Solubility properties of alkaline phosphatase from matrix vesicles

Alkaline phosphatase has been extracted from matrix vesicles of a calcifying cartilage with 0.15 M KCl, 0.4 M guanidinium chloride and 0.05 M deoxycholate/50% butanol mixture. The catalytic properties of the three extracts have been compared. Although the highest amount of enzyme activity is extracted with the latter reagent (55%), some of it is also extracted with KCl (11%) and guanidinium (7%). By submitting isolated matrix vesicles to a short time sonication the distribution pattern of the alkaline phosphatase activity in the extracts is clearly modified, as the amount extracted with KCl increases from 14 to 50% and the portion extracted with deoxycholate decreases from 55 to 27% of the total enzyme activity of matrix vesicles. The enzymatic preparations were comparable on the basis of specific activities, affinity for the substrates (p-nitrophenylphosphate, ATP), thermostability, sensitivity to inhibitors and activators. By electrofocusing a value of pI = 4.15 was found for the alkaline phosphatase of matrix vesicles independently of the extraction medium. These results contradict the concept that alkaline phosphatase is exclusively an intrinsic membrane protein.     

Interaction of cholesterol with photoactivable phospholipids in sonicated vesicles

Photoactivable phospholipids containing either α-diazo-β-trifluoropropionyloxy or m-diazirinophenoxyl groups in the ω-positions of sn-2 fatty acyl chains were synthesized and incorporated into sonicated vesicles containing 33 mol% of cholesterol. Photolysis of the vesicles at 350 nm produced covalent cross-links between the synthetic phospholipids and cholesterol. The cross-linked products obtained using [¹⁴C]cholesterol were characterized by their chromatographic behavior, cleavage on phospholipase A₂ treatment, base-catalyzed transesterification and mass spectral measurements. The cross-linking was shown not to involve the 3-β-hydroxyl group of cholesterol, and it was concluded that the reactive carbene intermediates formed from the photolabels inserted into the hydrocarbon skeleton of cholesterol in the bilayer. The extent of cross-linking obtained was comparable to that observed previously using phospholipids alone, indicating that no lateral phase separation occurred. The present approach is promising for further precise studies of the molecular interactions between cholesterol and phospholipids in biological membranes.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles

We have investigated the contribution of various phospholipids to membrane fusion induced by divalent cations. Fusion was followed by means of a new fluorescence assay monitoring the mixing of internal aqueous contents of large (0.1 μm diameter) unilamellar liposomes. The rate and extent of fusion induced by Ca²⁺ in mixed phosphatidylserine/phosphatidylcholine vesicles were lower compared to those in pure phosphatidylserine vesicles. The presence of 50% phosphatidylcholine completely inhibited fusion, although the vesicles aggregated upon Ca²⁺ addition. When phosphatidylserine was mixed with phosphatidylethanolamine, however, rapid fusion could be induced by Ca²⁺ even in mixtures that contained only 25% phosphatidylserine. Phosphatidylethanolamine also facilitated fusion by Mg²⁺ which could not fuse pure phosphatidylserine vesicles. In phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine mixtures, in which the phosphatidylcholine content was kept at 25%, phosphatidylethanolamine could not substitute for phosphatidylserine, and the fusogenic capacity of Mg²⁺ was abolished by the presence of merely 10% phosphatidylcholine. The initial rate of release of vesicle contents was slower than the rate of fusion in all the mixtures used. The presence of phosphate effected a considerable decrease in the threshold concentration of Ca²⁺ and also enhanced     

The effect of surface curvature on the head-group structure and phase transition properties of phospholipid bilayer vesicles

Proton nuclear magnetic resonance spectra at 360 MHz of small sonicated distearoyl phosphatidylcholine vesicles show easily distinguishable resonances due to choline N-methyl head-group protons located in the inner and outer bilayer halves. A study of the chemical shift of these resonances as a function of temperature reveals that the splitting between them increases below the phase transition. This occurs as a result of an upfield shift of the inner layer resonance at the phase transition. Consideration of the possible causes of this effect results in the conclusion that, at the phase transition, there is a change in the organization of the inner layer head-groups which does not occur for the outer layer head-groups.     

Temperature acclimation and phospholipid phase transition in hypothalamic membrane phospholipids of garden lizard, Calotes versicolor

Garden lizards, Calotes versicolor, were acclimated to three different temperatures, i.e., 16°C, 26°C and 36°C for a period of 30 days in ‘walk-in-environmental chambers’. The phospholipid profile and fatty acid pattern were analysed in the hypothalamus and brain of the acclimated animals. Hypothalamic and brain membrane phospholipids were prepared and their phase-transition temperatures were recorded using differential scanning calorimetry. Acclimation temperature, phospholipid composition, fatty acids of these phospholipids and the physical state and fluidity of the specific model membranes of hypothalamus (and brain) are intimately inter-related. Evidence is presented for the first time to show a possible correlation between acclimation temperature and phase-transition temperature of hypothalamic phospholipid membrane. A direct physico-chemical basis is suggested for the thermoregulatory process of hypothalamus leading to a better understanding of our knowledge on the origin of thermoregulation.     

Cation transport properties of a synthetic Ca2+-selective peptide ionophore in phospholipid and sarcoplasmic reticulum vesicles

Transport by the synthetic cyclic peptide ionophore CYCLEX-2E (Deber, C.M., Young, M.E.M., and Tom-Kun, J. (1980) Biochemistry 19, 6194–6198), which in contrast to Ca²⁺ ionophore A23187 contains no ionizable protons, has been studied with respect to Ca²⁺ and Na⁺ transport, and the involvement of exchanged, or counter-transported ions during the transport process. CYCLEX-2E was found to equilibrate Na⁺ and Ca²⁺ gradients across phospholipid vesicle membranes. Experiments using the indicator dye Arsenazo III established that calcium ions were indeed reaching the aqueous intravesicular compartments. Absence of metal cations in the external buffer slowed, but did not eliminate, the efflux of Ca²⁺ from phosphatidylcholine vesicles. As an example of its activity in a biological membrane, CYCLEX-2E was shown to be capable of producing Ca²⁺ efflux from sarcoplasmic reticulum vesicles which had been loaded with Ca²⁺ in an ATP-dependent manner. The overall results suggest that in transport by synthetic peptide ionophores typified by CYCLEX-2E, electroneutrality is achieved either through (a) peptide-mediated compensating (but not coupled) fluxes of other cations, or where this is not an option, by (b) transmembrane diffusion of permeant ions such as H⁺, OH^?, or Cl^?.     

Fusogenic properties of reconstituted hybrid vesicles containing Sendai and influenza envelope glycoproteins: fluorescence dequenching and fluorescence microscopy studies

Co-reconstitution of influenza and Sendai virus phospholipids and glycoproteins resulted in the formation of membrane vesicles containing the envelope glycoproteins from both viruses within the same membrane. Reconstituted influenza-Sendai hybrids (RISH) were able to lyse human erythrocytes and fuse with their membranes or with living cultured cells at pH 5.0 as well as at pH 7.4, thus exhibiting the fusogenic properties of both viruses. This was also inferred from experiments showing that the fusogenic activity of RISH was inhibited by anti-influenza as well as by anti-Sendai virus antibodies. Fusion of FISH and of reconstituted influenza (RIVE) or reconstituted Sendai virus envelopes (RSVE) with recipient membranes was determined by the use of fluorescently labeled envelopes and fluorescence dequenching methods. Observations with the fluorescence microscope were used to study localization of fused reconstituted envelopes within living cells. Incubation of RISH and RSVE with living cells at pH 7.4 resulted in the appearance of fluorescence rings around the cell plasma membranes and of intracellular distinct fluorescent spots indicating fusion with cell plasma membranes and with membranes of endocytic vesicles, respectively. The fluorescence microscopy observations clearly showed that RIVE failed to fuse, at pH 7.4, with cultured cell plasma membranes, but fused with membranes of endocytic vesicles.     

Transfer properties of the bovine brain phospholipid transfer protein. Effect of charged phospholipids and of phosphatidylcholine fatty acid composition

The monolayer technique has been used to study the transfer of [¹⁴C]phosphatidylinositol from the monolayer to phosphatidylcholine vesicles. An equivalent transfer rate was found for egg phosphatidylcholine, dioleoylphosphatidylcholine, dielaidoylphosphatidylcholine and dipalmitoylphosphatidylcholine. A reduced transfer rate was found for a shorter-chain derivative, dimyristoylphosphatidylcholine, and for species with two polyunsaturated fatty acid chains such as dilinoleoylphosphatidylcholine, diheptadecadienoylphosphatidylcholine, dilinolenoylphosphatidylcholine and diether and dialkyl derivatives. No activity was found for 1,3-dipalmitoylphosphatidylcholine. The presence of up to 5 mol% phosphatidylinositol in egg phosphatidylcholine vesicles had no effect on the transfer rate. Introduction of more than 5 mol% phosphatidylinositol or phosphatidic acid into the phosphatidylcholine vesicles gradually decreased the rate of phosphatidylinositol transfer from the monolayer. 20 mol% acidic phospholipid was nearly completely inhibitory. Transfer experiments between separate monolayers of phosphatidylcholine and phosphatidylinositol showed that the protein-bound phosphatidylcholine is readily exchanged for phosphatidylinositol, but the protein-bound phosphatidylinositol exchange for phosphatidylcholine occurs at a 20-times lower rate. The release of phosphatidylinositol is dependent on the lipid composition and the concentration of charged lipid in the acceptor membrane, but also on the ratio between donor and acceptor membranes. The main transfer protein from bovine brain which transfer phosphatidylinositol and phosphatidylcholine transfers also phosphatidylglycerol, but not phosphatidylserine or phosphatidic acid. The absence of significant changes in the surface pressure indicate that the phosphatidylinositol and phosphatidylcholine transfer is not accompanied by net mass transfer.     

Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry

Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid-liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.     

Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol

We use fluorescence microscopy to directly observe liquid phases in giant unilamellar vesicles. We find that a long list of ternary mixtures of high melting temperature (saturated) lipids, low melting temperature (usually unsaturated) lipids, and cholesterol produce liquid domains. For one model mixture in particular, DPPC/DOPC/Chol, we have mapped phase boundaries for the full ternary system. For this mixture we observe two coexisting liquid phases over a wide range of lipid composition and temperature, with one phase rich in the unsaturated lipid and the other rich in the saturated lipid and cholesterol. We find a simple relationship between chain melting temperature and miscibility transition temperature that holds for both phosphatidylcholine and sphingomyelin lipids. We experimentally cross miscibility boundaries both by changing temperature and by the depletion of cholesterol with beta-cyclodextrin. Liquid domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. To date, we have not observed macroscopic separation of liquid phases in only binary lipid mixtures.     

Resonance Raman resolution of a-, b- and c-type cytochromes in membrane vesicles of alkalophilic bateria

Resonance Raman spectroscopy has been used to obtain complete spectra of each individual cytochrome type — a, b and c — in the reduced state within membrane vesicle preparations from two species of obligately alkalophilic bacteria: Bacillus alcalophilus and Bacillus firmus RAB. The vibrational spectra, in the range 250–1700 cm^?1, were obtained with tunable dye laser excitation in the wavelength range 550–600 nm tuned to resonance with the appropriate reduced alpha band maximum for the cytochrome type of interest. The spectra reveal details which serve to characterize the specific type of cytochrome as well as to confirm the similarity of the heme prosthetic group to previously well-characterized cytochromes of the the a- b- or c-type. Preliminary evidence in support of heterogeneity of b-type, and possibly a-type cytochromes, or of heme-heme interaction within the membrane is presented.     

The effects of high concentrations of sodium or calcium ions on the lipid composition and properties of Tetrahymena membranes

Tetrahymena pyriformis cells have been grown in media varying in NaCl concentration from 3.7 mM (normal medium) to 0.3 M and varying in CaCl₂ from 0.2 mM (normal medium) to 0.1 M. Tetrahymena grown in 0.3 M NaCl showed relatively few alterations in phospholipid composition, with significant changes being found only in the cell surface membranes (pellicle), which increased in phosphatidylethanolamine content from 39% (low Na⁺) to 48% (high Na⁺) of the total phospholipids. The small decrease in fatty acid unsaturation and increase in shorter chain fatty acids in pellicle phospholipids were not statistically significant. No significant changes in phospholipid head group composition or fatty acid distribution were observed in high Ca²⁺-grown cells. Complementary studies of membrane fluidity, as inferred from freeze-fracture electron microscopy analysis, indicated that membranes of high Na⁺-acclimated cells were similar to those of control cells, when each was measured in its respective medium. However, the outer alveolar membrane of the pellicle and the food vacuolar membrane were considerably less fluid in high-Ca²⁺ cells. The lower fluidity in vacuolar membranes may have been responsible for alterations in the cells' capacity to form food vacuoles.     

NMR studies of glycerol permeability in lipid vesicles, erythrocytes and the alga Dunaliella

Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T₁ relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (ΔH^‡) were determined for glycerol at 25°C: for phosphatidylcholine vesicles 5·10⁻⁶ cm/s and 11±2 kcal/mol; for pig erythrocytes 7·10⁻⁸ cm/s and 18±3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17°C was found to be exceptionally low and only a lower limit (P<5·10⁻¹¹cm/s) could be calculated. At temperatures above 50°C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.     

Effect of succinylphosphatidylcholine on phosphatidylcholine vesicles. Structural studies by gel chromatography,electron microscopy and nuclear magnetic resonance

The effect of an aqueous dispersion of succinylphosphatidylcholine on an aqueous suspension of phosphatidylcholine vesicles was studied by gel chromatography, freeze-fracture electron microscopy and proton nuclear magnetic resonance with Mn²⁺ (broadening paramagnetic reagent). Total phospholipid concentrations were in the range 10–20 mM.Succinylphosphatidylcholine is in micellar form and behaves as a detergent. The structures obtained depend on the molar percentage of succinylphosphatidylcholine.Above a succinylphosphatidylcholine molar percentage of 60%, mixed micelles are formed, assumed to be essentially spherical.Below a succinylphosphatidylcholine molar percentage of 30%, principally mixed vesicles are observed, with an external diameter of 215–240 Å, and an almost constant internal volume.Between 30 and 60% of succinylphosphatidylcholine, a mixture of these structures is obtained; rod-shaped profiles are also observed in electron microscopy, which may correspond to sections of leaky vesicles or to a new kind of cylindrical micelle.     

Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity

(1) ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether (C₁₂E₈) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. (2) About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of ‘non-oriented’ (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. (3) The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the diffrent orientations is the same. (4) The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ration of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. (5) The ATP-dependent, K⁺-stimulated Na⁺-influx is found to be about 35 μmol Na⁺ per mg (i/o)-protein per min at 22°C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na⁺ transported per ATP molecule hydrolyzed.     

Effects of divalent metal ions on the calcium pump and membrane phosphorylation in human red cells

In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Fe²⁺. This activation is based on the formation of Me²⁺-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me²⁺ in this transport process. Higher Me²⁺ concentrations inhibit calcium transport with various efficiencies. Mn²⁺ directly competes with Ca²⁺ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [γ-³²P]ATP is induced by Ca²⁺ while rapid dephosphorylation requires the presence of Mg²⁺. At higher concentrations Mn²⁺ and Ni²⁺ inhibit predominantly the formation of EP, while Co²⁺ and Fe²⁺ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from [γ-³²P]ATP is significantly stimulated by Mn²⁺ and Co²⁺, as compared to that produced by Mg²⁺, Fe²⁺ and Ni²⁺. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.     

Effect of K and H on sodium/citrate cotransport in renal brush-border vesicles

The uptake of citrate by renal brush-border vesicles, prepared according to the method of Vannier, occurs by Na⁺-linked cotransport. It is ‘positive rheogenic’, i.e., stimulated by an (inside) negative, and inhibited by an (inside) positive electrical potential. The question arises whether, besides Na⁺, other ions (e.g., K⁺ and H⁺) participate in the cotransport. As to K⁺, neither an inward nor an outward directed K⁺ gradient has a significant effect on the citrate movement, but at equal concentrations of K⁺ inside and outside, equilibrium exchange of citrate, and to a smaller extent, the Na⁺-linked net uptake of citrate, are significantly stimulated. This observation is consistent with a hypothetical model in which K⁺ acts by accelerating both the empty and the fully loaded translocator. As to H⁺, citrate uptake is also stimulated by decreasing extravesicular pH, an effect previously attributed to protonization of the citrate anion in the assumption that the resulting secondary citrate anion is more acceptable to the translocator site. It was found, however, that the pH effect is still apparent if the concentration of the secondary citrate is kept constant by adjusting the total citrate concentration. This is taken as an argument against the above assumption and as being consistent with H⁺-linked cotransport. After the overshoot peak citrate exits slowly, and even after several hours does not attain equilibrium distribution, presumably owing to trapping by vesicular calcium.     

Effects of methylbenzenes on microsomal enzymes in rat liver,kidney and lung

The effects of pretreatment with toluene, o-, m-, p-xylene and mesitylene were investigated on the microsomal enzymes of liver, kidney and lung in rats. The activities of aminopyrine N-demethylase, aryl hydrocarbon hydroxylase, aniline hydroxylase, NADPH-cytochrome c reductase, as well as the concentrations of cytochrome P-450 and cytochrome b₅ were determined. The effects were most marked in the liver, where toluene caused increase in aniline hydroxylase and cytochrome P-450; o-xylene in aminopyrine N-demethylase and cytochrome b₅; m-xylene and mesitylene in all the enzymes investigated. In kidneys, all the compounds increased the activity of aniline hydroxylase; m-xylene induced cytochrome P-450 and b₅ as well as NADPH-cytochrome c reductase; p-xylene induced cytochrome P-450, and mesitylene cytochrome P-450 and b₅. Aminopyrine N-demethylase activity was decreased by toluene. In lungs, only mesitylene caused any significant differences from the controls: increase in aminopyrine N-demethylase and aryl hydrocarbon hydroxylase, decrease in aniline hydroxylase. The methylbenzenes tested induced the microsomal enzymes in a rough correlation to the number of their methyl groups and their hydrophobic properties.