Cholesterol-dependent modification of microsomal dynamics and UDPglucuronyltransferase kinetics

The effect of both in vitro incorporation and removal of cholesterol in guinea pig liver microsomes on the lipid composition, dynamic properties of the membrane, and kinetic constants of UDPglucuronyltransferase was studied. No significant changes either in the fatty acid composition or in the distribution of phospholipid classes were observed upon cholesterol incorporation and removal. Lateral and rotational mobility measured by the efficiency of pyrene excimer formation and fluorescence of 1,6-diphenylhexatriene decreased with cholesterol incorporation and increased in parallel to cholesterol removal. These changes were associated with alterations in the kinetic properties of UDPglucuronyltransferase. Whereas Vmax increased, the Km of the different steps of the reaction decreased with cholesterol incorporation. The negative homotropic effect and apparent cooperativity of UDP-glucuronic acid decreased when cholesterol was incorporated and increased after cholesterol removal. Moreover, the UDP-N-acetylglucosamine-dependent activation of the enzyme decreased in correlation with an increase of cholesterol concentration in microsomes. It has been demonstrated that both the shift of the non-Michaelian kinetics of the enzyme to Michaelian and the decrease of the UDP-N-acetylglucosamine-dependent activation of the enzyme are evoked by a change of the physical state of the UDPglucuronyltransferase milieu from a gel phase to a liquid-crystalline phase. Therefore, we must admit that cholesterol incorporation in the microsomes while producing an increased packing of the bulk lipids would also cause the separation of more fluid phospholipids, which increase the proportion of molecules in the liquid-crystalline state within the enzyme environment.     

Allosteric inhibition of the water-soluble C-terminal fragment of Sendai virus neuraminidase.

Trypsin solubilized hemagglutinin-neuraminidase of Sendai virus (cHN) displays michaelian kinetics, with native fetuin as substrate, at 37 degrees C. Vmax and Km values are only marginally altered, as compared to intact viral neuraminidase. At lower temperatures, cHN follows non-michaelian kinetics, with marked substrate inhibition at 4 degrees C. With denaturated fetuin, michaelian kinetics are observed in all conditions, while asialo fetuin was an uncompetitive inhibitor of cHN, with native fetuin or sialyl lactose as substrates. These results can be explained assuming that the protein moiety of fetuin acts as an allosteric inhibitor of cHN.     

Phospholipid fatty acid modification of rat liver microsomes affects acylcoenzyme A:cholesterol acyltransferase activity

The effect of phospholipid fatty acyl composition on the activity of acylcoenzyme A:cholesterol acyltransferase was investigated in rat liver microsomes. Specific phosphatidylcholine replacements were produced by incubating the microsomes with liposomes and bovine liver phospholipid-exchange protein. Although the fatty acid composition of the microsomes was modified appreciably, there was no change in the microsomal phospholipid or cholesterol content. As compared to microsomes enriched for 2 h with dioleoylphosphatidylcholine, those enriched with dipalmitoylphosphatidylcholine exhibited 30-45% less acyl-CoA:cholesterol acyltransferase activity. Enrichment with 1-palmitoyl-2-linoleoylphosphatidylcholine increased acyl-CoA:cholesterol acyltransferase activity by 20%. By contrast, dilinoleoylphosphatidylcholine abolished microsomal acyl-CoA:cholesterol acyltransferase activity almost completely. Addition of cofactors that stimulated microsomal lipid peroxidation inhibited acyl-CoA:cholesterol acyltransferase activity by only 10%, however, and did not increase the inhibition produced by submaximal amounts of dilinoleoylphosphatidylcholine. Certain of the phosphatidylcholine replacements produced changes in palmitoyl-CoA hydrolase, NADPH-dependent lipid peroxidase, glucose-6-phosphatase and UDPglucuronyl transferase activities, but they did not closely correlate with the alterations in acyl-CoA:cholesterol acyltransferase activity. Electron spin resonance measurements with the 5-nitroxystearate probe indicated that microsomal lipid ordering was reduced to a roughly similar extent by dioleoyl- or by dilinoleoylphosphatidylcholine enrichment. Since these enrichments produce widely different effects on acyl-CoA:cholesterol acyltransferase activity, changes in bulk membrane lipid fluidity cannot be the only factor responsible for phospholipid fatty acid compositional effect on acyl-CoA:cholesterol acyltransferase. The present results are more consistent with a modulation resulting from either changes in the lipid microenvironment of acyl-CoA:cholesterol acyltransferase or a direct interaction between specific phosphatidylcholine fatty acyl groups and acyl-CoA:cholesterol acyltransferase.     

INCORPORATION OF LIPIDS INTO SUBCELLULAR FRACTIONS OF BRAIN OF QUAKING MUTANT

The synthesis of lipids and their assembly into subcellular membrane fractions of the myelin deficient Quaking mutant and control brains was studied in 18-, 24- and 41-day-old animals using a double label methodology with¹⁴C and ³H acetate as precursors. As a general procedure, Quaking mutants were injected intracranially with 50 μCi [¹⁴C]acetate and their littermate controls with 300 μCi [³H]acetate. The animals were killed 3 h post-injection, their brains were pooled and subcellular fractions prepared from the common homogenate. An 80-90% decrease in the incorporation of acetate into eleven lipids of myelin in the Quaking mutant was found. This occurred in the face of apparent normal incorporation (relative to microsomes) into lipids of the other main subcellular fractions (nuclear. mitochondrial and synaptosomal) with the exception of decreased incorporation into the myelin-like fraction at 18 and 24 days. Cholesterol and cerebroside were less readily incorporated into Quaking myelin than the other lipids. Although the microsomal synthesis of cholesterol and cerebroside was depressed by about 30% in the Quaking mutant, the incorporation of cholesterol into nuclear, synaptosomal and mitochondrial fractions was unaffected in the mutant. This indicates that sufficient cholesterol is synthesized for the normal assembly of these organelles. In contrast the incorporation of acetate into cholesterol and cerebroside of Quaking myelin was decreased much more than microsomal synthesis. This latter result is consistent with a defect in the process of myclin membrane assembly     

Cholesterol-induced microsomal changes modulate desaturase activities

The effect of 1% dietary cholesterol and 0.5% cholate on the rat liver microsomal composition and fatty acid desaturase activities was studied over various periods of time. The cholesterol content of liver microsomes increased as well as that of phosphatidylcholine. Cholesterol/phosphatidylcholine and phosphatidylcholine/phosphatidylethanolamine ratios were also elevated. Phosphatidylinositol decreased, but it recovered its original values at the end of the experimental period. Phosphatidylserine and sphingomyelin slightly decreased with time. Fatty acid composition changes were expressed by a saturated acid decrease and monounsaturated acid increase. Arachidonic acid content was also reduced. A similar pattern appeared in the main phospholipids: phosphatidylcholine and phosphatidylethanolamine. Delta 9-Desaturase activity was enhanced as early as 48 h after cholesterol administration, whereas delta 5- and delta 6-desaturases were depressed during the same period and this enzymatic behaviour remained after 21 days of diet administration. The microsomal membrane was rigidized, as demonstrated by the increase of the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene.     

In vitro modification of cholesterol content of rat liver microsomes. Effects upon membrane 'fluidity' and activities of glucose-6-phosphatase and fatty acid desaturation systems

The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver.     

Non-michaelian behavior of 5 alpha-reductase in human prostate

An in-depth analysis of the kinetics of 5 alpha-reductase in human prostatic tissue gave findings inconsistent with the claim that the enzyme is michaelian. In both hyperplastic and malignant tissue, the time-course of the conversion of testosterone (T) into dihydrotestosterone (DHT) was non-linear under conditions ensuring less than 15% conversion of substrate and cofactor. An initial rapid phase of conversion was followed by a long steady-state phase. This time-dependent change in conversion rate was not due to enzyme denaturation, fast inhibition by substrate or product effects. It resulted from a true slow transient kinetic process induced in the reactive enzyme by the substrates. Under our experimental conditions at pH 5.5, 5 alpha-reductase appeared to undergo a conformational change from an initially highly reactive form to a less reactive form. Since this "hysteretic" behavior was correlated with apparently negative cooperativity in enzyme kinetics, we postulate that, as previously described for other key metabolic enzymes, regulation of 5 alpha-reductase activity in the prostate depends on the molecular flexibility of the enzyme and on changes in the cooperativity of different enzyme forms over time. This original non-michaelian behavior may explain the conflicting kinetics reported so far in the literature for this enzyme. The clinical implications of 5 alpha-reductase hysteresis and its involvement in the damping of DHT production within the prostate are discussed.     

Effects of chronic consumption of ethanol and low-thiamin, low-protein diets on the lipid composition of rat whole brain and brain membranes

Four groups of rats were used in a nutritionally-controlled study of effects of chronic ethanol consumption on brain membrane lipid composition. Rats chronically consuming ethanol were fed high-nutrient or low-thiamin, low-protein diets. After 4 months, lipid analyses were performed on brains, brain microsomes and myelin from each group and from pair-fed, non-ethanol controls. Among the effects of ethanol was an increase of the relative proportion of cholesterol in microsomal lipids while there was decrease of it in myelin. Ethanol also increased plasmenylethanolamine while decreasing phosphatidylethanolamine proportions in myelin and in whole brain lipids, decreased the total lipid phosphorus of whole brain, and elevated the proportion of phosphatidylserine in microsomal and whole brain lipids. Effects of poor diet generally did not interfere with ethanol effects except in the case of microsomal lipids, where it apparently prevented an ethanol-induced increase in proportion of cholesterol. These changes may be adaptive responses to the fluidizing effect of ethanol on membranes.     

Effect of probucol on the lipid composition of blood plasma, erythrocyte ghosts and liver membranes in mice

1. Probucol treatment of mice (0.6 g/kg) induced a decrease of cholesterol (CH) and total phospholipids (PLs) in blood plasma, erythrocyte ghosts, liver plasma and microsomal membranes. 2. The incorporation of [14C]acetate in the microsomal lipids of probucol-treated mice was lowered by 23% compared to controls. 3. Probucol administration induced a reduced specific activity of PLs, CH and CH esters, whereas in triacylglycerols it was augmented. 4. Phospholipase A2 and neutral sphingomyelinase activities were not enhanced, indicating that the catabolism of the membrane PL was not elevated.     

Lipid peroxidation increases the molecular order of microsomal membranes

The effect of enzymatic lipid peroxidation on the molecular order of microsomal membranes was evaluated by ESR spectroscopy using the spin probes 5-, 12-, and 16-doxyl-stearic acid. Rat liver microsomal membranes were peroxidized by the NADPH-dependent reaction in the presence of the chelate ADP-Fe3+. Peroxidation resulted in a preferential depletion of polyenoic fatty acids and an increase in the percentage composition of shorter fatty acyl chains. There was no change in the cholesterol/phospholipid ratio of the peroxidized microsomes. The molecular order of both control and peroxidized membranes decreased toward the central region of the bilayer, and the order parameter (S) of each probe was temperature dependent. Peroxidation of the microsomal membrane lipids resulted in an increase in the order parameter determined with the three stearic acid spin probes. Of the three probes, 12-doxylstearic acid was the most sensitive to the changes in membrane organization caused by peroxidation. These data indicate that ESR spectroscopy is a sensitive method of detecting changes in membrane order accompanying peroxidation of membrane lipids.     

Influence of dietary lipids on microsomal membranes from chick breast muscle

1. The effect of different dietary fat intake on the lipid composition and fluidity of microsomal membranes as well as in the enzymatic activity of the Ca2+-ATPase from chick breast muscle was investigated. 2. When a standard diet was supplemented with 10% sunflower seed oil, an increase in the relative amounts of unsaturated fatty acids and membrane fluidity and a decrease in the cholesterol content was observed. 3. The presence of 6% cholesterol in the diet does not modify the fatty acid composition and the fluidity of the membrane but increased, in a low extension, the cholesterol content. 4. The provision of the sunflower seed oil-rich diet supplemented with cholesterol just 48 hr before death promoted an increase in the relative amounts of unsaturated fatty acids and cholesterol content whereas the membrane fluidity decreased in a significant extent. 5. Despite that dietary lipids gave rise in some cases to changes in lipid composition and in the physical state of the microsomal membrane, neither the Ca2+ uptake capacity nor the ATPase activity were significantly affected.     

The influence of fatty acid unsaturation and physical properties of microsomal membrane phospholipids on UDP-glucuronyltransferase activity.

The relationship between lipid composition, the physical properties of microsomal phospholipids and the kinetics of liver UDP-glucuronyltransferase was studied in microsomes from guinea pigs supplied with a normal or a fat-free diet for 28 days. Fatty acid deficiency did not modify either the cholesterol/phospholipid molar ratio or the polar head group composition, but exclusively redistributed the unsaturated fatty acid pattern, by partially exchanging oleic for linoleic acid. This phenomenon accounts for the decrease of both rotational and translational mobilities of the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and pyrene respectively. When the thermotropic behaviour of the different systems was assessed, no transition temperature (gel-liquid-crystalline) between 10 and 40 degrees C was seen as a consequence of the lower degree of unsaturation, either in the microsomal membranes or in the total lipid or total phospholipid extracts from the treated animals. In spite of this, the polarization ratio of trans-parinaric acid and the fluorescence intensity of merocyanine 540 revealed that a significant lateral phase separation occurred at 20-22 degrees C in the extracted phospholipids, which was smoother in the total lipid fractions and in the native microsomal membranes. Fatty acid deficiency caused an upward shift of the midpoint temperature of the lateral phase separation. Furthermore, the phosphatidylcholine extracted from the 'normal' microsomes showed a lateral phase separation centred at a lower temperature than that extracted from 'fat-deficient' microsomes. In contrast, the Arrhenius plot of UDP-glucuronyltransferase from 'normal' microsomes exhibited a change in slope at a higher temperature than that from treated microsomes. These results would suggest that fatty acid deficiency in guinea-pig liver microsomes, while rigidizing the bulk lipids, would segregate the most unsaturated phosphatidylcholine molecules towards the UDP-glucuronyltransferase microenvironment, in accordance with our previous results with cholesterol incorporation [Castuma & Brenner (1986) Biochemistry 25, 4733-4738].     

Membrane homeostasis: thermotropic behaviour of microsomal membrane lipids isolated from livers of rats fed cholesterol-supplemented diets

Differential scanning colorimetry (DSC) has been applied to study the phase transition properties of isolated lipids from liver microsomal membranes of rats fed high cholesterol diets with or without high levels of either saturated (coconut oil) or unsaturated (sunflower seed oil) fat. DSC of aqueous buffer dispersions of liver microsomal lipids exhibited two independent, reversible phase transitions. The dietary cholesterol treatments had their major effect on the temperature at which the lower phase transition (T1) occurred. This transition occurred at a lower temperature when cholesterol was added to the diet, irrespective of the nature of the fatty acid supplement. However the magnitude of decrease was more when cholesterol was fed with sunflower seed oil. Inclusion of cholesterol into the rat diets also lowered the enthalpy values for the lower phase transition (T1). No appreciable effect on the temperature of the higher phase transition (T2) was observed, however the enthalpy values were slightly decreased by cholesterol feeding. These results suggest that certain domains of microsomal lipids, probably containing some relatively higher melting-point lipids, independently undergo solidus or gel formation and this transition (T2) is not greatly affected by dietary cholesterol. On the other hand, domains representing the bulk of the microsomal lipids undergo a phase change (T1) at temperatures which are dependent on cholesterol content and fatty acid profiles of the membrane, which are in turn, modified by dietary cholesterol intake.     

Effect of fatty acid deficiency on microsomal membrane fluidity and cooperativity of the UDP-glucuronyltransferase

The effect of fat deprivation on microsomal membrane fluidity of guinea-pig livers and the kinetic cooperativity of UDP-glucuronyl transferase towards its natural substrate, the UDP-glucuronic acid, were studied. Fat deprivation in the diet of weanling guinea-pigs evoked a typical essential fatty-acid-deficient pattern in the composition of the microsomal membrane. The unsaturated:saturated fatty acid ratio progressively declined in the membrane during the 21-day period tested. This decline determined a gradual increase in the fluorescence anisotropy (rs) of the membrane labeled with diphenylhexatriene and the apparent microviscosity of the lipid bilayer calculated from these values increased from 1.1 to 1.8 poise. In addition, when the infinitely slow decaying fluorescence anisotropy ((r infinity), which is proportional to the square of the lipid order parameter, was calculated from rs data, a significant increase in these parameters was also obtained. Furthermore, this decrease of the double bond index:saturated acid ratio of the membrane was associated with a parallel increase in Hill coefficients of the UDP-glucuronyl transferase that gradually lost the negative homotropic effect and cooperativity of UDP-glucuronic acid. The Hill coefficient varied from 0.39 to 0.98 during the 21-day period studied. Our observations indicate on one side that changes in the fat composition of the diet are accompanied by modifications in the lipid composition and fluidity of the microsomal membrane, and the apparent cooperativity of the enzyme. On the other side, they suggest that the evaluation of Hill coefficients of UDP-glucuronyl transferase might be used as a sensitive test to investigate conformational changes in the microsomal membrane of the liver.     

Effect of aflatoxin B1 on lipids of rat tissues.

The effect of aflatoxin B1 on lipids of liver, kidney, adipose and plasma of rats was studied. A single dose administration (6 mg/kg body weight) increased liver and kidney weights and their total lipids within 24 h. Increase in liver lipids was confined mainly to phospholipid and cholesterol, whereas triglycerides showed a significant decrease. Adipose tissue triglycerides were, however, increased. Plasma showed decreases in triglycerides, free fatty acids and cholesterol. Incorporation studies with palmitate-1-14C revealed increased incorporation in adipose tissue lipids and decreased incorporation in liver and plasma lipids, thereby indicating an increased synthesis of lipids in adipose. Their mobilization to plasma was, however, inhibited, hence the low levels of triglyceride in liver. But the adrenals showed hypo-activity upon aflatoxin B1 administration.     

7-Dehydrocholesterol 5,6 beta-oxide as a mechanism-based inhibitor of microsomal cholesterol oxide hydrolase

7-Dehydrocholesterol 5,6 beta-oxide covalently modifies and inactivates the rat liver microsomal enzyme cholesterol oxide hydrolase. The covalent modification is presumed to occur at the active site of the enzyme since 5,6 alpha-iminocholestanol, a potent competitive inhibitor of the enzyme, blocks incorporation of 3-[3H]-7-dehydrocholesterol 5,6 beta-oxide into the protein. Kinetics of the inactivation were measured both by following the loss of catalytic activity and by monitoring incorporation of 3-[3H]-7-dehydrocholesterol 5,6 beta-oxide into microsomal protein. Both the loss of catalytic activity and the incorporation of label followed first order kinetics. Linear plots of the reciprocal of the pseudo-first order rate constants for the loss of catalytic activity and for the incorporation of radioactivity versus reciprocal of inhibitor concentrations indicated saturation kinetics. The kinetic parameter kinac is found to be (2.83 +/- 0.43)10(-3) s-1 measured either by incorporation of tritium (300 mM potassium phosphate buffer, pH 8.0, 2.4 mg of microsomal protein/ml at 37 degrees C) or by the loss of catalytic activity (300 mM potassium phosphate buffer, pH 7.5, 0.99 mg of microsomal protein/ml at 37 degrees C). Unlike xenobiotic microsomal epoxide hydrolase (EC 3.3.2.3) which is not inactivated or inhibited by 7-dehydrocholesterol 5,6 beta-oxide, cholesterol oxide hydrolase appears to hydrolyze cholesterol oxides via a positively charged transition state.     

Lipid peroxidation contributes to age-related membrane rigidity

The aim of this work was to assess the relative contributions of lipid peroxidation and cholesterol content to the increase in membrane rigidity observed during senescence. Membrane fluidity was manipulated through exposure to peroxidized or cholesterol-loaded liposomes. Small unilamella liposomes were prepared and either peroxidized by Fe⁺⁺-ADP-ascorbic acid or loaded with cholesterol. After incorporation of the liposomes into rat liver microsomal membranes, membrane fluidity was quantitated by measuring changes in polarization. Membranes exhibited a greater sensitivity to peroxidation than cholesterol in that incorporation of peroxidized liposomes induced microsomal membrane rigidity substantially more than did cholesterol-loaded liposomes. Thus it is proposed, based on data from the present and earlier studies, that membrane fluidity can be modulated readily by lipid peroxidation of membrane phospholipids, irrespective of the influences of cholesterol. These results support the proposal that alterations of lipid structure are more potent and effective than compositional changes in cholesterol in inducing age-related increases in membrane rigidity.     

Use of fluorescence polarization to monitor intracellular membrane changes during temperature acclimation. Correlation with lipid compositional and ultrastructural changes.

Fluorescence polarization of 1,6-diphenylhexatriene (DPH) was used to study the effects of temperature acclimation on Tetrahymena membranes. The physical properties of membrane lipids were found to be highly dependent on cellular growth temperature. DPH polarization in lipids from three different membrane fractions correlated well with earlier freeze-fracture and electron spin resonance observations showing that membrane fluidity progressively decreases in the order microsomes greater than pellicles greater than cilia throughout a wide range of growth temperatures. Changes in membrane lipid fluidity following a shift from high to low growth temperatures proceed rapidly in the microsomes, whereas there is a pronounced lag in the changes of peripheral cell membrane lipids. These data support previous observations that adaptive changes in membrane fluidity proceed via lipid modifications in the endoplasmic reticulum, followed by dissemination of lipid components to other cell membranes. The rapid changes in polarization observed in the microsomal lipids following a temperature shift correspond closely with the time-dependent alterations in both lipid fatty acid composition and freeze-fracture patterns of membrane particle distribution, suggesting that, in the endoplasmic reticulum, lipid phase separation is the primary cause of membrane particle rearrangements.     

Vitamin A and microsomal membranes: the effect of retinol deficiency on lipid microviscosity and phospholipid turnover in rat liver microsomes

Studies with the use of the fluorescent probe pyrene revealed that vitamin A deficiency in maturing male rats results in the increased microviscosity of liver lipids. This effect seems to be due to changes in the lipid composition of microsomal membranes (increased cholesterol/phospholipid ratio and lowered polyunsaturated fatty acid content) as well as to the low level of retinol. Analysis of microsomal phospholipids labeled with [3H]palmitate and [14C]glycerol revealed that vitamin A deficiency accelerates the turnover of the glycerol skeleton but sharply decelerates that of fatty acid residues. It is concluded that the observed effect of retinol on the structural and functional properties of biological membranes is due to its ability to control the microviscosity and turnover of membrane lipids.     

Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration.

A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5'-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5'-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration.