Characterization of a novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86 was purified to homogeneity by ammonium sulfate precipitation and Sephacryl S-300 gel filtration chromatography. The purified xylanosome appeared as a single protein band on the non-denaturing (native) polyacrylamide gel electrophoresis (PAGE) gel with a molecular mass of approximately 1200 kDa. The optimal temperature and pH for xylanase activity was 60 °C and pH 6.0, respectively. The xylanase activity was stable within pH 4.1–10.3. It was stable up to 60 °C at pH 6.0. The xylanosome was highly specific towards oat-spelt xylan, and showed low activity towards corncob powder, but exhibited very low activity towards lichenan, CMC and p-nitrophenyl derivatives. Apparent K_m values of the xylansosome for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 2.5, 3.6, 1.7 and 4.9 mg ml⁻¹, respectively. The main hydrolysis products of birchwood xylan were xylotriose, xylobiose and xylose. Analysis of the products from wheat arabinoxylan degradation by xylanosome confirmed that the enzyme had endoxylanase and debranching activities, with xylotriose, xylobiose, xylose and arabinose as the main degradation products. These unique properties of the purified xylanosome from Streptomyces olivaceoviridis E-86 make this enzymatic complex attractive for biotechnological applications.     

Study of the compatibility/incompatibility of gelatin/iota-carrageenan/water mixtures

The incompatibility of acid gelatin/iota-carrageenan mixtures has been studied. Both these biopolymers undergo a conformational coil-helix transition under suitable conditions of temperature and salt. The aim of this work was to study the concentration at which mixtures are incompatible and the influence of pH, salt and temperature on the phase diagram. Incompatibility occurred over a wide range of concentrations for mixtures prepared in deionized water. Compatibility was increased by increasing the pH or the salt concentration. Temperature did not greatly influence the size of the incompatible region. This is in agreement with the hypothesis that attractive electrostatic interactions lead to associative phase separation (traditionally called complex coacervation).     

Studies on the Critical Micellar Concentration and Phase Transitions of Stearoylcarnitine

The critical micellar concentration (CMC) of stearoylcarnitine was determined at different pH values at room temperature by fluorescence spectroscopy, monitoring the spectral changes of 8-anilinonaphthalene-1-sulfonate (ANS). The CMC was found to vary with pH, increasing from about 10 μM at pH 3.0 to ca. 25 μM at pH 7.0, but decreasing slightly with further increase in pH to approximately 19 μM at pH 10.0. Differential scanning calorimetry (DSC) shows that stearoylcarnitine dispersed in water at low concentration undergoes a broad thermotropic phase transition at 44.5°C, with a transition enthalpy of 15.0 kcal/mol. The transition temperature (T _t) shifts to ca. 50.5°C in the presence of 1 mM EDTA or when the concentration is increased significantly. The turbidity of aqueous dispersions of stearoylcarnitine was found to be considerably high at low temperatures, which decreases quite abruptly over a short temperature range, indicating that a transition occurs from a phase of large aggregates to one of much smaller aggregates, most likely micelles. The phase transition temperature was determined as 29.1°C at pH 3.0, which increased with increasing pH up to a value of 55.3°C at pH 8.6 and remains nearly constant thereafter up to pH 11.2. The pH dependence of CMC and T _t suggest that the pK _a of the carboxyl group of long chain acylcarnitines shifts to higher temperatures upon aggregation (micelles or bilayer membranes).     

Carboxymethylcellulase produced by facultative bacteria from the hind-gut of the termite Reticulitermes hesperus.

Bacillus cereus RW1 and Serratia marcescens RW3, isolated from the hind-gut of the termite Reticulitermes hesperus, both grew well on mesquite wood and produced moderate amounts of carboxymethylcellulase. Carboxymethylcellulose (CMC) gels were depolymerized rapidly by B. cereus RW1 and slowly by S. marcescens RW3. The depolymerization of CMC was pH and temperature sensitive. Depolymerization of gels by growing cultures of B. cereus RW1 and the action of cell-free extracts of B. cereus RW1 on CMC sols were optimum at pH 6.0 and 5.5, respectively. Glucose and cellobiose increased the rate of CMC gel depolymerization. Enzyme synthesis rather than growth was stimulated by the addition of glucose to a culture of RW1 growing on a non-cellulosic substrate. Bacillus cereus RW1 produced both cell-free and cell-bound carboxymethylcellulase.     

Gum arabic-chitosan complex coacervation

The formation of electrostatic complexes of gum Arabic (GA) with chitosan (Ch), two oppositely charged polysaccharides, as a function of the biopolymers ratio (RGA/Ch), total biopolymers concentration (TBconc), pH, and ionic strength, was investigated. The conditions under which inter-biopolymer complexes form were determined by using turbidimetric and electrophoretic mobility measurements in the equilibrium phase and by quantifying mass in the precipitated phase. Results indicated that optimum coacervate yield was achieved at RGA/Ch = 5, independently of TBconc at the resulting pH of solutions under mixing conditions. High coacervate yields occurred in a pH range from 3.5 to 5.0 for RGA/Ch = 5. Coacervate yield was drastically diminished at pH values below 3.5 due to a low degree of ionization of GA molecules, and at pH values above 5 due to a low solubility of chitosan. Increasing ionic strength decreased coacervate yield due to shielding of ionized groups.     

Complex formation between polyadenylic acid and formycin B

Polyadenylic acid forms a 2:1 complex with the C-nucleoside formyein B at both pH 7.0, 0.15 m-Na⁺ and pH 6.0, 0.15 M-Na⁺. The formation of this complex has been followed by equilibrium dialysis, and by optical rotatory dispersion measurements in the range 333 to 450 nm. At pH 7, melting curves for thermal dissociation of the complex (followed by the optical rotation at 345 nm) show a strongly co-operative helix-coil transition. From the variation of T_m with the free formyein B concentration at this temperature, the partial molar enthalpy of formation of the complex, at the mid-point of the transition, has been calculated as -12.8 kcal./mol of formyein B. Viscometry and optical rotatory dispersion measurements indicate that the structure of the complex at pH 6 is the same as at pH 7, and that it may be formed in preference to the double-helical acid form of poly (A). The structure and properties of the complex are discussed.     

pH dependence of the reversible and irreversible thermal denaturation of gamma interferons

Heated at pH 6.0 and at 50 degrees C, human interferon gamma (HuIFN-gamma) is inactivated via the formation of insoluble aggregates. At pH 6.0, the aggregation rate increases with temperature from 40 to 65 degrees C. There is a temperature-dependent time lag to aggregate formation observed in the generation of light-scattering particles at pH 6.0, and this correlates with the fast phase observed in the kinetics of reversible thermal unfolding. In addition, the dependence of aggregation kinetics on temperature closely follows the reversible melting curve. These observations suggest that at pH 6.0 irreversible thermal denaturation and aggregation depend on partial or complete unfolding of the molecule. At pH 5.0, also at 50 degrees C, the molecule is stable to irreversible aggregation. In reversible unfolding in 0.25 M guanidine hydrochloride, the Tm for HuIFN-gamma increases from 30.5 degrees C at pH 4.75 to 41.8 degrees C at pH 6.25, in analogy to the behavior of other globular proteins. These observations suggest that the relative instability of HuIFN-gamma to irreversible denaturation via aggregation at pH 6.0 compared to pH 5.0 is not due to an increased stability toward unfolding at the lower pH. Alternatively, stability at pH 5.0 must be due either to the improved solution properties of the unfolded state or to the improved solubility/decreased kinetic lifetime of an unfolding intermediate. Aggregation of HuIFN-gamma at 50 degrees C is half-maximal at pH 5.7, suggesting that protonation of one or both of the histidine residues may be involved in this stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complex coacervation of silk fibroin and hyaluronic acid

This study aimed to investigate the pH-induced complexation of silk fibroin (SF) and hyaluronic acid (HA). SF-HA complex coacervation was investigated by monitoring turbidity of the SF-HA system under slow acidification. Gravimetric analysis was performed to determine the yield of complex coacervation and viscosity of the system was measured to study the formation of the complexes at different pH values. The influences of total biopolymer concentration and biopolymer weight ratio on complex coacervation were examined during the analyses. Formation of the complexes was evidenced by the minimum viscosity and the maximum turbidity observed in the system. SF-HA complexes were formed within the pH-window of 2.5-3.5 regardless of the total biopolymer concentration or biopolymer ratio. Complex coacervation of SF-HA showed a reversible behavior and coacervation could be handled even in excess amounts of the biopolymers, which pointed out a non-stoichiometric complexation.     

Phase separation and gel formation in kinetically trapped gelatin/maltodextrin gels

The kinetics of phase separation and gel formation of gelatin/maltodextrin mixtures have been studied using confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), stereological image analysis and rheology. The quantified microstructural parameters were the volume-weighted mean volume and the interfacial area. The temperature of phase separation was defined as the temperature where the first signs of phase separation were observed in CLSM. The gelatin concentration varied between 4 (wt.) and 5% and the maltodextrin concentration varied between 2 and 6%. Maltodextrin was labelled covalently with RITC to improve the contrast between the gelatin phase and the maltodextrin phase. The solutions were cooled from 60 to 10 degrees C, and the cooling rates used were 0.4, 1 and 3 degrees C/min. All systems were found to be gelatin continuous under the experimental conditions used. The results showed that the temperature of phase separation (TPS) increased both with the gelatin concentration and the maltodextrin concentration, but particularly with the former. The size of the maltodextrin inclusions increased with TPS, and the interfacial area decreased with increasing TPS. The diameter of the maltodextrin inclusions varied between 1.6 and 8.5 microm at 1 degrees C/min. The size of the maltodextrin inclusions was found to increase with decreasing cooling rate and was largest at 0.4 degrees C/min. The TPS was compared with the gelation temperature (Tgel) at three different concentrations of gelatin and maltodextrin (4/3, 4/5, 5/5%). CLSM micrographs and TEM micrographs showed that these three concentrations of gelatin and maltodextrin had different microstructures. At a TPS above Tgel (5/5%), the phase separation proceeded faster than the gel formation and the microstructure had few, large maltodextrin inclusions and a clean continuous gelatin phase. At a TPS comparable with Tgel (4/5%), phase separation occurred during gel formation, which led to a varying microstructure and competition between the phase separation and the gel formation. At a TPS below Tgel (4/3%), gel formation proceeded faster than the phase separation and the microstructure had many, small inclusions and a diffuse microstructure, and the phase separation was incomplete. It was established that the microstructure was determined by the relative rates of the phase separation and the gel formation. Three different zones of phase separation could be distinguished based on comparisons of TPS and Tgel, and results from CLSM, TEM and image analysis.     

Complex Coacervates Formed between Whey Protein Isolate and Carboxymethylcellulose for Encapsulation of β-Carotene from Sacha Inchi Oil: Stability,In Vitro Digestion and Release Kinetics

The present work aimed to study the influence of the pH and protein ratio on the formation of complex coacervates of carboxymethylcellulose (CMC) and whey protein isolated nanoparticles (WPIN). These biopolymers and transglutaminase, as a cross-linking agent, were used to encapsulate sacha inchi oil (SIO) containing β-carotene (β-C). The stability of β-C from SIO microcapsules (β-SIO microcapsules) was evaluated under in vitro digestion using an INFOGEST 2.0 in vitro digestion protocol. The release of β-C in a simulated food model was studied, and mathematical models were used to determine the mechanism. A ratio of 1:6 (CMC/WPIN) at pH 3.5 was used for the formation of the complex. Chemical and morphological analyses suggested that SIO was microencapsulated and that a high encapsulation efficiency was obtained. The β-C from β-SIO microcapsules was preserved in vegetable oil (food model), and Fickian diffusion occurred. The β-C from β-SIO microcapsules was preserved under oral and gastric conditions, and higher release occurred during intestinal digestion when samples were subjected to in vitro digestion simulation. After in vitro digestion, the β-C from β-SIO microcapsules presented higher stability (83.37%) and acceptable bioaccessibility (31.16%). There are few studies in the literature of encapsulated SIO using the CMC/WPIN complex or studies of the release of β-carotene from SIO during in vitro digestion and in food simulants. The knowledge obtained in this study will facilitate the use and applications of β-C-loaded microcapsule delivery systems.

     

Synthesis and rheological properties of responsive thickeners based on polysaccharide architectures

New thermothickening copolymers were synthesized by grafting responsive poly(ethylene oxide-co-propylene oxide) [PEPO] onto three different polysaccharide backbones: carboxymethylcellulose [CMC], alginate [ALG], and carboxylated dextran [DEX]. The coupling reaction between carboxylic groups of biopolymers and the terminal amine of PEPO was activated at low temperature ( T < 10 degrees C) in water by using carbodiimide and N-hydroxysuccinimide. In these conditions it was shown that the formation of amide bonds strongly depends on the concentration of reactive groups, which is limited by the viscosity of the polymer sample. While a full conversion was obtained for the low molecular weight dextran, the efficiency of grafting remains low (between 30 to 40%) for CMC and alginate, which give a solution of high viscosity even at low concentration. When studied in the semidilute regime, all the copolymer solutions clearly exhibit thermothickening behavior with a large and reversible increase of viscosity upon heating. The association temperature and the gelation threshold were shown to depend on polymer concentration as it is expected from the phase diagram of PEPO precursor. Similarly, the influence of added salt on PEPO solubility in water has been used to control the self-assembling behavior of copolymer formulations. The relative comparison between the three copolymers reveals that the amplitude of the viscosity jump induced by heating mainly depends on the proportion of responsive material inside the macromolecular architecture rather than the dimensions of the main chain. The high increase of viscosity, which can reach several orders of magnitude between 20 degrees C and body temperature, clearly demonstrates the potentiality of these copolymers in biomedical applications like injectable gels for tissue engineering.     

Emulsion-stabilizing properties of chitosan in the presence of whey protein isolate: Effect of the mixture ratio, ionic strength and pH

The emulsion-stabilizing properties of a chitosan preparation were compared as a function of the whey protein isolate/chitosan mixture ratio (WPI/CNI) and the ionic strength (μ), at pH 5.5 and 6.0. At both pH conditions, general decreases in emulsion stability towards charge neutralization flocculation and syneresis were observed at WPI/CNI > 5. This was particularly evident at pH 6.0, due to a lower surface net charge (lower electrostatic stabilization). In counterpart, when μ was increased, the higher load of chitosan at pH 6.0 produced higher stabilities (higher steric stabilization), in spite of comparable decreases of surface net charge at both pH conditions. The transition from soluble to insoluble protein–chitosan complex formation in mixtures at pH 6.0 and WPI/CNI > 5.0 was due to an emulsion destabilization towards syneresis, whereas soluble complex formation at pH 5.5 also produced syneresis. It showed that soluble protein–chitosan adsorbing complex formation prior homogenization is not essentially linked to emulsion stabilization.     

Studies on surfactant-biopolymer interaction. I. Microcalorimetric investigation on the interaction of cetyltrimethylammonium bromide (CTAB) and sodium dodecylsulfate (SDS) with gelatin (Gn), lysozyme (Lz) and deoxyribonucleic acid (DNA)

The interaction of the surfactants cetyltrimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) with the biopolymers gelatin (Gn), lysozyme (Lz) and deoxyribonucleic acid (DNA) was studied by isothermal titration microcalorimetry at varied biopolymer concentration, pH and temperature. The nature of interaction of the surfactants with the biopolymers was assessed from the observed enthalpy-[surfactant] profiles. The biopolymer-induced aggregation of the surfactants was observed. The enthalpies of aggregation of amphiphiles, binding of aggregates with macromolecules, organisational change of bound aggregates, and threshold concentrations for micelle formation of surfactants in the presence of biopolymers were estimated. The results collected on the three biopolymers were analysed and compared.     

Temperature—composition phase diagram and gel properties of the gelatin—starch—water system

The gelatin-starch-water system has been studied at different temperatures, at a total biopolymer concentration of 5.0 wt%. The weight ratios (W) of gelatin/ starch used were 9:1, 8:2.4. 2:8, 1:9, with pH values between 5.82 (at W = 9:1) and 6.50 (at W = 1:9). The systems were characterized rheologically and by turbidity measurements to construct a phase diagram in the temperature (T) and composition (W) variables. The T-W quadrant consists of three regions: a singlephase solutions region (A) and regions of complete and incomplete phase separation (B and C, respectively). The system in region C is a gel. Region B, lying between A and C, corresponds to two co-existing liquid phases. The transition from A to C (obtained by cooling the system at constant W) involves crossing region B. The properties of the resulting gels depend on the rate of this intersection. Gels formed on rapid cooling have an even distribution of turbidity, whereas slow cooling gives two gel layers of different turbidity. The gelation temperature and gel strength of the mixed systems are dominated by the gelatin component, with no indication of network formation by starch.     

Effect of extraction parameters on the chemical structure and gel properties of kappa/iota-hybrid carrageenans obtained from Mastocarpus stellatus

Extraction parameters (temperature, pH, duration) and alkaline pre-treatment duration have been systematically varied in the aim of exploring their impact on both chemical structure and gelling properties of carrageenan biopolymers obtained from Mastocarpus stellatus seaweeds, collected on the Northern coast of Portugal. Increasing the alkaline pre-treatment duration PT leads to kappa/iota-hybrid carrageenans containing less sulphate groups and biological precursor monomers. As a result, gel properties in the presence of KCl are improved as demonstrated by the increase in the Young's modulus with parameter PT. Increasing the extraction duration t ameliorates the biopolymer yield with no significant change in the complex kappa/iota-hybrid carrageenan chemical structure. However, smaller molecular weights are obtained and gel properties are seen to be negatively affected. Extraction temperature and pH have dramatic effects on the biopolymer gel strength, and a set of extraction parameters optimized with respect to extraction yield and gel properties is reported. In addition, kappa/iota-hybrid carrageenans obtained throughout this study exhibit a wide range of gel strengths in KCl, and allow us to present correlations between gel thermal properties and the kappa/iota-hybrid carrageenans chemical structure.     

Interaction between cytochrome P-450 (P-450C21) and NADPH-cytochrome P-450 reductase from adrenocortical microsomes in a reconstituted system

The interaction between P-450C21 and NADPH-cytochrome P-450 reductase, both purified from bovine adrenocortical microsomes, has been investigated in a reconstituted system with a nonionic detergent, Emulgen 913, by kinetic analysis and gel filtrations. Steady state kinetic data in progesterone 21-hydroxylation showed formation of an equimolar complex between the two enzyme proteins at low Emulgen concentration. Steady state kinetic studies on the electron transfer from NADPH to P-450C21 via the reductase showed that a stable complex formation between the two enzyme proteins was not involved in the steady state electron transfer at high Emulgen concentration. In stopped flow experiments, a time course of the P-450C21 reduction showed biphasic kinetics composed of fast and slow phases. The dependence of kinetic parameters on Emulgen concentration indicates that the fast phase corresponds to the electron transfer within the complex and the slow phase to the electron transfer through a random collision between P-450C21 and the reductase. The stable complex formation between P-450C21 and the reductase has been clearly demonstrated by gel filtration. The stable complex was composed of several molecules of the two enzyme proteins at an equimolar ratio, which was active for progesterone 21-hydroxylation and had a tendency to dissociate at high Emulgen concentration.     

The lipid charge density at the bilayer surface modulates the effects of melittin on membranes

The influence of melittin on two DMPA membrane systems at pH 4.2 and 8.2 has been investigated by solid-state 31P and 2H NMR, as a function of temperature and peptide concentration. Melittin promotes greater morphological changes for both systems in the fluid phase, the effect being larger at pH 4.2. Close inspection of fatty acyl chain dynamics suggests that some parallels can be drawn between the DMPA/melittin at pH 8.2 and PC/melittin systems. In addition, at pH 8.2 a direct neutralization at the interface of one of the lipid negative charges by a positive charge of the peptide occurs, as can be monitored by 31P NMR at the molecular level. For the system at pH 4.2 and at high temperature, a lipid-to-peptide molar ratio of 30 is sufficient to transform the whole system into an isotropic phase, proposed to be inverted micelles. When the system is cooled down towards the gel phase one observes an intermediate hexagonal phase in a narrow range of temperature.     

Aqueous micellar two-phase system composed of hyamine-type hydrophobically modified ethylene oxide and application for cytochrome P450 BM-3 separation

The hydrophobically modified ethylene oxide polymer, HM-EO, was modified with an alkyl halide to prepare a hyamine-type HM-EO, named N-Me-HM-EO, which could be used for forming N-Me-HM-EO/buffer aqueous micellar two-phase system. The critical micelle concentration of N-Me-HM-EO solution and the phase diagrams of N-Me-HM-EO/buffer systems were determined. By using this novel aqueous micellar two-phase system, the separation of cytochrome P450 BM-3 from cell extract was explored. The partitioning behavior of P450 BM-3 in N-Me-HM-EO/buffer systems was measured. The influences of some factors such as total proteins concentration, pH, temperature and salt concentration, on the partitioning coefficients of P450 BM-3 were investigated. Since the micellar aggregates in the N-Me-HM-EO enriched phase were positively charged, it was possible to conduct the proteins with different charges to top or bottom phases by adjusting pH and salt concentration in the system. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: the first extraction with N-Me-HM-EO/buffer system at pH 8.0, and the second extraction in the same system at pH 6.0. The recovery of P450 BM-3 was 73.3% with the purification factor of 2.5. The results indicated that the aqueous micellar two-phase system composed of hyamine modified polysoap has a promising application for selective separation of biomolecules depending on the enhanced electrostatic interactions between micelles and proteins.     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

Characterization of the activation of rat liver beta-glucosidase by sialosylgangliotetraosylceramide

We show that sialosylgangliotetraosylceramide (GM1) is a potent activator of delipidated (sodium cholate- and 1-butanol-extracted) lysosomal rat liver glucocerebroside:beta-glucosidase. Stimulation of 4-methylumbelliferyl-beta-D-glucopyranoside hydrolysis by the beta-glucosidase was markedly dependent upon the concentration of GM1 in the assay medium. Estimations of critical micellar concentration (CMC) performed fluorometrically using the dye N-phenylnaphthylamine revealed two CMC values of GM1 above 18 degrees C; the CMC of the primary micelles (3.32 microM) was temperature-independent whereas that of the secondary micelles decreased with decreasing temperature (17.2 and 10.8 microM at 37 and 20 degrees C, respectively). In the temperature range of 18-39 degrees C, beta-glucosidase activity increased sharply when the GM1 concentration was above the CMC of the secondary micelles. Although a heat-stable factor, purified from the spleen of a patient with Gaucher's disease, had a profound effect on the activation of beta-glucosidase by GM1, it decreased the CMC only slightly (14.8 versus 17.2 microM at 37 degrees C). The heat-stable factor (8 micrograms/ml) changed the shape of the activation curve from sigmoidal to hyperbolic, suggesting that the heat-stable factor permits beta-glucosidase to be activated by primary micelles or monomers. The results of gel filtration chromatography and sucrose gradient centrifugation in H2O and D2O revealed that the activation of beta-glucosidase by GM1 was associated with an increase in the size of the enzyme from 45,800 to 178,500 daltons and an increase in the partial specific volume from 0.697 to 0.740 ml/g. The active, reconstituted beta-glucosidase appears to consist of 50% protein and 50% ganglioside (56 molecules/178,500 g). Concentrations of GM1 below the CMC of secondary micelles increased the rate of inactivation of the enzyme by the irreversible inhibitor conduritol B epoxide at 37 degrees C, indicating that GM1 monomers or primary micelles do interact with the enzyme, even though they do not increase the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside by the enzyme.