintaining the B820 subunits in the dimeric state in solution. Further measurements on individual alpha- and beta-polypeptides exhibited a marked difference in aggregation property between the two polypeptides. The alpha-polypeptides appear to be uniformly dissolved in OG solution in a monomeric form, whereas the beta-polypeptides favor a self-associated form and tend to form large aggregates even in the presence of detergent. The difference in aggregation tendency was discussed in relation to the different behavior between alpha- and beta-polypeptides in reconstitution with bacteriochlorophyll a molecules.     

Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale.

We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.     

Light scattering measurements on gangliosides: dependence of micellar properties on molecular structure and temperature

Static and dynamic laser light scattering measurements on micellar aqueous solutions of gangliosides GM2, GM1, GD1a are reported. The aggregation number, the hydrodynamic radius and the micellar shape depend on the type of ganglioside and the unsaturation degree of the hydrocarbon chains. At a temperature of 25 degrees C the molecular weights of GM2, GM1 and GD1a are 740,000, 470,000 and 418,000 DA respectively. A significant decrease of micellar size with temperature has been found for saturated GM1 in the region 25 degrees-40 degrees C. The strong sensitivity of the micellar parameters to the ganglioside structure is explained by making reference to some simple model which takes into account geometrical packing considerations. By measuring the scattered light intensity at low ionic strength of the solution (0.1-30 mE) it was possible to evaluate the ganglioside micellar charge, 100 electronic units for GM2, 48 for GM1 and 60 for GD1a.     

Isolation of untransformed bovine estrogen receptor without molybdate stabilization

A low concentration estrogen-derivatized affinity resin has been used in a rapid, single step purification of the untransformed estrogen receptor from calf uterine cytosols prepared without sodium molybdata. The procedure isolates the Mr 65,000 estrogen receptor in association with the bovine heat shock protein hsp90. Small amounts of proteolyzed receptor ranging in size from Mr 50,000 to 60,000 are also present in the purified extracts. Results from affinity chromatography of receptor cytosols either untreated or presaturated with estradiol suggest that two proteins of Mr 22,000 and 38,000 are co-purified with the untransformed receptor complex and may represent additional nonhormone-binding components of the native receptor form. Some indication of the stability of protein-protein interactions within the oligomeric complex has been derived from differential salt elution studies with heparin-sepharose and affinity gel-immobilized untransformed receptor. On size exclusion high performance liquid chromatography the untransformed complex eluted with a Stokes radius of 75 +/- 2 A (n = 18), but was shown to be sensitive to extended ultracentrifugal analysis dissociating to the receptor homodimer, sedimentation coefficient 5.3 +/- 0.3 s (n = 5). Preliminary data on urea- and heat-induced transformation of the isolated receptor to the DNA-binding state is presented.     

Synthesis of alkyl glycosides, catalyzed by beta-glycosidases in a reversed micelle system]

A basic possibility of enzymic synthesis of alkyl glycosides in a system of the Aerosol-OT (AOT) reverse micelles was studied. Octyl beta-D-galactopyranoside and octyl beta-D-glucopyranoside were synthesized from the corresponding sugars (lactose or glucose) and octyl alcohol under catalysis with glycolytic enzymes, beta-galactosidase and beta-glucosidase, respectively. The transglycosylation/hydrolysis ratio was shifted toward transglycosylation by using octyl alcohol, one of the substrates, as an organic solvent. The alkyl glycosides were thus obtained in one step from a hydrophilic mono- or disaccharide and a hydrophobic aliphatic alcohol. The direction of the reaction was shown to depend on the pH of aqueous solution immobilized in nerves micelles. The maximum yields were 45% and 40% for octyl galactoside and octyl glucoside, respectively; they markedly exceeded the yields of enzymic syntheses in a two-phase system reported previously.     

Hydrodynamic properties of the purified glutamate-binding protein subunit of the N-methyl-D-aspartate receptor.

The hydrodynamic properties of the previously purified glutamate-binding protein from rat synaptic membranes were determined in order to estimate the molecular size of the protein in its native state. This protein is apparently a subunit of a multisubunit complex that forms the N-methyl-D-aspartate subtype of glutamate receptor and has a molecular size of approximately 70 kDa based on electrophoretic migration under denaturing conditions. On the basis of results obtained from H2O/D2O sucrose density gradient sedimentation and gel filtration chromatography of the purified glutamate-binding protein we calculated the partial specific volume of the protein-detergent complex to be 0.766 cc3/g, the Stokes radius of the complex as 4.9 nm, the Mc of the complex as 203,000 +/- 22,000 and the Mr of the protein as 182,000 +/- 19,000. These results are indicative of stable self-association of the glutamate-binding protein and are in agreement with recent studies indicating that more than one molecule of glutamate may be required to activate the N-methyl-D-aspartate receptor-associated ion channel.     

Purification and characterization of urease from Helicobacter pylori

Urease was purified 112-fold to homogeneity from the microaerophilic human gastric bacterium, Helicobacter pylori. The urease isolation procedure included a water extraction step, size exclusion chromatography, and anion exchange chromatography. The purified enzyme exhibited a Km of 0.3 +/- 0.1 mM and a Vmax of 1,100 +/- 200 mumols of urea hydrolyzed/min/mg of protein at 22 degrees C in 31 mM Tris-HCl, pH 8.0. The isoelectric point was 5.99 +/- 0.03. Molecular mass estimated for the native enzyme was 380,000 +/- 30,000 daltons, whereas subunit values of 62,000 +/- 2,000 and 30,000 +/- 1,000 were determined. The partial amino-terminal sequence (17 residues) of the large subunit of H. pylori urease (Mr = 62,000) was 76% homologous with an internal sequence of the homohexameric jack bean urease subunit (Mr = 90,770; Takashima, K., Suga, T., and Mamiya, G. (1988) Eur. J. Biochem. 175, 151-165) and was 65% homologous with amino-terminal sequences of the large subunits of heteropolymeric ureases from Proteus mirabilis (Mr = 73,000) and from Klebsiella aerogenes (Mr = 72,000; Mobley, H. L. T., and Hausinger, R. P. (1989) Microbiol. Rev. 53, 85-108). The amino-terminal sequence (20 residues) of the small subunit of H. pylori urease (Mr = 30,000) was 65 and 60% homologous with the amino-terminal sequences of the subunit of jack bean urease and with the Mr = 11,000 subunit of P. mirabilis urease (Jones, B. D., and Mobley, H. L. T. (1989) J. Bacteriol. 171, 6414-6422), respectively. Thus, the urease of H. pylori shows similarities to ureases found in plants and other bacteria. When used as antigens in an enzyme-linked immunosorbent assay, neither purified urease nor an Mr = 54,000 protein that co-purified with urease by size exclusion chromatography was as effective as crude preparations of H. pylori proteins at distinguishing sera from persons known either to be infected with H. pylori or not.     

Molecular characterisation of soybean polysaccharides: an approach by size exclusion chromatography, dynamic and static light scattering methods

Water soluble polysaccharides from soybean (SSPS) have a pectin-like structure and are used as stabilisers in acidified beverages. Physicochemical properties such as structure, molecular weight and shape or conformation are primary factors controlling their functional properties. Two soybean polysaccharides, a native SSPS and a modified SSPS treated with beta-(1-->4)-D-galactosidase (GPase/SSPS) were studied by dynamic and static light scattering (DLS, SLS) and size exclusion chromatography (SEC). Consecutive filtrations using a range of membrane pore size removed a small fraction of macromolecular aggregates from dilute polysaccharide solutions with relatively little effect on the major component molecules as monitored by DLS and SEC measurements. Access to aggregate-free dilute solutions of SSPS and GPase/SSPS allowed the direct measurement of molecular characteristics. SLS results showed that SSPS had a weight average molecular weight of (645+/-11)x 10(3)g/mol and a radius of gyration, Rg, of (23.5+/-2.8)nm. By comparing R(g) with the hydrodynamic radius, Rh (21.1+/-0.5 nm) obtained from DLS, the structural parameter rho (Rg/Rh) was found to be 1.1, suggesting that SSPS has an overall globular shape due to a highly branched structure. The modified SSPS had a significantly lower molecular weight (287+/-18)x 10(3)g/mol but a similar radius of gyration (23.2+/-1.7 nm). The structure parameter rho of GPase/SSPS was higher (rho=1.3) because of a smaller hydrodynamic radius (17.7+/-1.8 nm). This suggests that GPase/SSPS has a much less branched structure yet still differs significantly from a linear random coil conformation (rho=1.7-2.0). The results derived from SLS and DLS are in agreement with the conclusions obtained from a chemical analysis where the reduction of molecular weight of GPase/SSPS was caused by the cleavage of galactan side chains.     

Characterization of lipase in reversed micelles formulated with cibacron blue F-3GA modified span 85

Sorbitan trioleate (Span 85) modified by Cibacron Blue F-3GA (CB) was prepared and used as an affinity surfactant to formulate a reversed micellar system for Candida rugosa lipase (CRL) solubilization. The system was characterized and evaluated by employing CRL-catalyzed hydrolysis of olive oil as a model reaction. The micellar hydrodynamic radius results reflected, to some extent, the redistribution of surfactant and water after enzyme addition, and the correlation between surfactant formulation, water content (W0), micellar size, and enzyme activity. An adequate modification density of CB was found to be important for the reversed micelles to retain enough hydration capacity and achieve high enzyme activity. Compared with the results in AOT-based reversed micelles, CRL in this micellar system exhibited a different activity behavior versus W0. The optimal pH and temperature of the encapsulated lipase remained unchanged, but the apparent activity was significantly higher than that of the native enzyme in bulk solution. Kinetic studies indicated that the encapsulated lipase in the reversed micelles of CB-formulated Span 85 followed the Michaelis-Menten equation. The Michaelis constant was found to decrease with increasing surfactant concentration, suggesting an increase of the enzyme affinity for the substrate. Stability of the lipase in the reversed micelles was negatively correlated to W0.     

Ultrastructure of human erythrocyte GLUT1

This study was undertaken to examine GLUT1 quaternary structure. Independent but complementary methodologies were used to investigate the influence of membrane-solubilizing detergents on GLUT1/lipid/detergent micelle hydrodynamic radii. Hydrodynamic size analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze-fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. GLUT1 forms a multimeric complex in octyl glucoside that dissociates upon addition of reductant. Some detergents (e.g., CHAPS and dodecyl maltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). These complexes do not reassemble as larger oligomers when dissociating detergents are subsequently replaced with nondissociating detergents such as octyl glucoside or cholic acid. When dissociating detergents are replaced with lipids, the resulting proteoliposomes catalyze protein-mediated sugar transport, and the subsequent addition of solubilizing, nondissociating detergents generates higher (tetrameric) GLUT1 aggregation states. These findings suggest that some detergents stabilize while others destabilize GLUT1 quaternary structure. GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer.     

Molecular Size of the γ-Aminobutyric AcidA Receptor Purified from Mammalian Cerebral Cortex

The hydrodynamic behaviour of both the soluble and purified gamma-aminobutyric acidA (GABAA) receptor of bovine or rat cerebral cortex has been investigated in solution in Triton X-100 or in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS). In all the hydrodynamic separations made, it was found that the binding activities for GABA, benzodiazepine, and (where detectable) t-butylbicyclophosphorothionate comigrated. Conditions were established for gel exclusion chromatography and for sucrose density gradient velocity sedimentation that maintain the GABAA receptor in a nonaggregated form. Using these conditions, the molecular weight of the bovine GABAA receptor in the above-mentioned detergents was calculated using the H2O/2H2O method. A value of Mr 230,000-240,000 was calculated for the bovine pure GABAA receptor purified in sodium deoxycholate/Triton X-100 media. A value of Mr 284,000-290,000 was calculated for the nonaggregated bovine or rat cortex receptor in CHAPS, but the Stokes radius is smaller in the latter than in the former medium and the detergent binding in CHAPS is underestimated. Thus the deduced Mr, 240,000, is the best estimate by this method.     

GM1-ganglioside-triton X-100 mixed micelles: Changes of micellar properties studied by laser-light scattering and enzymatic methods

The micellar properties of mixtures of GM1 ganglioside and the non-ionic amphiphile Triton X-100 in 25 mM Na phosphate-5 mM di Na EDTA buffer (pH = 7.0) were investigated by quasielastic light scattering in a wide range of Triton/GM1 molar ratios and in the temperature range 15–37°C. These measurements: (a) provided evidence for the formation of mixed micelles; (b) allowed the determination of such parameters as the molecular weight and the hydrodynamic radius of the mixed micelles; (c) showed the occurrence of statistical aggregates of micelles with increasing temperature and micelle concentration. Galactose oxidase was chosen for studying the relation between enzyme activity and micellar properties. The action of the enzyme on GM1 was found to be strongly dependent on the micellar structure. In particular: (a) galactose oxidase acted very poorly on homogeneous GM1 micelles, while affecting mixed GM1/Triton X-100 micelles; (b) at fixed GM1 concentration the oxidation rate increased by enhancing Triton X-100 concentration and followed a biphasic kinetics with a break at a certain Triton X-100 concentration; (c) the formation of statistical micelle aggregates was followed by inhibition of the enzyme activity.     

Micelles of cerebroside sulphate

The critical micelle concentration of cerebroside sulphate in water is 0-01 mM: it increases with increasing concentrations of buffer to 0-07 mM in 0-1 M sodium acetate and formate buffers, pH 5-6 and 4-5 respectively. The partial specific volume of the micelles is about 0-94. The behaviour of the micelles in the ultracentrifuge and on Sephadex G-200 shows them to be grossly heterogeneous with respect to size. In 0-1 M buffer s20,w is about 26 S; in water or 0-01 M buffer smaller micelles with an s20,w of about 6 S are also present. In 0-01 M formate, pH 4-5, the smallest species detectable by equilibrium ultracentrifugation had a micellar weight of about 180,000 corresponding to an aggregation number of about 180. Much larger aggregates were also present. It is suggested that the smallest micelles are the substrate for sulphatase A when this is acting as a cerebroside sulphatase in buffers of low ionic strength.     

A new strategy for the study of oligomeric enzymes: gamma-glutamyltransferase in reversed micelles of surfactants in organic solvents

A heterodimeric enzyme (gamma-glutamyltransferase) was studied in the reversed micellar medium of Aerosol OT (AOT) in octane. As was shown earlier, the size (radius) of inner cavity of the AOT-reversed micelles is determined by their hydration degree, i.e., [H2O]/[AOT] molar ratio, in the system. Owing to this, the dependence of hydrolytic, transpeptidation and autotranspeptidation activities of the enzyme on the hydration degree was investigated using L- and D-isomers of gamma-glutamyl(3-carboxy-4-nitro)anilide and glycylglycine as substrates. For all of the reaction types, the observed dependences are curves with three optima. The optima are found at the hydration degrees, [H2O]/[AOT] = 11, 17 and 26 when the inner cavity radii of reversed micelles are equal to the size of light (Mr 21,000) and heavy (Mr 54,000) subunits of gamma-glutamyltransferase, and to their dimer (Mr 75,000), respectively. Ultracentrifugation experiments showed that a change of the hydration degree resulted in a reversible dissociation of the enzyme to light and heavy subunits. The separation of light and heavy subunits of gamma-glutamyltransferase formed in reversed micelles was carried out and their catalytic properties were studied. The two subunits catalyze hydrolysis and transpeptidation reactions; autotranspeptidation reaction is detected only in the case of the heavy subunit. These findings imply that the reversed micelles of surfactants in organic solvents function as the matrices with adjustable size permitting to regulate the supramolecular structure and the catalytic activity of oligomeric enzymes.