Isolation from neutrophil membranes of a complex containing active NADPH oxidase and cytochrome b-245

The lipid composition and fluidity of basolateral membranes prepared from the mucosa of the proximal, middle and distal thirds of the rat small intestine were determined. Fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and a series of anthroyloxy fatty acid derivatives, is decreased in the distal third as compared to the proximal segments. This pattern is similar to that described previously for microvillus membranes. The decrease in fluidity of the distal as compared to the proximal membranes results from an increase in cholesterol content, cholesterol/phospholipid molar ratio and degree of saturation of the fatty acid residues. In the middle and distal thirds of the gut, the degree of saturation of the fatty acid residues is higher in microvillus as compared to basolateral membranes, accounting in part for the characteristically lower fluidity of the luminal membranes. The specific activity of the basolateral membrane (Na+ + K+)-dependent adenosine triphosphatase is significantly lower in the distal as compared to the proximal and middle thirds of the intestinal mucosa. Studies of the binding of [3H]ouabain indicate that this pattern results from fewer enzyme sites in the distal membranes.     

Regional differences in the lipid composition and fluidity of rat colonic brush-border membranes

The lipid composition and fluidity of brush-border membranes prepared from rat proximal and distal colonocytes were determined. Fluidity, as assessed by steady-state fluorescence polarization techniques using the fluorophores 1,6-diphenyl-1,3,5-hexatriene, DL-2(9-anthroyl)stearic acid and DL-12(9-anthroyl)stearic acid, was decreased in distal compared to proximal plasma membranes. This pattern was similar to that previously described for both antipodal plasma membranes in rat enterocytes of the small intestine. The decrease in fluidity of the distal as compared to the proximal membranes resulted from an increase in cholesterol content, cholesterol/phospholipid molar ratio and degree of saturation of the fatty acid residues in the distal membranes. The specific activities of total alkaline phosphatase and cysteine-sensitive alkaline phosphatase, enzymes previously shown to be functionally dependent on the physical state of the colonic brush-border membrane's lipid, were also significantly lower in distal as compared to proximal clonic plasma membranes. These studies, therefore, demonstrate that differences in the lipid fluidity, lipid composition and certain enzymatic activities exist in brush-border membranes prepared from rat proximal and distal colonocytes. The regional variation in rat colonic luminal membrane lipid fluidity and composition may, at least partially, be responsible for differences in these enzymatic activities as well as in sodium and water absorption along the length of this organ.     

Isolation and lipid composition of apical and basolateral membranes of colonic segments of guinea Pig

In adapting several methods of membrane isolation we established a successful way to purify apical and basolateral membranes of guinea pig colon in a parallel procedure. The conventional purification control by marker enzymes was applied. In addition, luminal membrane proteins were stained with Texas Red. Apical and basolateral enterocyte membranes were enriched 10- to 12-fold by differential precipitation and via a continuous sorbitol gradient. The membrane fractions were examined with regard to their phospholipid (PL) and fatty acid patterns and to their cholesterol content. Fluorescence polarization studies were carried out using 1,6-diphenyl-1,3, 5-hexatrien. Remarkable differences in the fatty acid pattern of the proximal and the distal colon were seen. Due to a higher content of oleic acid the saturation index of the apical membranes of the proximal colon is lower compared to that of the apical membranes of the distal colon (0.34 +/- 0.03 vs 0.42 +/- 0.05). The cholesterol content of the apical membranes of the proximal colon is markedly higher than that of the apical membranes of the distal colon (3.42 +/- 0.14 vs 1.88 +/- 0.29 mol/mol PL). There are no differences in the fluidity of these apical membranes. We assume a balancing mechanism between the cholesterol content and the amount of saturated PL-fatty acids.     

Lipid fluidity and composition of intestinal microvillus membranes isolated from rats of different ages

The lipid composition and fluidity of microvillus (luminal) membranes isolated from the small intestines of Fisher 344 rats aged 6, 17, and 117 weeks were compared. Lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, was significantly greater in rats aged 6 weeks as compared to 17 or 117 weeks. A lipid thermotropic transition was observed at 17.5 +/- 1.3 degrees C in the membranes of the youngest group, approx. 5-6 degrees C lower than that of the older animals. The differences in lipid composition which account for the higher fluidity of the youngest preparations include a decreased cholesterol/phospholipid molar ratio in both the proximal and distal halves of the small intestine and, in the proximal half alone, increases in the lipid/protein ratio and double bond index. The foregoing reduction in cholesterol/phospholipid ratio derives mainly from a higher content of total phospholipid, and the increment in double bond index results from an increase in arachidonic acid residues. The results demonstrate an age-dependent decrease in fluidity of intestinal microvillus membranes in the early post-weaning period in the rat. This pattern was unlike that of the microvillus membrane p-nitrophenylphosphatase, whose specific activity declined progressively in the older age groups.     

Variations in dietary triacylglycerol saturation alter the lipid composition and fluidity of rat intestinal plasma membranes

Rats were maintained on nutritionally complete diets enriched in unsaturated (corn oil) or saturated (butter fat) triacylglycerols. After 6 weeks, significant differences in the lipid composition and fluidity of a number of intestinal membranes were observed. The corn oil diet (enriched mainly in linoleic acid) increased the overall unsaturation of the acyl chains and enhanced the lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, of enterocyte microvillus and basolateral membranes and of colonocyte basolateral membranes. Concomitantly, the cholesterol content and the cholesterol/phospholipid molar ratio were increased in the microvillus but not in the basolateral membranes. The increased cholesterol in ileal microvillus membranes can result from enhanced cellular biosynthesis, since ileal slices from rats fed the unsaturated diet incorporated [14C]octanoate more rapidly into digitonin-precipitable sterol. Increased fluidity of the enterocyte microvillus and basolateral membranes, respectively, enhanced the enzyme specific activities of p-nitrophenylphosphatase and (Na+ + K+)-dependent adenosine triphosphatase. The results indicate that the lipid composition, fluidity and enzyme activities of intestinal plasma membranes can be altered by dietary means. Moreover, rat enterocytes possess regulatory mechanisms which modulate the cholesterol content of the microvillus membranes so as to mitigate changes in lipid fluidity.     

Isolation and characterization of brush-border membrane from trout intestine. Regional differences

The isolation of brush-border membranes from trout enterocytes is described for both middle and posterior intestine. Both procedures are based on differential centrifugations combined with calcium precipitation. Classical marker enzymes are quantified and indicate a valuable purification of the membranes (13-18-fold). No difference appears when comparing the relative amounts of phospholipids, cholesterol and proteins in microvillus membranes isolated from either middle or posterior intestine. In contrast, the membranes isolated from middle intestine are more unsaturated than those from the posterior one, and their sphingomyelin/phosphatidylcholine ratio is lower. These differences are reflected by fluorescence anisotropy studies with diphenylhexatriene as lipid fluorophore which indicate a higher fluidity of the microvillus membranes from the middle intestine as compared with those from the posterior intestine. These results point out the importance of the fatty acyl chains and that of the relative amounts of phosphatidylcholine and sphingomyelin in controlling the fluidity of biological membranes in relation with their transport properties.     

Renal cortical brush-border and basolateral membranes: Cholesterol and phospholipid Composition and relative turnover

Summary A new procedure for the rapid isolation of renal cortical brush-border and basolateral membranes from the same homogenate is described. Brush-border membranes isolated using Mg²⁺-EGTA precipitation were enriched 18-fold for leucine aminopeptidase and had a recovery of 32.5%. Basolateral membrane fractions were isolated using a discontinuous sucrose gradient and showed an enrichment of 10.7-fold and recovery of 12.8% using (Na⁺, K⁺)-ATPase as a marker enzyme. Lipid analysis using two-dimensional TLC separation of phospholipids and gas liquid chromatography for cholesterol showed marked differences in the lipid composition of the brush-border and basolateral membranes. The brush-border membrane had increased sphingomyelin, phosphatidylserine, ethanolamine plasmalogens, and an increased cholesterol-to-phospholipid and sphingomyelin-to-phosphatidylcholine ratio compared to the basolateral membrane. The relative turnover of total membrane and individual phospholipid species using a double isotope ratio method was carried out. Phospholipids were labeled with either phosphorus 32 and 33 or acetate (³H, 1-¹⁴C). The relative turnover of phospholipid species and cholesterol differed strikingly. Phosphatidylcholine showed a high turnover, phosphatidylethanolamine and phosphatidylinositol had intermediate values and sphingomyelin, phosphatidylserine and cholesterol had low relative turnover rates. The order of phospholipid class relative turnover was independent of the labeled precursor used. The brush-border membrane had a significantly reduced relative turnover rate for total membrane phospholipids, sphingomyelin and cholesterol compared to the basolateral membrane. These data show marked differences in the lipid composition and relative turnover rates of the phospholipid species of the brush-border and basolateral membranes. They provide a biochemical basis for the recently reported differences in brush-border and basolateral membrane fluidity and suggest independent cellular regulation of brush-border and basolateral membrane lipids.     

Ontogeny of proximal colon basolateral membrane lipid composition and fluidity in the rabbit.

Basolateral membranes from rabbit proximal colon were prepared from isolated colonocytes throughout postnatal maturation, using a modification of published techniques. In suckling (14-20 day) and post-weaning/mature (35-49 day) animals, membranes were purified approx. 10-fold, based upon the enrichment of ouabain-sensitive, sodium-potassium dependent adenosine triphosphatase activity. Membrane lipid analyses demonstrated age-dependent increases in total cholesterol and the cholesterol/phospholipid molar ratio, as well as decreases in phosphatidylethanolamine content and the fatty acid unsaturation index. Fluidity of basolateral membranes and membrane liposomes, determined from fluorescence anisotropy measurements using the lipid probes 1,6-diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid, demonstrated significant, ontogenic decreases in fluidity; and, additional studies showed that fluidity changes occurred early in the weaning period (by day 24 postnatally). Arrhenius plots of liposome anisotropies suggested a bilayer lipid thermotropic transition temperature of 22 degrees C in sucklings 26 degrees C in mature rabbits. These findings demonstrate that ontogeny of colonic basolateral membranes is associated with significant modulations in lipid composition and fluidity.     

Cholesterol affects physical properties and (Na+,K+)-ATPase in basolateral membranes of renal and intestinal epithelia from thermally acclimated rainbow trout

Previous work has shown that cholesterol levels are modulated in plasma membranes from some but not all tissues of poikilotherms over the course of temperature change. To gain a better understanding of tissue and membrane domain-specific cholesterol function during thermal adaptation we examined effects of cholesterol on membrane physical properties and (Na⁺,K⁺)-ATPase in native and cholesterol-enriched basolateral membranes from kidney and intestine of thermally acclimated trout (Oncorhynchus mykiss). Membrane order (as indicated by fluorescence depolarization studies) is increased, whereas its thermal sensitivity is decreased by elevated cholesterol levels in mem branes with relatively low endogenous amounts of cholesterol (intestinal membranes and renal membranes from cold-acclimated fish). Thermal sensitivities of membrane order in kidney are 1.5-fold higher in native compared with cholesterol-enriched basolateral membranes. For renal plasma membranes, (Na⁺,K⁺)- ATPase activity is lowest near the transition between native and surpraphysiological cholesterol levels. Endogenous cholesterol levels (relative to phospholipid contents) in intestinal basolateral membranes from cold-acclimated fish vary more than 1.5-fold; membranes with cholesterol/phospholipid molar ratios of 0.3 have activities of (Na⁺,K⁺)-ATPase that are twofold lower than native membranes having a ratio of 0.2. These results suggests that maintenance of cholesterol levels in intestinal basolateral membranes during thermal acclimation may ensure sufficient activity of (Na⁺,K⁺)-ATPase. Membrane function in kidney, with its high native cholesterol content, is less likely to be affected by temperature change. Accepted: 21 January 1997     

Lipid dynamics and protein-lipid interactions in rat colonic epithelial cell basolateral membranes

Lipid dynamics and lipid-protein interactions were examined in basolateral membranes prepared from rat proximal and distal colonic epithelial cells. The results demonstrate that: (1) these membranes have a high lipid fluidity, as assessed by steady-state fluorescence polarization studies using seven fluorescent probes; (2) lipid compositional differences exist between these membranes but their fluidity is similar; (3) fluorescence polarizations studies, using diphenylhexatriene (DPH), detect a thermotropic transition at 22–23°C in each membrane; (4) several membrane protein activities, including adenylate cyclase and sodium-potassium dependent adenosine triphosphatase ($\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$) appear to be functionally dependent on the physical state of the proximal basolateral membrane's lipid.     

Basolateral membrane lipid dynamics alter Na-K ATPase activity in rabbit small intestine.

The role of basolateral membrane fluidity in regulating Na-K ATPase activity along the crypt-villus axis in rabbit distal small intestine was assessed. Basolateral membranes were prepared from isolated villus and crypt enterocytes at 24- to 28-fold enhancement. Villus basolateral membranes were significantly (p < 0.001) more fluid than crypt basolateral membranes as measured by 1,6-diphenyl-1,3,5-hexatriene. No difference was seen between the two groups as measured by either 2-(9-anthroyloxy)-stearic fatty acid or 16-(9-anthroyloxy)-palmitic acid. Fluidity alterations were accompanied by an increased phospholipid content in villus membranes, which resulted in a decreased cholesterol:phospholipid ratio and an increased lipid:protein molar ratio. Na-K ATPase activity was significantly (p < 0.01) greater in villus basolateral membranes than in crypt membranes, and demonstrated a greater sensitivity to ouabain inhibition. Ouabain inhibition curves calculated from villus data fit well (p < 0.001) with a two binding site model, with a high affinity (Ki 16 nM) and a low affinity (Ki 4.2 microM) ouabain binding site. In crypt basolateral membranes, only a low affinity site was apparent (Ki 3.0 microM). Fluidizing crypt basolateral membranes in vitro with benzyl alcohol to levels seen in villus basolateral membranes resulted in the appearance of a high affinity ouabain binding site (Ki 110 nM) and an increased sensitivity of Na-K ATPase to ouabain inhibition. The fluidization of villus basolateral membranes eliminated the binding associated with the high affinity site. Treatment with methanol, as a control, did not alter Na-K ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of cholesterol in the colonic epithelium of the guinea pig: regional differences and functional implications

Summary It is generally accepted that variations in membrane cholesterol content affect the fluidity of the bilayer, thus altering its permeability. In the biological membranes, in physiological conditions, a high cholesterol content rigidifies the bilayer decreasing its permeability, a lower cholesterol content induces the opposite effect by increasing the permeability. Since differences in the epithelial permeability for short chain fatty acids have previously been demonstrated in the proximal and distal colon of the guinea pig, these two regions were investigated to establish whether differences in membrane cholesterol content of the absorbing cells can be demonstrated. Freeze-fracture replicas of filipin-treated colonic tissue were used. The results show that in the proximal colon the density of filipin cholesterol complexes located on the luminal plasma membrane of the columnar absorbing cells was significantly higher (about twice) than in the distal colon. Therefore the lower amount of cholesterol present in the membrane of the absorbing cells in the distal colon indicates a greater fluidity of the membranes of the epithelial cells in this region. Such fluidity could be correlated to the higher absorption rates of shortchain fatty acids characteristic of this region.     

Correction of abnormal lipid fluidity and composition of rat ileal microvillus membranes in chronic streptozotocin-induced diabetes by insulin therapy

Diabetes was induced in rats by administration of streptozotocin. After 90-120 days, one group of chronic diabetic animals was treated with insulin for chronic diabetic animals was treated with insulin for 10 days. The lipid fluidity and composition of microvillus membranes prepared from ileal enterocytes of control, diabetic, and insulin-treated diabetic animals were determined. Lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the probes 1,6-diphenyl-1,3,5-hexatriene, DL-2-(9-anthroyl)stearic acid and DL-12-(9-anthroyl)stearic acid, was decreased in membranes of diabetic animals compared to membranes of control and insulin-treated diabetic membranes. The differences in fluidity resulted from an increased cholesterol content and cholesterol/phospholipid molar ratio in membranes of diabetic animals. The activities of sucrase and alkaline phosphatase were also found to be higher in membranes of diabetic animals. Insulin treatment, however, failed to significantly influence the enzymatic activities of these membranes. These studies, therefore, demonstrate that alterations in the lipid fluidity, lipid composition, and certain enzymatic activities exist in microvillus membranes of enterocytes prepared from chronic streptozotocin-induced diabetic rats. Administration of insulin for 10 days to these animals restored membrane fluidity and lipid composition but not enzymatic activities to control membrane levels.     

Rabbit distal colon epithelium: I. Isolation and characterization of basolateral plasma membrane vesicles from surface and crypt cells

Summary A method has been developed for the simultaneous isolation of basolateral plasma membrane vesicles from surface and crypt cells of rabbit distal colon epithelium by sequential use of differential sedimentation, isopycnic centrifugation and Ficoll 400 barrier centrifugation. The protein yield was high (total 0.81 mg/g mucosa) and surface and crypt cell-derived basolateral membrane fractions have been purified 34- and 9-fold with respect to the homogenate. The pattern of marker enzyme enrichments revealed only minor contamination by subcellular organelles. Latency of ouabain-sensitive (Na⁺, K⁺)-ATPase activity prior and after trypsin treatment of membranes indicated a vesicle configuration of sealed right side-out: sealed inside-out: leaky of approximately 211. The presence of sealed vesicles was also evident from the osmotic sensitivity of thed-[1-¹⁴C] mannitol equilibrium space determined with either fraction. Although considerably different in protein profile, surface and crypt basolateral membranes were similar in cholesterol to phospholipid molar ratio and membrane fluidity as determined by steady-state fluorescence polarization.Stopped-flow light scattering experiments revealed a rather low water permeability of the membranes with a permeability coefficient of 6 m/sec at 35°C, which is one order of magnitude lower than reported for small intestinal plasma membranes. Both membrane fractions have been shown to effectively generate outward uphill potassium ion gradients, a process that is energized by ATP and inhibited by the membrane-permeant cardiacglycoside digitoxin. These characteristics are consistent with the activity of a (Na⁺, K⁺) pump operating in inside-out vesicles.     

Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells

The lipid fluidity in purified plasma membranes (PM) of murine leukemic GRSL cells, as measured by fluorescence polarization, is much higher than in PM of normal thymocytes. This was found to be due to relatively low contents of cholesterol and sphingomyelin and a high amount of unsaturated fatty acyl chains, especially linoleic acid, in the phospholipids. PM from GRSL cells contain markedly more phosphatidylethanolamine than those from thymocytes. For both GRSL cells and thymocytes the detailed lipid composition of isolated PM was compared with that of the corresponding shed extracellular membranes (ECM), which were isolated from the ascites fluid and from thymus cell suspensions, respectively. The somewhat decreased lipid fluidity of thymocyte ECM as compared to their PM, can be ascribed to the increased cholesterol/phospholipid molar ratio (0.88 vs. 0.74). No other major differences were found between the lipid composition of these membranes. In contrast, significant differences were found between PM and ECM from GRSL cells. In this system a much lower lipid fluidity of the shed ECM was found, due to the much increased cholesterol/phospholipid molar ratio (3.5-fold) and sphingomyelin (9-fold) content, as compared to the PM. Further, the ECM contain relatively more lysophosphatidylethanolamine and less phosphatidylcholine and -inositol. ECM contain a higher amount of polyunsaturated fatty acids, especially in the phosphatidylethanolamine and lysophosphatidylethanolamine classes. On the other hand, the fatty acids of phosphatidylcholine and lysophosphatidylcholine are more saturated than in PM. In particular, ECM of GRSL cells contain less oleic and linoleic acid residues and more arachidonic acid and 22:polyunsaturated fatty acid residues than PM. The possible relevance of these differences with respect to the mechanism of shedding of vesicles from the cell surface, is discussed.     

Localization of Na+, K+-ATPase to the inside of the basolateral cell membranes of epithelial cells of proximal and distal tubules in rabbit kidney

Summary Cysteine-sensitive alkaline phosphatase and/or ouabain-sensitive Na⁺, K⁺-ATPase were studied by ultrastructure cytochemistry in epithelial cells of proximal and distal kidney tubules. Alkaline phosphatase reactivity was confined to the surface of the microvillous luminal cell membrane of proximal tubule cells, whereas distal tubules and collecting ducts were unreactive. The Na⁺, K⁺-ATPase reactivity was localized evenly along the cytoplasmic side of the basolateral cell membrane of cells of proximal and distal tubules and in collecting ducts. In the proximal tubules, where the activity was strongest, the Na⁺, K⁺-ATPase deposits were also found in the 10–50 nm gap between the cell membrane and the cisternae of tubulo-cisternal endoplasmic reticulum (TER) underlying a major part of the basolateral cell membrane. The restriction of Na⁺, K⁺-ATPase sites, which are involved in extrusion of Na⁺ from the cell, to a narrow cytoplasmic compartment located between the cell membrane and the cisternae of TER, is consistent with a transport role for the TER.     

Isolation and partial characterization of basolateral membranes from rat proximal colonic epithelial cells

The isolation of basolateral membranes from rat proximal colonic epithelial cells is described. Cells were harvested using a technique combining chelation of divalent cations with mechanical dissociation. After homogenization, differential centrifugation yielded a 'crude' membrane fraction which was further purified using sucrose density centrifugation. The final membrane fraction was enriched 10-14-fold over homogenate in ouabain-sensitive sodium-potassium dependent adenosine triphosphatase and ouabain-sensitive potassium-dependent phosphatase specific activities. SDS-polyacrylamide gel electrophoresis of this membrane revealed at least 18 protein bands with molecular weights of 14600-200000. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, free cholesterol and fatty acids were the major lipid components of this membrane. The predominant fatty acids were palmitic (16:0), oleic (18:1), stearic (18:0) and linoleic (18:2) acid. Membranes and their liposomes were studied, using the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH), by steady-state fluorescence polarization. The fluorescence anisotropy was greater in the intact membranes compared to their liposomes, indicating greater fluidity in the liposomes. Compositional studies suggested that the high fluidity of this membrane was due to its low ratios of protein/lipid (w/w), cholesterol/phospholipid (mol/mol), and sphingomyelin/phosphatidylcholine (mol/mol).     

Permeability properties of apical and basolateral membranes of the guinea pig caecal and colonic epithelia for short-chain fatty acids

Unidirectional fluxes of short-chain fatty acids (SCFA) indicated marked segmental differences in the permeability of apical and basolateral membranes. The aim of our study was to prove these differences in membrane permeability for a lipid-soluble substance and to understand the factors affecting these differences. Apical and basolateral membrane fractions from guinea pig caecal and colonic epithelia were isolated. Membrane compositions were determined and the permeability of membrane vesicles for the protonated SCFA was measured in a stopped-flow device. Native vesicles from apical membranes of the caecum and proximal colon have a much lower permeability than the corresponding vesicles from the basolateral membranes. For the distal colon, membrane permeabilities of native apical and basolateral vesicles are similar. In vesicles prepared from lipid extracts, the permeabilities for the protonated SCFA are negatively correlated to cholesterol content, whereas no such correlation was observed in native vesicles. Our findings confirm that the apical membrane in the caecum and proximal colon of guinea pig is an effective barrier against a rapid diffusion of small lipid-soluble substances such as SCFAH. Besides cholesterol and membrane proteins, there are further factors that contribute to this barrier property.     

Cholesterol modulates alkaline phosphatase activity of rat intestinal microvillus membranes

Experiments were conducted, using a nonspecific lipid transfer protein, to vary the cholesterol/phospholipid molar ratio of rat proximal small intestinal microvillus membranes in order to assess the possible role of cholesterol in modulating enzymatic activities of this plasma membrane. Cholesterol/phospholipid molar ratios from 0.71 to 1.30 were produced from a normal value of 1.05 by incubation with the transfer protein and an excess of either phosphatidylcholine or cholesterol/phosphatidylcholine liposomes for 60 min at 37 degrees C. Cholesterol loading or depletion of the membranes was accompanied by a decrease or increase, respectively, in their lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the lipid-soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Increasing the cholesterol/phospholipid molar ratio also decreased alkaline phosphatase specific activity by approximately 20-30%, whereas decreasing this ratio increased this enzymatic activity by 20-30%. Sucrase, maltase, and lactase specific activities were not affected in these same preparations. Since the changes in alkaline phosphatase activity could be secondary to alterations in fluidity, cholesterol, or both, additional experiments were performed using benzyl alcohol, a known fluidizer. Benzyl alcohol (25 mM) restored the fluidity of cholesterol-enriched preparations to control levels, did not change the cholesterol/phospholipid molar ratio, and failed to alter alkaline phosphatase activity. These findings, therefore, indicate that alterations in the cholesterol content and cholesterol/phospholipid molar ratio of microvillus membranes can modulate alkaline phosphatase but not sucrase, maltase, or lactase activities. Moreover, membrane fluidity does not appear to be an important physiological regulator of these enzymatic activities.     

Membrane lipid fatty acids and regulation of membrane-bound enzymes. Allosteric behaviour of erythrocyte Mg2+-ATPase, (Na+ + K2+)-ATPase and acetylcholineasterase from rats fed different fat-supplemented diets

Studies were carried out to determine the Hill coefficients for the inhibition by F^? of the erythrocyte membrane-bound Mg²⁺-ATPase, $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase from rats fed with seven different diets. Five groups were fed with different natural fats or oil supplements, one with a hydrogenated fat supplement and the other with fat-free diet. The responses of the red cell fatty acids to dietary fats were recorded. The value of $\text{n}$ for the inhibition by F^? of the three enzymes revealed a particular and different behaviour in each group. Correlations between the fatty acid compositions of erythrocyte membranes and cooperativity of each enzyme were calculated. The results indicate that neither the essential fatty acid family nor the non-essential ones are particularly involved in the allosteric phenomena. The increase of the double bond index/saturation ratio of fatty acids, which is taken as indicative of membrane fluidity, was accompanied in an inverse manner by changes in allosteric transitions of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and acetylcholinesterase, whereas the Mg²⁺-ATPase was not dependent on this ratio. Diminution of membrane fluidity, carried out by in vitro increase of its cholesterol content, yields confirmatory results of this regulatory mechanism since the value of $\text{n}$ for acetylcholinesterase shifted as predicted.These facts indicate that the membrane fluidity is a physiological regulator for the allosteric behaviour of the membrane-bound enzymes and that each enzyme exhibits a particular behaviour in this phenomenon.