Liver plasma membranes from essential fatty acid-deficient rats: Isolation,fatty acid composition,and activities of 5′-nucleotidase,ATPase and adenylate cyclase

To determine whether changes in unsaturation of fatty acids in rat liver plasma membranes might alter activities of membrane-associated enzymes, liver plasma membranes were prepared from rats fed purified diets lacking or supplemented with essential fatty acids. Two methods of membrane purification were used. A similar degree of purification was obtained with both methods for both depleted and control membranes, as indicated by marker enzyme purification. The proportion of essential fatty acids of the linoleate series was significantly lower in phospholipids from depleted rats. The specific activity of 5′-nucleotidase was lower, and the activity, $\text{V}$ and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for total $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ were higher in the depleted liver plasma membranes. Arrhenius plots of total ATPase activity showed a discontinuity at the same temperature for both the depleted and control membranes. Activity with the depleted membranes was higher at all temperatures tested. Supplementation of deficient rats with a source of essential fatty acids (corn oil) restored $\text{V}$ and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values to normal. Adenylate cyclase activity in the presence of fluoride, glucagon or glucagon plus GTP was significantly lower in the depleted plasma membranes.     

Phosphatidic acid phosphatase and phospholipase A activities in plasma membranes from fusing muscle cells

Plasma membranes from fusing embryonic muscle cells were assayed for phospholipase A activity to determine if this enzyme plays a role in cell fusion. The membranes were assayed under a variety of conditions with phosphatidylcholine as the substrate and no phospholipase A activity was found. The plasma membranes did contain a phosphatidic acid phosphatase which was optimally active in the presence of Triton X-100 and glycerol. The enzyme activity was constant from pH 5.2 to 7.0, and did not require divalent cations. Over 97% of the phosphatidic acid phosphatase activity was in the particulate fraction. The subcellular distribution of the phosphatidic acid phosphatase was the same as the distibutions of the plasma membrane markers, $\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase and the acetylcholine receptor, which indicates that this phosphatase is located exclusively in the plasma membranes. There was no detectable difference in the phosphatidic acid phosphatase activities of plasma membranes from fusing and non-fusing cells.     

Comparison of plasma membranes and endoplasmic reticulum fractions obtained from whole white adipose tissue and isolated adipocytes

The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and 5′-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome $\text{c}$ reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADHPH cytochrome $\text{c}$ reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome $\text{c}$ reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis.     

Plasma membranes from rat intestinal epithelial cells at different stages of maturation. I. Preparation and characterization of plasma membrane subfractions originating from crypt cells and from villous cells

To determine the mechanism of the maturation of the brush border membrane in intestinal epithelial cells, purification of the plasma membrane from undifferentiated rat crypt cells and of the basal-lateral membrane from villous cells has been performed. The method is based on density perturbation of the mitochondria to selectively disrupt their association with the membrane. With both cell populations, two membrane subfractions displaying the same respective density on sucrose gradient have been obtained with an overall yield of 15–20% and a 10-fold enrichment of the plasma membrane markers 5′-nucleotidase and $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$, ouabain-sensitive ATPase chosen to follow their purification. The four fractions constituted by sheets and apparently closed vesicles of various sizes. Each fraction was characterized by a distinct protein composition and different levels of enzyme activities. The cells, used for the preparation of the membranes, were isolated as a villus to crypt gradient. This separation and that of the membranes led to the conclusion that the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase is localized principally in the plasma membrane of all cells whatever their state of maturation, while 5′-nucleotidase is predominantly located in the basal-lateral membrane of the villous cells and may serve as a specific marker for the purification of this membrane. Finally it has been shown that aminopeptidase, disaccharidases and alkaline phosphatase do not appear simultaneously in the maturation process of the cells, alkaline phosphatase being absent from the crypt cells and aminopeptidase being the first to be synthesized. This enzyme seems to appear in the crypt cells membrane before being integrated into the mature brush border membrane.     

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes

EL4 cells were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membrane phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the $\text{P}$ value of both probes compared to the $\text{P}$ value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, $\text{P}$ values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in $\text{P}$ values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization for monitoring physical properties of membranes (such as ‘fluidity’) is discussed.     

Effects of insulin on the lipid structure of liver plasma membrane measured with fluorescence and ESR spectroscopic methods

Insulin increased the lipid order of rat and mouse liver plasma membrane domains sampled by the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in a concentration-dependent saturable manner. The ordering is half maximal at $\text{5.1 · 10}{}^{\mathrm{?11}}\text{M}$ and fully saturated at $\text{1.7 · 10}{}^{\mathrm{?10}}\text{M}$ insulin. Membranes prepared from obese hyperglycemic (ob / ob) mice demonstrated a right-shift in the dose-dependent ordering induced by insulin, such that ordering was half maximal at $\text{1.2 · 10}{}^{\mathrm{?10}}\text{M}$ and fully saturated at $\text{2.0 · 10}{}^{\mathrm{?10}}\text{M}$. Insulin also increased the order of rat liver plasma membranes labeled with the cis- and trans-parinaric acid methyl esters. The ordering caused by insulin as detected with cis methyl parinarate was complete within approx. 15 min. after hormone addition at 37°C, and the ordering was approximately double that observed with the trans isomer. Additional ESR experiments demonstrated that the addition of insulin increased the outer hyperfine splittings of spectra recorded from membranes labeled with the steroid-like spin labels, nitroxide cholestane and nitroxide androstane, but not the fatty acid spin probe, 5-nitroxide stearate. Studies utilizing model membrane systems strongly suggest that the 5-nitroxide stearate samples a cholesterol-poor domain of the membrane, while the steroid-like probes preferentially sample cholesterol-rich regions of the membrane. Finally, insulin-induced membrane ordering was dose-dependently inhibited by cytochalasin B in the range 1–50 μM. From these results, we conclude that (1) the ordering effect of insulin addition to isolated liver plasma membrane fractions occurs within the physiological range of hormone concentration, and the dose-response is right-shifted in membranes from ‘insulin resistant’ animals; (2) the relative responses of the fluorescent and spin probes suggest that the effects of insulin are confined to specific domains within the membrane matrix; and (3) the direct effects of insulin on the membranes may involve protein components having cytochalasin B binding sites.     

The relationship between plasma membrane lipid composition and physical-chemical properties. II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive (Na+ + K+)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant differences was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane 'fluidity' based on lipid modulation of biological membranes.     

Separation of plasma membrane markers by glycerol-induced blistering of muscle cells

Glycerol (50%, w/w) was found to cause blistering of chick primary myoblast and fibroblast plasma membranes and extensive blistering of 5–6-day-old-myotube plasma membranes in tissue culture. The tips of myoblasts and fibroblasts appeared to be the most sensitive portion of the plasma membrane to the blistering effect of glycerol. The glycerol-induced blistering of myotubes was reduced and delayed by brief EDTA pretreatment.Glycerol treatment (50, 15 and 8% sequentially) of myotubes was used to remove plasma membrane blisters and a plasma membrane-enriched fraction was isolated from these blisters using a modified Dextran T500-polyethylene-glycol 6000 aqueous two-phase polymer system. This fraction was found to be enriched 4.1-fold for 5′-nucleotidase activity, but not for other putative plasma membrane markers, ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity or $\text{α-[}{}^{125}\text{I}\text{]}\text{bungarotoxin}$ binding material. Autoradiographs of $\text{α-[}{}^{125}\text{I}\text{]}\text{bungarotoxin}$, glycerol-treated (50%, w/w) myotubes showed the plasma membrane blisters to be devoid of reduced silver grains.5′-Nucleotidase was shown to be an ectoenzyme on myoblasts and 5-day-old myotubes and the total cellular activity was present on the cell surface. During the period of myoblast fusion and myotube formation, cell surface activity decreased to a low level while total cellular activity was elevated.     

The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase

The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient.The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase in purified basolateral plasma membranes was 13-fold. F^?-activated adenyl cyclase co-purified with ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K⁺-stimulated phosphatase and 5′-nucleotidase also followed ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase during fractionation.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

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.     

The isolation and analysis of the luminal plasma membrane of calf urinary bladder epithelium

The luminal plasma membrane of calf urinary bladder epithelium (urothelium) has been isolated by a method designed to preserve enzymic activity as well as structural integrity. The yield was about 80 μg per calf bladder. Low levels of 5′ nucleotidase, Mg²⁺-ATPase and ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activities were found in the luminal membrane fraction. Cerebroside was the major lipid present and dodecyl sulphate gel electrophoresis revealed a complex protein and glycoprotein composition in the whole membrane. A membrane fraction consisting of only the plaque areas was shown to have a simpler protein composition with major polypeptides of apparent $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 12 000 and 22 000. These may associate to form a 30 000 apparent $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ complex which could represent the individual ‘particles’ of the dodecameric subunits seen by electron microscopy in the plaque regions.     

Isolation of plasma membrane vesicles,derived from transverse tubules,by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$, Mg²⁺-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca²⁺-stimulated, Mg²⁺-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell Cell segments in an amber low-speed ($\text{800 × g}$) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at $\text{100 000 × g}$ for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ ATPase compared with the other fractions, but it had essentially no Ca²⁺-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate.Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined by a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basement membrane, could not be detected in this fraction.     

Isolation of a rat liver plasma membrane fraction of probable canalicular origin Preparative technique,enzymatic profile,composition, and solute transport

A technique currently used for isolation of brush border membranes from renal and intestinal epithelium that involves vigorous tissue homogenization and sedimentation of non-luminal membranes in the presence of Mg²⁺ has been adapted to rat liver. Liver plasma membranes so prepared consisted almost exclusively of vesicles by electron microscopy, showed some contamination with endoplasmic reticulum and minimal contamination with mitochondria or Golgi by marker enzymes, were highly enriched in alkaline phosphatase, Mg²⁺-ATPase, and 5′-nucleotidase activity compared with homogenate, and showed little enrichment in (Na⁺,K⁺)-ATPase. Comparison of this enzymatic profile with cytochemical studies localizing (Na⁺,K⁺)-ATPase and alkaline phosphatase to the sinusoidal/lateral and canalicular membranes, respectively, suggested that these membranes were predominantly of canalicular origin. They had a lower ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ specific activity, lower lipid content, and higher cholesterol to phospholipid molar ratio than a conventional plasma membrane preparation believed to be enriched in canaliculi. Moreover, it was possible to measure movement of d-[³H]glucose into an osmotically sensitive space bounded by these membrane vesicles.     

Plasma membrane NADH dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species

Ca²⁺-dependent K⁺ transport and plasma membrane NADH dehydrogenase activities have been studied in several ‘high-K⁺’ (human, rabbit and guinea pig) and ‘low-K⁺’ (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca²⁺-dependent K⁺ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome $\text{c}$ reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca²⁺-dependent K⁺ channel activities in the species studied. Nor were any of the above activities correlated with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity.     

Isolation of a synaptic membrane fraction enriched in cholinergic receptors by controlled phospholipase A2 hydrolysis of synaptic membranes

A procedure is described for the isolation of synaptic membrane fragments that retain such functionally important proteins as acetylcholine receptors, acetylcholinesterase, 3′,5′-cyclic nucleotide phosphodiesterase, and $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The method is based on the observation, made in brain slices, that junctional membranes are more resistant to phospholipase A₂ attack than mitochondrial or plasma membranes. Hydrolysis by phospholipase A₂ was controlled by addition of fatty acid-free bovine serum albumin. The membrane fraction obtained represents approximately a 15-fold enrichment of the postsynaptic marker proteins muscarinic and nicotinic acetylcholine receptor and 3′,5′-cyclic nucleotide phosphodiesterase over an ordinary synaptic plasma membrane preparation, and is devoid of mitochondrial and microsomal contaminations. The membranes appear on the electron micrographs as rigid fragments (average length 2500–4000Å), which do not form vesicles.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg²⁺, the plasma membrane preparation exhibits a Ca²⁺-dependent ATPase activity of 2 μmol P_i per h per mg protein. It is suggested that this $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ activity is related to the measured Ca²⁺ transport which was characterized by $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for ATP and Ca²⁺ of $\text{44 ± 9 μ}\text{M}$ and $\text{0.25 ± 0.10 μ}\text{M}$, respectively. Phosphorylation of plasma membranes with [γ-³²P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca²⁺-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and from the catalytic subunit of $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca²⁺-pump in plasma membranes isolated from nucleated cells.     

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.     

Fractionation of membrane vesicles. II. A method for separation of membrane vesicles bearing different enzymes by free-flow electrophoresis

Free-flow electrophoresis was used to subfractionate membrane vesicles from calf thymocyte plasma membranes. The fractionation resulted in a separation of vesicle populations bearing four different enzymes: alkaline nitrophenylphosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1), γ-glutamyltransferase (EC 2.3.2.2), $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) and acyl-CoA:lysophosphatidylcholine acyltransferase ($\text{acyl-CoA:1-acylglycero-3-phosphocholine}\text{-O-}\text{acyltransferase}$, EC 2.3.1.23). The specific content of cholesterol and total phospholipid coincided with the distribution of membrane-bound protein. However, vesicles migrating towards the cathode had a higher molar ratio of cholesterol to phospholipid (0.75) compared to those migrating to the anode (0.55). Sodium dodecyl sulphate-gel electrophoresis of pooled vesicle fractions also demonstrates distinct differences in their protein pattern. Electron-micrographic thin sections show that the vesicle populations have a similar morphology and size distribution.These results are discussed in terms of heterogeneity of the original thymocytes, contamination with intracellular membranes and a heterogeneous structure of the plasma membrane.