Showing papers on "Membrane lipids published in 1975"

Physical properties of membrane lipids: biological relevance and regulation.

Abstract: Introduction..................................................................... 232 Pertinent Properties of the Cell Envelope ......... ............................. 233 E. coli Fatty Acid Auxotrophs .............. ................................... 233 Specific inhibitors of Fatty Acid Synthesis ......... ............................ 234 LIPID BILAYER PHASE PROPERTIES ........ ............................ 234 Empirical Rules Deduced from Model Systems ........ ......................... 234 Physical Properties of the E. coli Membrane Lipids ....... ..................... 236 Phase transitions............................................................ 236 (i) X-ray diffraction ......................................................... 236 (ii) DSC ..................................................................... 237 (iii) Fluorescent probes ..................................................... 237 (iv) Spin labeling .............. ............................................. 237 Phase separations. (i) Physical studies ......... .............................. 237 (ii) Freeze-fracture electron microscopy ...................................... 239 Lateral movement of lipids ............... ................................... 240 CORRELATIONS BETWEEN MEMBRANE-ASSOCIATED PHYSIOLOGY AND LIPID PHYSICAL PROPERTIES ........ ............................ 240 General Physiology ............................................................ 240 Lactose Transport System ................ ..................................... 240 Relevant properties of lactose transport system ....... ....................... 240 Transport system induction and lipid synthesis ....... ........................ 241 Transport and lipid phase changes ........... ................................ 241 Other Transport Systems .................. .................................... 243 Sugar transport systems ................ ..................................... 243 Amino acid transport systems ............. .................................. 243 Other Physiological Processes ................................................ 243 Lipid requirement for alkaline phosphatase derepression ...... .............. 243 Chemotaxis ............. . .................................................... 244 Initiation of DNA synthesis ............... ................................... 244 Methylgalactoside permease induction ......... .............................. 244 Cell integrity ................. ............................................... 244 Enzymatic activity ........................................................... 244 Requirement for Lipids with Specific Physical Properties ...... ................ 245 Requirement for membrane liquidity ......................................... 245 Minimum UFA content...................................................... 246 Minimum saturated fatty acid content ......... ............................... 246 Significance of temperature-induced alterations in fatty acids ..... ........... 247 REGULATION OF LIPID PHYSICAL PROPERTIES ........................... 247 Possible Lipid Alterations Producing Altered Phase Transitions ..... ........... 247 Sites of Control ................................................................ 247 In Vitro Studies ............................................................... 247 (i) Acyltransferase specificity ............. ................................... 248 (ii) Fatty acid synthetase ................ .................................... 249 In Vivo Studies ................................................................ 249 (i) Determination of fatty acid chain length ........ ........................... 249 (ii) Regulation of the saturated-to-unsaturated fatty acid ratio ..... ........... 250 (iii) Temperature control of fatty acid composition ....... ..................... 250 CONCLUSION.................................................................. 251 LITERATURE CITED .............. ............................................ 252

Pores formed in lipid bilayer membranes by nystatin, Differences in its one-sided and two-sided action.

Abstract: Nystatin and amphotericin B induce a cation-selective conductance when added to one side of a lipid bilayer membrane and an anion-selective conductance when added to both sides. The concentrations of antibiotic required for the one-sided action are comparable to those employed on plasma membranes and are considerably larger than those required for the two-sided action. We propose that the two-sided effect results from the formation of aqueous pores formed by the hydrogen bonding in the middle of the bilayer of two "half pores," whereas the one-sided effect results from the half pores alone. We discuss, in terms of the flexibility of bilayer structure and its thickness, how it is possible to have conducting half pores and "complete pores" in the same membrane. The role of sterol (cholesterol and ergosterol) in pore formation is also examined.

Drug-induced phase change in bilayer as possible mode of action of membrane expanding drugs.

Abstract: MANY small molecules modulate membrane functions. Substances such as neurotransmitters interact directly with specific protein receptor sites, whereas others such as anaesthetics, interact with membrane lipids or with hydrophobic regions of membrane. This is consistent with a correlation of their activity with their lipid solubility and lipid–water partition coefficient1. Several lipid-soluble drugs are antihaemolytic2, which correlates not only with their pharmacological activity but also with their ability to expand the lipid bilayer of a biomembrane3. Although expansion of bilayers implies a drug-induced reorganisation of lipids, the mechanism involved remains to be established. We now have evidence that the lipid-soluble drugs may induce a transition of lipid acyl chains from organised gel to randomised liquid crystalline phase. Such drug-induced changes in the lipid phase in a bilayer may affect the function of various membrane-bound proteins4.

Thermotropic lipid clustering in tetrahymena membranes

Abstract: The effect of temperature on the core structure of endoplasmic reticulum membranes has been visualized directly in cells of the poikilothermic eukaryote Tetrahymena pyriformis by freeze-etch electron microscopy. Moreover, the effect of temperature on the smooth microsomal membrane vesicles isolated from these cells, as well as on the extracted membrane lipids, has been examined by fluorescence probing, electron spin resonance, proton nuclear magnetic resonance, and calorimetry. Freeze-etch electron microscopy of T. pyriformis cells, equilibrated at different temperatures between 28 and 5 degrees, reveals the emergence of smooth areas on the fracture faces of endoplasmic reticulum membranes at temperatures below similar to 17 degrees. In this temperature range, we also find discontinuities in the glucose 6-phosphatase activity, in the fluorescence intensity of 8-anilino-1-naphthalensulfonate, in the partition of 4-doxyldecane, and in the separation of the outer hyperfine extrema of 5-doxylstearic acid in the microsomal membranes. These membranes apparently contain at least two lipid environments of different fluidity as indicated by the 12-doxylstearic acid spin-label. Proton nuclear magnetic resonance of the extracted membrane lipids indicates an abrupt change of the fatty acid chain mobilities at temperatures below similar to 17 degrees. This, however, is not due to a true thermal liquid crystalline in equilibrium crystalline phase transition. Calorimetric measurements also support this conclusion. The thermotropic alterations observed within the membranes are interpreted to be due primarily to a clustering of "rigid" liquid crystalline lipid environments which exclude membrane-intercalating proteins.

Drug resistance and membrane alteration in mutants of mammalian cells.

Abstract: Independent colchicine-resistant (CHR) mutants of Chinese hamster ovary cells displaying reduced permeability to colchicine have been isolated. A distinguishing feature of these membrane-altered mutants is their pleiotropic cross-resistance to a variety of unrelated compounds. Genetic characterization of the CHR lines indicate that colchicine resistance and cross-resistance to other drugs are of a dominant nature in somatic cell hybrids. Revertants of CHR have been isolated which display decreased resistance to colchicine and a corresponding decrease in resistance to other drugs. These results strongly suggest that colchicine resistance and the pleiotropic cross-resistance are the result of the same mutation(s). Biochemical studies indicate that although colchicine is transported into our cells by passive diffusion, no major alterations in the membrane lipids could be detected in mutant cells. However, there appears to be an energy-dependent process in these cells which actively maintains a permeability barrier against colchicine and other drugs. The CHR cells might be altered in this process. A new glycoprotein has been identified in mutant cell membranes which is not present in parental cells, and is greatly reduced in revertant cells. A model for colchicine-resistance is proposed which suggests that certain membrane proteins such as the new glycoprotein of CHR cells, are modulators of membrane fluidity (mmf proteins) whose molecular conformation regulates membrane permeability to a variety of compounds and that the CHR mutants are altered in their mmf proteins. The possible importance of the CHR cells as models for investigating aspects of chemotherapy related to drug resistance is discussed.

Relationships between the Transition of the Physical Phase of Membrane Lipids and Photosynthetic Parameters in Anacystis nidulans and Lettuce and Spinach Chloroplasts.

Abstract: The transition of the physical phase of lipids in membrane fragments of a blue-green alga Anacystis nidulans was studied by a spin labeling technique. The maximum hyperfine splitting of the electron spin resonance spectrum of the N-oxyl-4', 4'-dimethyloxazolidine derivative of 5-ketostearic acid plotted against the reciprocal of the absolute temperature gave a discontinuity point that was characteristic of a transition of the physical phase of the hydrocarbon region of membrane lipids. The phase transition appeared at approximately 13 or 24 C in the organisms grown at 28 or 38 C, respectively.The temperature dependence curve of chlorophyll a fluorescence in intact cells, membrane fragments, and extracted lipids of Anacystis cells suspended in a buffer solution showed that the fluorescence yield became maximum near the phase transition temperatures. These findings suggest that chlorophyll a in the thylakoid membrane works as a native fluorescence probe for the detection of phase transition.The temperature dependence of photosynthetic electron transport reactions was studied by measuring the oxidoreductive reactions of P700 and by measuring O(2) evolution. Each of the Arrhenius plots of the reaction rates was composed of two straight lines with a break near the phase transition temperatures. The activation energy was always lower above than below the transition temperatures. It is proposed to explain these phenomena that a reaction involving plastoquinone is influenced by the physical state of membrane lipids.The shift between the pigment state 1 and state 2 measured by fluorescence transients also showed a characteristic break in the Arrhenius plots near the phase transition temperatures; below the transition temperatures the shift almost disappeared. This suggests that the configurational change of the thylakoid membrane related to the state 1 and state 2 shift is dependent on the physical state of membrane lipids. In the chloroplasts of lettuce and spinach, on the other hand, there was no break in the Arrhenius plot of the electron transport reactions or of Mg(2+)-induced changes of chlorophyll a fluorescence.It is suggested that the transitions of the hyperfine splitting of the ESR signal, electron transport, and the configurational change, as well as the appearance of the maximum of chlorophyll a fluorescence, in the thylakoid membranes of Anacystis nidulans are all related to the transition of the physical phase of membrane lipids between the liquid crystalline state and the mixed liquid crystal-solid state.

Biogenesis of membrane lipids: mutants of Escherichia coli with temperature-sensitive phosphatidylserine decarboxylase

Abstract: Phosphatidylserine decarboxylase catalyzes the last step in the pathway leading to phosphatidylethanolamine, the principal membrane lipid of E. coli. Mutants of E. coli have now been isolated in which this enzyme is theramolabile. The structural gene for phosphatidylserine decarboxylase (psd gene) is closely linked to the pur A locus at about 83 min on the standard map of the E. coli chromosome. When a mutant with thermolabile decarboxylase is incubated at 42 degrees, growth ceases, but only after a substantial fraction (20-40%) of the total phospholipid of the cell has been replaced by phosphatidylserine. Examination of such mutants with altered content of phospholipids may shed light on the role of specific phospholipids in membrane function.

Fluorescence polarization and viscosities of membrane lipids of 3T3 cells.

Abstract: Studies of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in membranes of normal, transformed, and revertant 3T3 cells allowed estimation of microviscosities (eta) of the lipid bilayers of these cells. Fluorescence polarization values observed with normal cells were significantly lower than those observed with cells transformed either by polyoma virus or by simian virus 40. The values of fluorescence polarization obtained with revertant Py6R1 cells were lower than those obtained with the normal cells. The calculated microviscosities (eta) show a 50% increase upon transformation. Possible correlations between the bilayer viscosity and the mobility of the receptors in the membrane are discussed.

A membrane-associated lipomannan in micrococci.

Abstract: Membranes of Micrococcus lysodeikticus, Micrococcus flavus and Micrococcus sodonensis contain acidic lipomannans. Lipoteichoic acids could not be detected in these organisms, and the suggestion that they are substituted for by the lipomannans is strengthened by the chemical and physical resemblances between the two polymers. The mannans contain glycerol, ester-linked fatty acids and mono-esterified succinic acid residues, giving them both hydrophobic and charged properties. The M. lysodeikticus mannan has a chain of about 60 hexose units with two branch points, and is joined at its reducing end to the 1-position of a glycerol moiety bearing two fatty acid residues. Succinic acid on the mannan enables it to bind Mg2+ efficiently, and the polymer is firmly associated with the cytoplasmic membrane, probably by intercalation of its fatty acids with those of the membrane lipids.

Regulation of Membrane Flexibility in Human Erythrocytes.

Abstract: We have used spin-labels to detect prostaglan- din E induced changes in erythrocyte membranes. The ob- served changes in spin-label resonance spectra can be mim- icked in erythrocyte ghosts by loading them with CAMP or cGMP. These changes can also be observed by adding ei- ther of these cyclic nucleotides to intact cells. This entry of cyclic nucleotides into intact cells is blocked by an inhibitor The morphology, chemical composition, and dynamical properties of the plasma membranes of most biological cells are doubtlessly subject to continuous regulation. The pres- ent paper is concerned with the membrane of the mature human erythrocyte. Recent studies by Allen and Rasmus- sen (1971) and by Kury et al. (1974) have shown that the rheological properties of erythrocytes at high hematocrit are affected by physiological concentrations of prostaglan- dins and epinephrine. These studies suggest but do not prove that the flexibility of an isolated erythrocyte can be controlled by these substances. Studies by Allen and Valeri (1974) indicate further that erythrocyte morphology is reg- ulated by low concentrations of the prostaglandins PGE1,' or PGE2, which can decrease, or increase, respectively, the internal volume of the cell by a small amount (-3%). The paramagnetic resonance spectra of fatty acid spin-labels bound nonspecifically to these membranes show small but reproducible changes on the addition of low concentrations ( 10-'o-10-'2 M; of the order of few molecules per cell) of the prostaglandins PGEl and PGE2 (Kury et al., 1974), and also show reproducible changes on the addition of physio- logical concentrations of adrenaline and carbamyl choline (Huestis and McConnell, 1974). The spin-label concentra- tion and resulting spectra suggest that the associated changes in membrane structure arise from small changes of the anion channel. We suggest that the observed changes in paramagnetic resonance spectra are due to changes in lipid "fluidity" that are brought about by changes in the biochemical state of membrane-associated proteins (such as spectrin) and in the direct or indirect biophysical interac- tions of these proteins with membrane lipids. throughout much of the membrane, rather than large changes localized to small regions of the membrane. The resonance spectra are a measure of the flexibility of the spin-label fatty acid chains in the bilayer region of the membrane. It is interesting that substances that increase the apparent single cell flexibility (e.g., PGE,) also increase this fatty acid chain flexibility, and vice versa (PGE2 and epinephrine) (Kury et al., 1974). Recent studies of erythro- cyte ghosts using circular dichroism also show changes in the presence of prostaglandins (Meyers and Swislocki, 1974).

Studies on mixed monolayers of phospholipids and fusogenic lipids.

Abstract: 1. The behaviour of mixed monolayers of 14 different lipids with preparations of erythrocyte lipids, purified natural and synthetic phospholipids, cholesterol and galactosylceramide was investigated. 2. The mean areas occupied per molecule in mixed films containing lipids that are fusogenic for hen erythrocytes were compared with those for corresponding films containing lipids that are inactive as fusogens. 3. Fusogenic lipids were found to exhibit interactions, which were not shown by non-fusogenic lipids, in mixed monolayers with several species of phospholipid, particularly those containing a choline head group. 4. Heterogeneity in the hydrophobic chains of phosphatidylcholine, their degree of unsaturation and the presence of cholesterol had little effect on the interaction of phosphatidylcholine with fusogenic lipids. 5. Fusogenic lipids showed little specific interaction with natural or synthetic preparations of phosphatidylethanolamine. 6. The possible significance of these observations in relation to the action of fusogenic lipids on biological membranes is discussed in the light of the asymmetrical distribution of phospholipids in erythrocyte membranes.

Cell surface lipids and adhesion. III. The effects on cell adhesion of changes in plasmalemmal lipids

Abstract: The two preceding papers of this series suggest that the state of the plasmalemmal lipids affects cell adhesion. Plasmalemmal composition was altered by the experimental incorporation of fatty acids into R1 and R2 positions in the phosphatidyl components of the cell surface. In this paper we report that: (1) If the incorporation is of long chain length fatty acids (saturated) cell adhesion rises. (2) If the incorporation is of unsaturated fatty acids cell adhesion falls as the unsaturation increases. (3) Incorporation has to be extensive to produce a large change in adhesion. (4) Changes in adhesion parallel the plasmalemmal incorporation but do not follow the total cell incorporation. Item (4) argues that it is plasmalemmal and not other membrane lipids that are involved in cell adhesion. Item (3) suggests that bulk membrane properties and not some very specific grouping are involved in the effects of lipids on adhesion. The similar extents of incorporation of the various different fatty acids and the negligible amounts of lysophospholipids in the membranes of cells that have incorporated fatty acids argue that the effects are not due to differential accumulations of these lysolipids when incubations are done with different fatty acids. The changes in adhesion cannot be accounted for by changes in surface charge density since the electrophoretic mobility of the cells is unchanged by these incubations. It is suggested that these effects on adhesion due to changes in plasmalemmal lipids can be explained either in terms of the action of intermembrane van der Waals--London (electrodynamic) forces in cell adhesion or of changes in surface fluidity. These alternatives are discussed.

The relation between lipid mobility and the specific hormone binding of thyroid membranes.

Abstract: 1. The specific binding of thyroid-stimulating hormone to isolated human thyroid membranes was examined under a variety of conditions. 2. In phosphate-saline buffer (in the presence of 0.14 M-NaCl) on increasing the temperature the binding of the hormone is increased, the plots of bound/free hormone against temperature showing a distinct break around 30 degrees C. 3. Detailed analysis showed that the increased binding is associated with an increase in the number of binding sites. 4. The motional characteristics of three membrane-bound fluorescent probes, 2-(9-anthroyl)palmitic acid, 12-(9-anthryl)stearic acid and N-1-naphthyl-N-phenylamine, were also examined as a function of temperature by measuring both fluorescence polarizations and lifetimes. 5. The results indicated that the 'fluidity' of membrane lipids also increased with temperature. The temperature-dependence of this property also shows a change at about 30 degrees C. 6. Bivalent cations decreased both membrane fluidity and hormone binding. 7. Similar correlations were found between the binding of adrenocorticotrophic hormone and the fluidity of the plasma membranes obtained from adrenal-cortical cells, with the discontinuity occurring in this case at 23 degrees C. 8. The possibility of lipid mobility being important in controlling hormone-receptor function is discussed.

Inhibition of leukocyte locomotion and chemotaxis by lipid-specific bacterial toxins

Abstract: SINCE a number of cytolytic bacterial protein toxins have specific actions on individual membrane lipids, these toxins, used at sub-toxic doses, should be useful for exploring the role of lipids in membrane-activated cell functions. I have suggested1–3 that leukocyte chemotactic factors initiate a locomotor response by virtue of their ability to penetrate the hydrophobic interior of the lipid bilayei of the cell membrane, and thus possibly to bring about changes in the intracytoplasmic concentration of divalent cations and activation of contractile systems in the cytoplasm. If this is so, it may be possible to modify locomotor and chemotactic responses in leukocytes using lipid-specific toxins. The oxygen-labile lysins such as streptolysin 0 or Clostridium perfringens θ toxin show a specific affinity for cell membrane cholesterol. Bacterial phospholipases C attack the polar heads of a number of membrane phospholipids4. Among those phospholipases, sphingomyelinase C, the β toxin of Staphylococcus aureus, shows a high substrate specificity for sphingomyelin5. Here I show that the locomotor responses of leukocytes to chemoattractants are modified by these toxins and that neutrophils show a different pattern of response from monocytes. The locomotor response of human blood monocytes is inhibited by Cl. perfringens phospholipase C (PLC) and by sphingomyelinase C but not by Cl. perfringens θ toxin. In contrast, the response of human blood neutrophils is inhibited by θ toxin but not by phospholipases C.

Molecular Dynamics in Biological Membranes

Abstract: 1 Introduction: Cell Structure and Function.- 2 Constituents of Biological Membranes.- Membrane isolation.- Membrane composition.- Membrane lipids.- Membrane proteins.- 3 Structure of Membranes and Serum Lipoprotein Complexes.- Soap molecules in aqueous solution.- Biological membranes.- Serum lipoprotein complexes.- Fluidity of membrane constituents.- Membrane action of anesthetics.- 4 Biological Consequences of Membrane Fluidity and Fusion.- Membrane biogenesis.- Intercellular junction formation.- Myogenesis.- Phagocytosis.- Secretion.- 5 Transmembrane Solute Transport Mechanisms.- Ion-transporting antibiotics.- Group translocation-the bacterial phosphotransferase system.- Group translocation-intestinal disaccharidases.- Active transport mechanisms in intestinal epithelial cells.- Energy interconversion and active transport in bacteria.- 6 Sensory Perception I: Chemoreception.- Bacterial chemoreception.- Chemotactic responses of solitary eukaryotic cells.- The chemical regulation of insect behavior.- The chemical senses of mammals.- 7 Sensory Perception II: Transmission Mechanisms.- The resting membrane potential and action potentials.- Excitability-inducing material.- Ion-conducting channels in excitable membranes.- Bioelectric control of ciliary activity in Paramecium.- Photoreception in the rod cell of the mammalian eye.- Bacterial photoreception and transmission.- 8 Hormonal Regulation of Cellular Metabolism.- The insulin receptor.- Possible mechanisms of hormone regulation.- Regulation of bacterial metabolism.- Regulation of cellular cyclic AMP levels.- 9 Cell Recognition.- Adhesion of bacteriophage to bacterial hosts.- Heterotypic adhesion-sexual agglutination in yeast.- Homotypic adhesion-sponge aggregation.- Animal cell adhesion and malignancy.- 10 Role of the Plasma Membrane in Growth Regulation and Neoplasia.- Growth of normal and transformed animal cells.- Positive growth control.- Negative growth control.- Changes in membrane structure and function associated with transformation.- Microbial systems for studying the molecular basis of neoplastic transformation.- Mammalian growth regulation and membrane biology.- Index 125.

Membrane-bound ribosomes of myeloma cells. I. Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of the ribosomes.

Abstract: A cell fractionation procedure is described which allowed, by use of MOPC 21 (P3K) mouse plasmocytoma cells in culture, the separation of the cytoplasmic free and membrane-bound ribosomes in fractions devoid of mutual cross-contamination, and in which the polyribosomal structure was entirely preserved. This was achieved by sedimentation on a discontinuous sucrose density gradient in which the two ribosome populations migrate in opposite directions. A variety of controls (electron microscopy, labeling of membrane lipids, further repurification of the isolated fractions) provided no evidence of cross-contamination of these populations. However, when an excess of free 60S or 40S subunits, labeled with a different isotope, was added to the cytoplasmic extract before fractionation, the possibility of a small amount of trapping and/or adsorption of free ribosomal particles by the membrane fraction was detected, especially in the case of the 60S subunits; this could be entirely prevented by the use of sucrose gradients containing 0.15 M KC1. EDTA treatment of the membrane fraction detached almost all the 40S subunits, and about 70% of the 60S subunits. 0.5 M KC1 detached only 10% of the ribosomal particles, which consist of the native 60S subunits and the monoribosomes, i.e. the bound particles inactive in protein synthesis. Analysis in CsC1 buoyant density gradients of the free and membrane-bound polyribosomes and of their derived 60S and 40S ribosomal subunits showed that the free and membrane-bound ribosomal particles have similar densities.

Membrane-lipid unsaturation and mitochondrial function in Saacharomyces cerevisiae.

Abstract: The lipid composition of yeast cells was manipulated by the use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. There was a 2-3-fold decrease in the concentration of cytochromes a+a3 when the unsaturated fatty acid content of the cells was decreased from 60-70% of the total fatty acid to 20-30%. The amounts of cytochromes b and c were also decreased under these conditions, but to a lesser extent. Further lipid depletion, to proportions of less than 20% unsaturated fatty acid, led to a dramatic decrease in the content of all cytochromes, particularly cytochromes a+a3. The ATPase (adenosine triphosphatase), succinate oxidase and NADH oxidase activities of the isolated mitochondria also varied with the degree of unsaturation of the membrane lipids. The lower the percentage of unsaturated fatty acid, the lower was the enzymic activity. Inhibition of mitochondrial ATPase by oligomycin, on the other hand, was not markedly influenced by the membrane-lipid unsaturation. Npn-linear Arrenius plots of mitochondrial membrane-bound enzymes showed transition temperatures that were dependent on the degree of membrane-lipid unsaturation. The greater the degree of lipid unsaturation, the lower was the transition temperature. It was concluded that the degree of unsaturation of the membrane lipids plays an important role in determining the properties of mitochondrial membrane-bound enzymes.

A Fluorescent Probe Study of the Lipid Mobility of Membranes Containing Sodium- and Potassium-Dependent Adenosine Triphosphatase

Abstract: The temperature-activity relationship of a membrane (Na+ + K+)-ATPase preparation [Mg2+-dependent, ouabain-sensitive, (Na+ + K+)-activated ATP phosphohydrolase, EC 3.6.1.3] obtained from sheep kidney cortex and medulla was determined and found to be very similar to that previously reported for preparations of this enzyme from either rabbit kidney or ox brain. These temperature-activity relationships can be shown as Arrhenius plots which characteristically are nonlinear and have transition temperatures near 22°. Two noncovalently bound fluorescent probes, 12-(9-anthroyl)stearic acid (12-AS) and N-phenyl-1-naphthylamine (NPN), were used to label the hydrophobic core of the partially purified membranes rich in (Na+ + K+)-ATPase. The fluorescence polarization of these probes was determined between 10° and 40°. The rotational relaxation times (ρ) for each probe were then calculated, and secondary plots of reciprocal relaxation time vs. reciprocal temperature were constructed. The plots for membranes labeled with 12-AS and NPN were nonlinear and showed transition temperatures near 22°, in good agreement with the transition temperature of the hydrolytic activity of the enzyme. A similar transition temperature was detected by right-angle light scattering of an unlabeled microsomal preparation of (Na+ + K+)-ATPase and of an aqueous suspension of liposomes made from a total lipid extract of the enzyme-containing membranes, thus excluding any direct effect of addition of the fluorescent probes to the membranes. The transition temperatures observed under all experimental conditions were very similar. We conclude that the nonlinear temperature-activity relationship of (Na+ + K+-ATPase and the nonlinear fluorescence polarization—temperature profile both arise from a temperature-dependent change in the molecular mobility of the membrane lipids in the immediate environment of the probes and the "active center" of the (Na+ + K+)-ATPase subunits. These changes illustrate the strong cooperative effect between the physical state of the membrane lipids and the functional state of the enzyme protein in this particulate membrane enzyme system, and suggest a powerful modulating effect of membrane lipids in regulating enzyme activity, or drug-receptor interactions more generally.

Changes in composition, biosynthesis, and physical state of membrane lipids occurring upon aging of Mycoplasma hominis cultures.

Abstract: During the progression of Mycoplasma hominis cultures from the early logarithmic phase to the stationary phase of growth, the total phospholipid content of the cell membranes decreased. Measurement of the amount of the various phospholipids during the growth cycle showed that a decrease in the phosphatidylglycerol (PG) content, accompanied by an increase in the phosphatidic acid content, occurred upon aging of the culture. Pulse labeling experiments revealed that the PG, once formed, is relatively stable, undergoing no detectable turnover in growing cultures of M. hominis. No changes in the fatty acid composition of the membrane phospholipids were observed on aging of the culture, with palmitic acid predominating throughout the growth cycle. The preferential incorporation of palmitate into the phospholipid fraction is apparently caused by the higher activity of the membrane-bound acyl-coenzyme A (CoA):alpha-glycerophosphate transacylase with palmityl-CoA rather than with oleyl-CoA as substrate. The activity of the soluble acyl-CoA synthetase was the same whether palmitate or oleate served as substate. M. hominis membrane preparations contained a PG-synthetase system catalyzing the incorporation of L-alpha-glycerol-3-phosphate into PG. The activity of the PG synthetase system was markedly dependent on the age of the culture, being highest in cells from the early exponential phase of growth while declining sharply thereafter, and thus probably responsible, in part, for the decrease in PG content upon aging of the cells. Electron paramagnetic resonance spectra of a spin-labeled fatty acid incorporated in M. hominis membranes revealed a marked decrease in the freedom of motion of the spin label on aging of the culture. It is proposed that this decrease is due primarily to the decrease in the lipid-to-protein ratio of the membranes and has a marked effect on the activity of membrane-associated enzymes and transport systems.

Liver cell plasma membrane lipids and the origin of biliary phospholipid.

Abstract: The liver cell plasma membranes of fed male Wistar rats were separated into a fraction rich in bile canaliculi and the remainder of the plasma membrane. Electron-microscopically, the bile canalicular fraction consisted almost exclusively of intact bile canaliculi with thier contiguous membranes. The remaining plasma membrane fraction consisted primarily of vesicles and sheets of membranes essentially free from the bile canaliculi. The bile canalicular membrane fraction contained relatively more total lipid, cholesterol, and phospholipid, and relatively less protein. Although the phospholipid composition of the two fractions was the same, the specific activity of the bile canalicular membrane phosholipids, up to 12 h following in vivo administration of [2-3H]glycerol, was always significantly greater than that of the remaining plasma membranes, and showed a biphasic response not found in the latter. The specific activity of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membranes rose to a peak within 40 min after administration of the label, fell sharply and then rose to a second peak after 120 min. The specific activity of the sphingomyelin and phosphatidylserine plus phosphatidylinositol of the bile canalicular membranes and of all the phospholipids of the remaining plasma membranes diphasic pattern but increased steadily to reach a maximum at 120 min. The specific activity of biliary phosphatidylcholine followed a pattern identical to that of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membrane fraction. These results show that the average rate of turnover of phospholipid in the bile canalicular membranes is considerably greater than that in the remaining plasma membrane and other cell membrane fractions; they indicate that the phospholipid of the bile canalicular membranes exists in two or more pools, turning over a different rates; and they support the concept that biliary phospholipid is derived from the bile canalicular membrane. The results also suggest that bile canalicular phospholipid may be derived from two different sources, in contrast to the remainong plasma membrane.

Reactions of fluorescent probes with normal and chemically modified myelin basic protein and proteolipid. Comparisons with myelin.

Abstract: Basic (encephalitogenic) protein and water-soluble proteolipid apoprotein isolated from bovine brain myelin bind 8-anilino-1-naphthalenesulfonate and 2-p-toluidinylnaphthalene-6-sulfonate with resulting enhancement of dye fluorescence and a blue-shift of the emission spectrum. The dyes had a higher affinity and quantum yield, when bound to the proteolipid (Kans=2.3x10--6,=0.67) than to the basic protein (Kans=3.3x10--5,=0.40). From the efficiency of radiationless energy transfer from trytophan to bound ANS the intramolecular distances were calculated to be 17 and 27 A for the proteolipid and basic protein, respectively. Unlike myelin, incubation with proteolytic enzymes (e.g., Pronase and trypsin) abolished fluorescence enhancement of ANS or TNS by the extracted proteins. In contrast to myelin, the fluorescence of solutions of fluorescent probes plus proteolipid was reduced by Ca-2+,not affected by La-3+, local anesthetics, or polymyxin B, and only slightly increased by low pH or blockade of free carboxyl groups. The reactions of the basic protein were similar under these conditions except for a two- to threefold increase in dye binding in the presence of La-3+, or after blockade of carboxyl groups. N-Bromosuccinimide oxidation of tryptophan groups nearly abolished native protein fluorescence, but did not affect dye binding. However, alkylation of tryptophan groups of both proteins by 2-hydroxy (or methoxy)-5-nitrobenzyl bromide reduced the of bound ANS (excited at 380 nm) to 0.15 normal. The same effect was observed with human serum albumin. The fluorescence emission of ANS bound to myelin was not affected by alkylation of membrane tryptophan groups with the Koshland reagents, except for abolition of energy transfer from tryptophan to bound dye molecules. This suggests that dye binding to protein is negligible in the intact membrane. Proteolipid incorporated into lipid vesicles containing phosphatidylserine did not bind ANS or TNS unless Ca-2+, La-3+, polymyxin B, or local anesthetics were added to reduce the net negative surface potential of the lipid membranes. However, binding to protein in the lipid-protein vesicles remained less than for soluble protein. Basic protein or bovine serum albumin dye binding sites remained accessible after equilibration of these proteins with the same lipid vesicles. It is proposed that in the intact myelin membrane the proteolipid is probably strongly associated with specific anionic membrane lipids (i.e., phosphatidylserine), and most likely deeply embedded within the lipid hydrocarbon matrix of the myelin membrane. Also, in the intact myelin membrane the fluorescent probes are associated primarily, if not solely with the membrane lipids as indicated by the binding data. This is particularly the case for TNS where the total number of myelin binding sites is three to four times the potential protein binding sites.

Glucose transport in Acholeplasma laidlawii B: dependence on the fluidity and physical state of membrane lipids.

Abstract: The uptake of D-glucose by Acholeplasma laidlawii B occurs via a mediated transport process, as shown by the following observations: (i) glucose permeates A laidlawii B cells at a rate at least 100 times greater than would be expected if its entry occurred only by simple passive diffusion; (ii) the apparent activation energy for glucose uptake in A laidlawii is significantly lower than that expected and observed for the passive permeation of this sugar; (iii) glucose uptake appears to be a saturable process; (iv) glucose uptake can be completely inhibited by low concentrations of phloretin and phlorizin; and (v) glucose uptake is markedly inhibited at temperatures above 45 C, whereas the passive entry of erythritol continues to increase logarithmically until at least 60 C The metabolism of D-glucose by this organism is rapid and, at low glucose concentrations, the intracellular radioactivity derived from D-[14-C]glucose is at any given time a reflection of the net effect of glucose transport, glucose metabolism, and loss from the cell of radioactive metabolic products Care must thus be taken when attempting to determine the rate of glucose transport by measuring the accumulation by the cells of the total radioactivity derived from D-[14-C]glucose The rate of uptake of D-glucose by A laidlawii B cells is markedly dependent on the fatty acid composition and cholesterol content of the plasma membrane and exhibits a direct dependence on the fluidity of the membrane lipids as measured by their reversible, thermotropic gel to liquie-crystalline phase transition temperatures In contrast to the transport rates, the apparent activation energy for glucose uptake above the phase transition temperature is not dependent on membrane lipid composition At the temperature range within the membrane lipid phase transition region, the apparent activation energy of glucose uptake is different from the activation energy observed at temperatures above the phase transition This may reflect the superimposed operation within the phase transition region of more than one temperature-dependent process