Detergent Effects on the Phosphatidylinositol-Specific Phospholipase C in Rat Brain Synaptosomes

In the presence of Ca2+ (2.5 mM) and using [14C]arachidonoyl phosphatidylinositol (PI) membrane as substrate, phosphatidylinositol-specific phospholipase C (PI-PLC) (EC 3.1.4.10) in rat brain synaptosomes was activated by deoxycholate but not taurocholate. Calcium stimulated enzymic hydrolysis by both detergents, but the stimulatory effect of taurocholate was less than that of deoxycholate. Peak stimulation for deoxycholate was observed at 1 mg/ml, whereas that for taurocholate was 4 mg/ml. When 1 mM EDTA was added to the taurocholate (4 mg/ml) and Ca2+ (3.5 mM) system, synaptosomal PI-PLC activity was greatly stimulated, to almost the same level as the deoxycholate + Ca2+ system. This system required the presence of all three factors, and EGTA could not effectively replace EDTA in the stimulatory action. The detergent-induced hydrolysis of synaptosomal PI by the deoxycholate + Ca2+ and the taurocholate + Ca2+ + EDTA systems was strongly inhibited by divalent metal ions such as Zn2+, Cu2+, Pb2+, and Fe2+, whereas Mg2+ and Ca2+ were ineffective. Nevertheless, only the deoxycholate + Ca2+ system was responsive to enzyme inhibition by membrane-perturbing agents such as lysophospholipids and free fatty acids. The specific requirement for EDTA in the taurocholate system may be due to the release of a pool of inhibitory divalent metal ions from the membranes.     

Kinetic properties of human pancreatic carboxylesterase

Fractionation of pancreatic juice by heparin-Sepharose and cholate-Sepharose affinity chromatography indicated that pancreatic carboxylesterase can be separated from pancreatic lipase with the former retained and the latter unretained by both columns. The chromatographic behavior of pancreatic carboxylesterase was found to be similar to that of human milk bile salt-activated lipase. The partially purified pancreatic carboxylesterase had a specific activity of 30 mumol/min per mg protein when assayed with p-nitrophenyl acetate. The reaction mechanism of human pancreatic carboxylesterase was studied using p-nitrophenyl acetate as substrate and taurocholate as activator. The reaction of the enzyme was found to follow a rapid-equilibrium random mechanism. Because of the presence of basal activity, the role of taurocholate can be considered as a non-essential activator and the dissociation constant for the enzyme-taurocholate binary complex was determined to be 0.20 mM. The activation effect of taurocholate consists in increasing the affinity of the enzyme to the substrate (5.6-fold) and in increasing the Vmax (2.3-fold). Based on the kinetic property of human pancreatic carboxylesterase and human milk bile salt-activated lipase with p-nitrophenyl acetate, cholesterol oleate and triolein as substrate, we conclude that they share common substrate specificity but show minor differences in kinetic parameters. Fluorescence studies indicated that both enzymes showed a decreased intrinsic tryptophanyl fluorescence upon incubation with taurocholate. This indicates that bile salt caused a conformational change of the enzymes, with a resultant decreased hydrophobicity in the microenvironment of tryptophan residues.     

Hydrolysis of insoluble cellulose to glucose catalyzed by cellulase‐containing liposomes in an aqueous solution of 1‐butyl‐3‐methylimidazolium chloride

The liposome containing cellulase from Trichoderma viride was prepared under the condition that an appreciable amount of cellulase was incorporated in lipid membranes. The liposomal cellulase and free enzyme were examined in their hydrolytic activities to insoluble cellulose powder CC31 in the acetate buffer solution (pH 4.8) of 15 w/w% [Bmim][Cl] (1‐butyl‐3‐methylimidazolium chloride). The mean diameter and size distribution of cellulase‐containing liposome were practically unchanged under the above condition. The free cellulase was deactivated more rapidly than the liposomal cellulase in catalyzing the hydrolysis of 2.0 g/l CC31 at 45°C in the presence of [Bmim][Cl] for 48 h. The activities of liposomal and free cellulase to cellobiose as soluble substrate were less susceptible to [Bmim][Cl] than their cellulolytic activities to CC31, meaning that β‐glucosidase is relatively stable among the three enzyme components of cellulase. The rate of glucose production could be appreciably improved by the pretreatment of CC31 with [Bmim][Cl] alone at 120°C for 30 min followed by the liposomal cellulase‐catalyzed hydrolysis of the substrate at 45°C at the [Bmim][Cl] concentration of 15 w/w%. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1190–1196, 2013     

A new method for the rapid isolation of basolateral plasma membrane vesicles from rat liver. Characterization, validation, and bile acid transport studies

Basolateral plasma membrane vesicles were prepared from rat liver by a new technique using self-generating Percoll gradients. The method is rapid (total spin time of 2.5 h) and protein yields were high (0.64 mg/g of liver). Transmission electron microscopy studies and measurements of marker enzyme activities indicated that the preparation was highly enriched in basolateral membranes and substantially free of contamination by canalicular membranes or subcellular organelles. High total recoveries for protein yield and marker enzyme activities during the fractionation procedure indicated that enzymatic activity was neither lost (inactivation) nor increased (activation). Thus, the pattern of marker enzyme activities found in the membrane preparation truly reflected substantial enrichment in membranes from the basolateral surface. Analysis of freeze-fracture electron micrographs suggested that approximately 75% of the vesicles were oriented "right-side-out." In order to assess the functional properties of the vesicles, the uptake of [3H]taurocholate was studied. In the presence of a Na+ gradient, taurocholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot"). In the absence of a gradient but in the presence of equimolar Na+ inside and outside of the vesicle, taurocholate uptake was faster than in the absence of Na+. These findings support a direct co-transport mechanism for the uptake of taurocholate and Na+. Kinetic studies demonstrated that Na+-dependent taurocholate uptake was saturable with a Km of 36.5 microM and a Vmax of 5.36 nmol mg-1 protein min-1. The high yield, enzymatic profile and retention of transport properties suggest that this membrane preparation is well suited for studies of basolateral transport.     

Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5

The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.     

Changing the efficiency and specificity of the esterase activity of human carbonic anhydrase II by site-specific mutagenesis.

Rates of hydrolysis of 4-, 3-, and 2-nitrophenyl acetate and 4-nitrophenyl propionate catalyzed by wild-type and mutant forms of human carbonic anhydrase II have been measured. The results show that the mutations Tyr7-->Phe and Ala65-->Leu lead to activity enhancements with all the investigated substrates, but there is no significant effect on the specificity. In contrast, some mutations at sequence position 200 have large effects on specificity. For example, while the mutation Thr200-->Gly results in a threefold increase of the rate of hydrolysis of 4-nitrophenyl acetate, the activity is enhanced 10 times with the meta-substituted substrate and 380 times with the ortho-substituted substrate. These results are interpreted in terms of the removal in the mutant of a steric interference between the 2-NO2 group, in particular, and the side chain of Thr200. Mutants involving residues lining a hydrophobic pocket near the catalytically essential zinc ion have also been investigated. The most pronounced effect on specificity was found for the Val143-->Gly mutant. This mutation leads to a sixfold decrease of the rate of hydrolysis of 4-nitrophenyl acetate but a 20-fold increase of the activity with the propionyl ester as substrate. These results suggest that the side chain of Val143 interferes sterically with the acyl moiety of 4-nitrophenyl propionate. Based on these results, we have constructed a hypothetical model of the location of these ester substrates in the enzymic active site.     

Hydrolysis of retinyl esters by non-specific carboxylesterases from rat liver endoplasmic reticulum.

The four most important non-specific carboxylesterases from rat liver were assayed for their ability to hydrolyse retinyl esters. Only the esterases with pI 6.2 and 6.4 (= esterase ES-4) are able to hydrolyse retinyl palmitate. Their specific activities strongly depend on the emulsifier used (maximum rate: 440 nmol of retinol liberated/h per mg of esterase). Beside retinyl palmitate, these esterases cleave palmitoyl-CoA and monoacylglycerols with much higher rates, as well as certain drugs (e.g. aspirin and propanidid). However, no transacylation between palmitoyl-CoA and retinol occurs. Retinyl acetate also is a substrate for the above esterases and for another one with pI 5.6 (= esterase ES-3). Again the emulsifier influences the hydrolysis by these esterases (maximum rates: 475 nmol/h per mg for ES-4 and 200 nmol/h per mg for ES-3). Differential centrifugation of rat liver homogenate reveals that retinyl palmitate hydrolase activity is highly enriched in the plasma membranes, but only moderately so in the endoplasmic reticulum, where the investigated esterases are located. Since the latter activity can be largely inhibited with the selective esterase inhibitor bis-(4-nitrophenyl) phosphate, it is concluded that the esterases with pI 6.2 and 6.4 (ES-4) represent the main retinyl palmitate hydrolase of rat liver endoplasmic reticulum. In view of this cellular localization, the enzyme could possibly be involved in the mobilization of retinol from the vitamin A esters stored in the liver. However, preliminary experiments in vivo have failed to demonstrate such a biological function.     

Activity of Pseudomonas cepacia lipase in organic media is greatly enhanced after immobilization on a polypropylene support

The purified lipase from Pseudomonas cepacia was used as free and immobilized enzyme preparation for hydrolysis of p-nitrophenyl palmitate (pNPP) and p-nitrophenyl acetate (pNPA) in organic media. The free enzyme was mixed with bovine serum albumin and lyophilized. Immobilization was on porous polypropylene. Conditions where diffusional limitations of the substrate were not limiting the reaction rate were defined. The specific activity of the lipase was greatly enhanced upon immobilization: 16.5- and 7.8-fold for pNPP and pNPA respectively. Both the free and immobilized lipases followed Michaelis–Menten kinetics in organic solvent despite the heterogeneity (solid/liquid) of the reaction mixture. For pNPP, the activation factor upon immobilization came mainly from a reduction in K _m, app while k _cat was increased for pNPA. Received: 30 January 1997 / Accepted: 14 February 1997     

Characterization of the bile acid transport system in normal and transformed hepatocytes. Photoaffinity labeling of the taurocholate carrier protein

The taurocholate transport system in normal and transformed hepatocytes has been characterized using transport kinetics and photoaffinity labeling procedures. A photoreactive diazirine derivative of taurocholate, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino [ 1,2-3H ]ethanesulfonic acid (7-ADTC), which has been shown to be a substrate for the bile acid carrier system, was photolyzed in the presence of intact hepatocytes, hepatoma tissue culture (HTC) cells, and plasma membranes derived from the hepatocyte sinusoidal surface. Irradiation of membranes in the presence of 7-ADTC resulted in the incorporation of the photoprobe into two proteins with Mr = 68,000 and 54,000. The specificity of labeling was confirmed by the significant inhibition of labeling observed when photolysis was carried out in the presence of taurocholate. The 68,000-Da protein was easily extracted with water and was shown to exhibit electrophoretic properties identical with rat serum albumin. The 54,000-Da protein required Triton X-100 for solubilization, indicating a strong association with the plasma membrane. Labeling of intact hepatocytes also resulted in specific labeling of the 54,000-Da protein. In contrast to hepatocytes, HTC cells derived from Morris hepatoma 7288C as well as H4-II-E cells derived from Reuber hepatoma H-35 exhibited a total loss of mediated bile acid uptake. Photolysis of 7-ADTC in the presence of HTC cells did not result in the labeling of any proteins, a result consistent with the loss of transport activity, and further supporting the specificity of the labeling reaction. The anion transport inhibitor N-(4-azido-2-nitrophenyl)-2-aminoethyl-[ 35S ]sulfonate, which has been shown to be a substrate for the bile acid carrier system also labeled the 54,000-Da plasma membrane protein when photolyzed in the presence of intact hepatocytes. These results suggest that the 54,000-Da protein is a component of the hepatocyte bile acid transport system and that the activity of this system is greatly reduced in several hepatoma cell lines.     

Isolation and Characterization of a GDSL Esterase from the Metagenome of a Marine Sponge-associated Bacteria

Using a metagenome library constructed from a bacterial associated with a marine sponge Hyrtios erecta, we identified a novel esterase that belongs to the SGNH hydrolase superfamily of esterases. The substrate specificity of EstHE1 was determined using p-nitrophenyl (pNP) ester (C2: acetate, C4: butylate, C6: caproate, C12: laurate, C16: palmitate). EstHE1 exhibited activity against C2 (5.6 U/mg), C4 (5.1 U/mg), and C6 (2.8 U/mg) substrates. The optimal temperature for EstHE1 esterase activity of the pNP acetate substrate was 40°C, and EstHE1 retained 60% of its enzymatic activity in the 30–50°C range. This esterase showed moderate thermostability, retaining 58% of its activity even after preincubation for 12 h at 40°C. EstHE1 also maintained activity in high concentrations of NaCl, indicating that this esterase is salt-tolerant. Thus, EstHE1 has the thermal stability and salt tolerance necessary for use as an industrial enzyme.     

Application of selectively acylated glycosides for the α-galactosidase-catalyzed synthesis of disaccharides

4-Nitrophenyl alpha-D-galactopyranosyl-(1-->3)-6-O-acetyl-alpha-D-galactopyranoside was prepared in a transglycosylation reaction catalyzed by alpha-D-galactosidase from Talaromyces flavus using 4-nitrophenyl alpha-D-galactopyranoside as a glycosyl donor and 4-nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside as an acceptor. 4-Nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside and 4-nitrophenyl 6-O-acetyl-beta-D-galactopyranoside were prepared in a regioselective enzymic transesterification in pyridine-acetone catalyzed by the lipase PS from Burkholderia cepacia. A series of water-miscible organic solvents (acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, 2-methoxyethanol, pyridine, 2-methylpropan-2-ol, tetrahydrofuran, propargyl alcohol) were used as co-solvents in this enzymic reaction. Their influence on the activity and stability of the alpha-galactosidase from T. flavus was established. 2-Methylpropan-2-ol and acetone (increasing the solubility of the modified substrate acceptors and displaying the minimum impairment of the activity and stability of the enzyme) were used as co-solvents in transglycosylation reactions.     

Regulation of enzyme activity in adsorptive enzyme systems

The kinetic behaviour of adsorptive enzyme systems with free and adsorbed enzyme forms in rapid equilibrium has been analysed. It has been shown that the dependences of enzymic reaction rate on substrate or “adsorptive effector” concentrations reveal the deviations from simple kinetic laws of Michaelis-Menten type (positive or negative kinetic co-operativity). Such kinetic anomalies should be observed when adsorption of the enzyme results in the changing catalytic properties and when the state of the equilibrium between free and bound enzyme forms depends on the presence of low molecular substances (substrates, coenzymes and various cellular metabolites). The physiological significance of adsorption-desorption processes for the enzyme activity regulation has been emphasized.     

The mechanism of liposomal damage by taurocholate

The stability of small unilamellar vesicles formed by egg-yolk phosphatidylcholine (PC) has been examined in the presence of sodium taurocholate. The permeability of the vesicular membrane changes as the total taurocholate concentration increases, until a transformation from mixed bile salt/PC vesicles to mixed micelles occurs. Based on experiments in which the bile salt-induced release of either hydrophilic (carboxyfluorescein) or hydrophobic (Bromothymol blue) probes was studied, and on fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene and turbidity measurements, a two-step process for the initial stage of liposomal damage by taurocholate is postulated.     

Reversal of part of the aldehyde dehydrogenase reaction pathway during the hydrolysis of an ester.

An aldehyde dehydrogenase from rabbit liver, a homogeneous protein on three distinct polyacrylamide-gel systems, has an associated 4-nitrophenyl esterase activity. At pH 7.0 in the presence of 80 micrometer-NADH and 800 micrometer-4-nitrophenyl acetate the enzyme produces NAD+ and a stoicheiometric amount of an aldehyde, as well as hydrolysing the ester. On this and other evidence it is proposed that ester hydrolysis occurs at the usual active site of the enzyme.     

Lipolytic and Esterolytic Activities in Posterior Lingual Glands of Rat: A Histochemical Study

Catalytic activities of lingual lipase were investigated by enzyme histochemistry in post-mortem tongues from male rats. Sections of fresh-frozen or formalin-calcium fixed tissue were incubated with naphthol-AS-nonanoate and α-naphthyl acetate substrate mixtures. The effects of pH level, sodium taurocholate activator and E600 inhibitor were also examined. The use of cryostat sections of tissues fixed in formalin-calcium and of nonanoate substrate within the range of pH 4.4–6.4, were optimal for localizing maximum reaction product, captured by Fast Blue BB, in acini and demilunes of the posterior deep and superficial lingual glands respectively. The reaction product corresponded with the distribution of secretory granules and failed to develop when taurocholate was omitted from the incubation medium. Similarly localized E600-resistant reaction product occurred with the acetate substrate and hexazotized New Fuchsin at pH 7.4, in the absence of taurocholate. Lipase and conventional esterase activities appear to be superimposed in posterior lingual glands of rat. The ability of their acini and demilunes to hydrolyse nonanoate substrate at an acidic pH optimum, when activated by sodium taurocholate, seems attributable to lipase destined for secretion into saliva – hence convenient for routine histochemical identification of the enzyme.     

Lipolytic and esterolytic activities in posterior lingual glands of rat: a histochemical study

Catalytic activities of lingual lipase were investigated by enzyme histochemistry in post-mortem tongues from male rats. Sections of fresh-frozen or formalin-calcium fixed tissue were incubated with naphthol-AS-nonanoate and alpha-naphthyl acetate substrate mixtures. The effects of pH level, sodium taurocholate activator and E600 inhibitor were also examined. The use of cryostat sections of tissues fixed in formalin-calcium and of nonanoate substrate within the range of pH 4.4-6.4, were optimal for localizing maximum reaction product, captured by Fast Blue BB, in acini and demilunes of the posterior deep and superficial lingual glands respectively. The reaction product corresponded with the distribution of secretory granules and failed to develop when taurocholate was omitted from the incubation medium. Similarly localized E600-resistant reaction product occurred with the acetate substrate and hexazotized New Fuchsin at pH 7.4, in the absence of taurocholate. Lipase and conventional esterase activities appear to be superimposed in posterior lingual glands of rat. The ability of their acini and demilunes to hydrolyse nonanoate substrate at an acidic pH optimum, when activated by sodium taurocholate, seems attributable to lipase destined for secretion into saliva--hence convenient for routine histochemical identification of the enzyme.     

Synthesis and transport characteristics of photoaffinity probes for the hepatocyte bile acid transport system

In an effort to characterize the hepatocyte bile acid transport system, a photoreactive derivative of taurocholate, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid (7-ADTC) has been synthesized and its transport properties compared to those of the natural substrate. Both the bile acid and its synthetic analog were shown to be transported against an electrochemical gradient as well as a chemical gradient. Transport as a function of concentration and the presence of sodium indicated that both substrates were taken up by a sodium-dependent and a sodium-independent route. Taurocholate had Km values of 26 and 57 microM and Vmax values of 0.77 and 0.15 nmol/mg of protein/min, respectively. In comparison, 7-ADTC had very similar kinetic properties with Km values of 25 and 31 microM and Vmax values of 1.14 and 0.27 nmol/mg of protein/min. Each compound was shown to inhibit competitively the transport of the other, suggesting that these substrates utilized a common membrane carrier. The transport properties of the photoreactive anion transport inhibitor, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) were also characterized in the hepatocyte system. Transport occurred via a sodium-dependent and a sodium-independent route with Km values of 210 and 555 microM and Vmax values of 0.57 and 1.62 nmol/mg of protein/min. As in the case of 7-ADTC, NAP-taurine and taurocholate were also shown to be mutual competitive inhibitors. In the absence of light, 7-ADTC was a reversible inhibitor of taurocholate uptake. Upon irradiation, irreversible photoinactivation of the taurocholate uptake system was observed. These results indicate that 7-ADTC and NAP-taurine can be utilized as photoaffinity probes for the identification of the bile acid carrier protein(s) in hepatocyte plasma membranes.     

Biochemical characterization of the retinoid isomerase system of the eye

We have previously shown that membranes from the retinal pigment epithelium can transform added all-trans-retinol into a mixture of 11-cis-retinoids, demonstrating the "missing reaction" in the visual cycle for the first time (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853). In this article, this isomerase activity is further characterized. Double-label experiments with [15-3H]- and [15-14C]all-trans-retinol as the substrate show that the tritium label is retained in the 11-cis-retinol and 11-cis-retinyl palmitate products. This requires that isomerization occur at the alcohol level of oxidation. All-trans-retinyl esters, such as the palmitate, acetate, butyrate, and hexanoate esters, are not directly transformed into their 11-cis counterparts by the membranes. The data are consistent with the presence of an all-trans-retinol isomerase enzyme system or enzyme complex, which produces 11-cis-retinol. Other isomeric retinols were tested for substrate activity. Neither 9-cis-retinol(al) nor 13-cis-retinol were processed by the isomerase. Since the membranes containing the isomerase possess other retinol metabolizing activities, such as retinyl ester synthetase and dehydrogenase activities, further purification was attempted. Appreciable quantities of all detergents tested led to the disappearance of isomerase activity, and high salt or EDTA did not dissociate isomerase activity from the membranes. However, extensive sonication of the membranes did produce a 100,000 x g supernatant fraction of light membranes depleted of other all-trans-retinol processing activities. The isomerase activity in these membranes was saturable with all-trans-retinol, as required for a biologically significant process, and showed a Vmax of 5 pmol/h/mg of protein, a KM of 0.8 microM, and a pH optimum of 8. The isomerase was destroyed by proteinase K, by phospholipase C, by heating, or by ethanol at concentrations greater than 1%. The addition of high energy compounds, such as MgATP, MgGTP, or palmitoyl-CoA, did not appear to stimulate isomerase activity in the 100,000 x g supernatant.     

Effects of divalent cations and sodium taurocholate on pancreatic lipase activity with gum arabic-emulsified tributyrylglycerol substrates.

The effects of Ca2+ and/or sodium taurocholate on lipase activity with gum arabic-emulsified tributyrylglycerol substrates were investigated. Calcium was found to slightly increase lipase activity while bile salts showed marked inhibition except at very low concentrations. Calcium eliminated inhibition seen with low concentrations of bile salts and reduced the inhibition seen at higher bile shift of the enzyme from the alkaline region in the absence of bile salt to the slightly acidic region in the presence of bile salt. Calcium was shown to eliminate the time lag periods between enzyme addition and maximum rate of hydrolysis seen at low substrate concentrations and the time lag noted when bile salts were included with normal (substrate concentration not limiting) assay concentrations of substrate. Zeta potential measurements indicated that Ca2+ reduced the negative charge on the gum arabic-emulsified particle while bile salts did not increase the negative charge. Commercial preparations of gum arabic were found to have significant concentrations of Ca2+ and Mg2+.     

Liposomal encapsulation of yeast alcohol dehydrogenase with cofactor for stabilization of the enzyme structure and activity

Yeast alcohol dehydrogenase (YADH) with its cofactor nicotinamide adenine dinucleotide (NAD+) could be stably encapsulated in liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine). The YADH- and NAD+-containing liposomes (YADH-NADL) were 100 nm in mean diameter. The liposomal YADH and NAD+ concentrations were 2.3 mg/mL and 3.9 mM, respectively. A synergistic effect of the liposomal encapsulation and the presence of NAD+ was examined on the thermal stability of YADH at 45 and 50 degrees C. The enzyme stability of the YADH-NADL was compared to the stabilities of the liposomal YADH (YADHL) containing 3.3 mg/mL YADH without NAD+ as well as the free YADH with and without NAD+. Free YADH was increasingly deactivated during its incubation at 45 degrees C for 2 h with decrease of the enzyme concentration from 3.3 to 0.01 mg/mL because of the dissociation of tetrameric YADH into its subunits. At that temperature, the coexistence of free NAD+ at 3.9 mM improved the stability of free YADH at 2.3 mg/mL through forming their thermostable complex, although the stabilization effect of NAD+ was lowered at 50 degrees C. The turbidity measurements for the above free YADH solution with and without NAD+ revealed that the change in the enzyme tertiary structure was much more pronounced at 50 degrees C than at 45 degrees C even in the presence of NAD+. This suggests that YADH was readily deactivated in free solution due to a decrease in the inherent affinity of YADH with NAD+. On the other hand, both liposomal enzyme systems, YADH-NADL and YADHL, showed stabilities at both 45 and 50 degrees C much higher than those of the above free enzyme systems, YADH/NAD+ and YADH. These results imply that the liposome membranes stabilized the enzyme tertiary and thus quaternary structures. Furthermore, the enzyme activity of the YADH-NADL showed a stability higher than that of the YADHL with a more remarkable effect of NAD+ at 50 degrees C than at 45 degrees C. This was considered to be because even at 50 degrees C the stabilization effect of lipid membranes on the tertiary and quaternary structures of the liposomal YADH allowed the enzyme to form its thermostable complex with NAD+ in liposomes.