Solubilization of the semliki forest virus membrane with sodium deoxycholate

The effects of increasing concentrations of sodium deoxycholate on Semliki Forest virus have been studied. Sodium deoxycholate begins to bind to the virus at less than 0.1 mM free equilibrium concentration and causes lysis of the viral membrane at $\text{0.9 ± 0.1}\text{mM}$ free equilibrium concentration when $\text{2.2 ± 0.2 ß 10}{}^3\text{mol}$ of sodium deoxycholate are bound per mol of virus. Liberation of proteins from the membrane begins at $\text{1.5 ± 0.1}\text{mM}$ sodium deoxycholate and the proteins released are virtually free from phospholipid above 2.0 mM sodium deoxycholate. The overall mechanism of sodium deoxycholate solubilization of the viral membrane resembles that of Triton X-100 and sodium dodecyl sulphate except that with sodium deoxycholate the various stages of membrane disruption occur at about 10-fold higher equilibrium free detergent concentrations. At sodium deoxycholate concentrations higher than 2.3 mM the viral spike glycoproteins can be separated by sucrose gradient centrifugation or gel filtration into constituent polypeptides E1, E2 and E3. E1 carries the haemagglutinating activity of the virus.     

Association of myelin basic protein with detergent micelles

Equilibrium measurements of the binding of central nervous system myelin basic protein to sodium dodecyl sulphate, sodium deoxycholate and lysophosphatidylcholine have been obtained by gel permeation chromatography and dialysis. This protein associates with large amounts of each of these surfactants: the apparent saturation weight ratios (surfactant/protein) being $\text{3.58 ± 0.12}\text{and}\text{2.30 ± 0.15}$ for dodecyl sulphate at ionic strengths 0.30 and 0.10, respectively, $\text{1.34 ± 0.10}$ for deoxycholate (at 0.12 ionic strength) and $\text{4.0 ± 0.5}$ for lysophosphatidylcholine. Binding to the ionic surfactants increases markedly close to their critical micelle concentrations. Sedimentation analysis shows that at 0.30 ionic strength in excess dodecyl sulphate the protein is monomeric. It becomes dimeric when the binding ratio falls below 1 at a free detergent concentration of approximately 0.25 mM: below this concentration much of the protein and detergent forms an insoluble complex. The amount of dodecyl sulphate bound at high concentrations and at both above-mentioned ionic strengths corresponds closely to that expected for interaction of a single polypeptide with two micelles. Variability of deoxycholate micelle size on interaction with other molecules precludes a similar analysis for this surfactant. Association was observed only with single micelles of lysophosphatidylcholine. The results provide strong evidence for dual lipid-binding sites on basic protein and indicate that lipid bilayer cross-linking by this protein may be effected by single molecules.     

On the metabolic function of heparin-releasable liver lipase

Intravenous administration of specific antibody against heparin-releasable liver lipase (liver lipase) induced a 75% inhibition of the enzyme activity $\text{in situ}$. Administration of the antibody resulted in an increase of high density lipoprotein (density range 1.050–1.13 g/ml; HDL₂) phospholipid levels (20% after 1 h; 54% after 4 h). Short-term (1 h) treatment with antibody had no significant effect on any of the other lipoprotein components. After long-term (4 h) treatment the free cholesterol level of HDL₂ and all components in the very low density lipoprotein (VLDL) + intermediate density lipoprotein (IDL) fraction were elevated (1.5–2.0 fold). In the low density lipoprotein (LDL) fraction only the phospholipid level was affected (increased by 72%). All lipid components in the HDL₃ fraction were decreased by the antibody treatment, but this decrease was only statistically significant for the cholesterolesters. The rate of removal of iodine-labeled high density lipoprotein (HDL) and LDL from serum was not affected by the antibody treatment.These results suggest that liver lipase may promote phospholipid removal $\text{in vivo}$ and show that a lowering of liver lipase $\text{in situ}$ has profound consequences for serum lipoprotein metabolism.     

Effect of sodium deoxycholate on 5′-nucleotidase

The 5′-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5′-nucleotidase exhibited the same properties as the 5′-nuelcotidase in plasma membranes. The 5′-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.     

Fluidity of the lipid phase of bovine serum high density lipoprotein from fluorescence polarization measurements.

The microviscosity of the lipid phase of bovine serum high density lipoprotein was determined by fluorescence polarization measurements on a lipophilic probe (1,6-diphenyl-1,3,5-hexatriene) dissolved in the lipoprotein. At 25°C the average microviscosity was 6.1 ± 0.5 poise, and the activation energy calculated from a plot of log η versus $\text{1}\text{T}$ was 13±3Kcal/mole. A constant slope for the Arrhenius plot from 0 to 46°C indicated no apparent phase transitions in this temperature range.Comparison of the present results with reported microviscosity values for rat lymphocyte membranes and liposomes [Shinitzky and Inbar (1974) $\text{J. Mol. Biol. 85}$, 603] indicates a more rigid environment of the probe in the high density lipoprotein system fluidity of the lipid appears to be considerably decreased in the lipoprotein relative to organic solvent or oil solutions of lipids, probably as a result of the anisotropic environment of the probe, high total cholesterol, and presence of protein in these particles.     

Isolation of bovine cytochrome c1 as a single non-denatured subunit using gel filtration or high pressure liquid chromatography in deoxycholate

The non-denatured cytochrome $\text{c}{}_1$ subunit of bovine ubiquinone-cytochrome $\text{c}$ reductase was isolated using either gel filtration or high pressure liquid chromatography in 1% deoxycholate. The preparation was a single band on polyacrylamide gel electrophoresis in dodecyl sulfate, had a heme content of 31 nmol heme/mg protein, had an absorbance ratio $\text{A}{}_{417}\text{A}{}_{278}\text{= 2.65}$, a visible spectrum with maxima at 553, 530, 523.5, 417, 317, and 277 nm for the reduced protein, and an amino acid analysis identical to that previously reported for the isolated denatured protein. The Stokes' radius of this non-denatured deoxycholate solubilized protein was 34Å, indicating that the protein either is a dimer in deoxycholate, is asymmetric, or binds large amounts of detergent.     

A new serum lipoprotein found in many rhesus monkeys.

A new serum lipoprotein was found in about 10 out of 30 rhesus monkeys ($\text{Macaca}\text{mulatta}$) which contained 28% by weight of protein, 42% total cholesterol, 22% phospholipid, and 8% triglyceride. This is in contrast to LDL (which the ten monkeys also contained) which had 24% protein, 46% total cholesterol, 24% phospholipid and 7% triglyceride. An S_{f, 1.063} in KBr of 3.0 to 3.7 and molecular weight of 3.5 – 3.7 million were observed compared to means of 8.1 and 3.0 million for normal rhesus LDL. The lower S_f was caused by its higher density. This new lipoprotein was most easily demonstrated and isolated by preparative ultracentrifugation of all serum lipoproteins at density 1.22 g/ml, followed by 6% agarose gel filtration at 6°. The new lipoprotein appeared as a distinct peak eluting before LDL.     

Identification of the covalently bound flavin of L-gulono-gamma-lactone oxidase.

A specific mRNA for a structural lipoprotein of the $\text{Escherichia}\text{coli}$ outer membrane was translated in a wheat germ cell-free protein synthesizing system, S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) had neither stimulative nor inhibitory effect on the translation. When the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two peaks appeared at the appearent molecular weights of about 15,000 and about 7,500. Both products were cross-reactive with antiserum against the lipoprotein.     

Inhibition of small intestinal mucosal and smooth muscle cell function by ricinoleic acid and other surfactants.

Sodium ricinoleate, dioctyl sodium sulfosuccinate, sodium dodecyl (lauryl) sulfate, polysorbate 80, sodium deoxycholate and chenodeoxycholate were found to produce depression of $\text{in vitro}$ mucosal and smooth muscle cell function. These actions were assessed by measuring net water and electrolyte transport from everted hamster gut sacs and contractile activity of the electrically stimulated guinea-pig ileum. All compounds were effective depressants of both systems at concentrations which were likely to be below their respective critical micellar concentration. Ricinoleic acid may produce its cathartic effect due to its amphipathic nature, possibly by hydrophobic interaction with membrane lipoproteins. Ricinoleic acid and the other surfactants may be acting through a common mechanism.     

Comparative studies of purified and reconstituted monoamine oxidase from bovine liver mitochondria

Monoamine oxidase, a strictly membrane-bound flavoenzyme, has been purified using a modified procedure recently developed. Probably similarly to other preparations known from the literature, the enzyme solubilizes to a clear suspension, which represents large clusters ranging in size from 5 to 50 nm containing appreciable amounts of residual lipids. The purified and reconstituted enzymes are inhibited differently by deoxycholate. In contrast to deoxycholate, Triton X-100 does not inhibit the purified enzyme, but rather disintegrates the lipid-enzyme clusters to the smallest active units. However, removal of the detergent leads to reconglomeration to larger lipid-enzyme aggregates. Using the irreversible destruction of the enzyme by deoxycholate as assay, reconstitution of the enzyme with exogeneous lipids has been studied. All basic enzyme properties, such as stability, maximal activity ($\text{V}$), Michaelis constant ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$), pH- and temperature-dependence of the purified and reconstituted systems, are significantly different.     

Association between plasma high density lipoprotein cholesterol and antipyrine metabolism in alcoholics

Seven patients entering an alcoholic detoxification and treatment unit exhibited elevated levels of plasma high density lipoprotein cholesterol (HDLC) and enhanced metabolic disposal of antipyrine. Followig a 2-week abstinence treatment, the HDLC levels were reduced by 28% (from 64 to 47 mg/100 ml) and the $\text{t}\text{1}\text{2}$ of antipyrine was extended from 12.4 to 13.7 hours. The extent of the HDLC reduction correlated with the antipyrine $\text{t}\text{1}\text{2}$ changes (r = ?0.753, P = 0.05).     

Activité γ-glutamyltransferase dans la membrane du globule rouge humainγ-Glutamyltransferase activity in the human red blood cell membrane

A γ-glutamyltransferase activity is found in the human red blood cell membrane.Membrane isolation was carried out according to the method of Dodge et al. (Dodge, J. T., Mitchell, C. and Hanahan, J. (1963) Arch. Biochem. Biophys. 100, 119–130) (modified) and proteins were solubilized either with 1 % sodium deoxycholate or 5 mM EDTA or 10 mM of its disodium salt, under various conditions of time and temperature. The γ-glutamyltransferase activity of the membrane preparations was investigated using two substrates, $\text{γ-}\text{l}\text{-glutamyl}\text{-p-}\text{nitroanilide}$ and γ-lglutamyl-α-naphthylamide.The specific enzymatic activities of the various preparations, expressed in munits per mg of protein, were found to have similar values under similar technical conditions. The chelating agents seem to allow a more specific isolation than the detergent.The presence of a γ-glutamyltransferase activity in the erythrocyte membrane is discussed in relation to the membrane association of this enzyme in other tissues.     

Interaction of cholesterol,phospholipid and apoprotein in high density lipoprotein recombinants

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of ³H cholesterol/¹⁴C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27°C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1 or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. $\text{160 × 55}\text{A}\text{?}$. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Shape and thermodynamic parameters of a Ca2+-dependent ATPase. A solution x-ray scattering and sedimentation equilibrium study

Solution X-ray scattering and sedimentation equilibrium experiments in solvents of variable density were used to determine some thermodynamic parameters of deoxycholate-solubilized ATPase monomers. Molecular weight and partial specific volume of the unsolvated deoxycholate-ATPase complexes were determined from X-ray scattering and/or sedimentation equilibrium data in $\text{H}{}_2\text{O}\text{H}{}_2$¹⁸O mixtures. Volume, mean density (electron density) and hydration were determined by analysis of both solution X-ray scattering and sedimentation equilibrium data recorded in solvents containing various concentrations of sucrose. Protein shape was investigated further with the help of the autocorrelation function corresponding to the scattering curve recorded in the absence of sucrose. This curve was shown to represent mainly the particle shape. The autocorrelation function is fitted adequately by a model formed by two adjacent and coaxial cylinders of different heights and diameters. One cylinder is assumed to bind deoxycholate, while the other one, which is larger, is considered to represent the portion of the ATPase molecule that, in sarcoplasmic reticulum membranes, is exposed to the outer medium.     

The mechanism of assimilation of constituents of chylomicrons, very low density lipoproteins and remnants - a new theory.

Purified remnant lipoproteins produced from chylomicrons $\text{in vivo}$ or $\text{in vitro}$ by the action of lipoprotein lipase (LPL) contain firmly bound LPL. The perfused rat liver removes the particulate bound LPL and triglyceride-labeled remnants at exactly the same rate, while purified chylomicrons are not removed. Once remnants are removed by the liver, they are not rereleased into the perfusate. These observations have led to the theory that the LPL attached to the remnant is the signal that allows the liver to “recognize” remnants from chylomicrons. This is followed by fusion of the particle with the cell surface and may be associated with the splitting off a low density lipoprotein particle. The remaining lipids of the remnant are further metabolized by the liver triglyceridase and the cholesterol esterase.     

Serum lipoproteins and albumin in the lecithin: Cholesterol acyltransferase reaction

The unique features of pig ovarian follicular fluids, i.e., presence of high density lipoprotein (HDL) only and lecithin: cholesterol acyltransferase (EC 2.3.1.43; LCAT) activity, provides a good model to study the effect of serum lipoproteins and serum albumin on the LCAT reaction. $\text{In}\text{vitro}$ cholesterol esterification is enhanced when very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions are added, but is inhibited when one or the other of these lipoproteins is absent. High concentrations of HDL₂ result in decreased activation which can be compensated for by the addition of the VLDL-LDL mixture. These findings suggest that the rate of cholesterol esterification in ovarian follicular fluid may be enhanced by providing the exogenous VLDL and LDL as the recipients of HDL-cholesteryl ester. The inhibition of LCAT activity caused by free fatty acid and lysophosphatidylcholine can be partially reversed by the addition of serum albumin, suggesting that serum albumin may regulate the LCAT reaction.     

Acyltransferases and the biosynthesis of pulmonary surfactant lipid in adenoma alveolar type II cells.

Acyltransferases are present in microsomes from alveolar type II cell adenomas (produced by urethan injections) that transfer palmitic acid in the presence of CoA, ATP, and Mg⁺⁺ to $\text{sn}$-glycerol-3-P to form phosphatidic acid, to dihydroxyacetone-P to form acyldihydroxyacetone-P, and to 1-acyl-$\text{sn}$-glycero-3-phosphocholine to form 3-$\text{sn}$-phosphatidylcholine. The data clearly demonstrate that the microsomal preparations can catalyze significant incorporation of palmitic acid into the 2-position of the disaturated species of 3-sn-phosphatidylcholine independently of phosphatidic acid formation as evidenced by the fact that $\text{sn}$-glycerol-3-P and calcium ions (which inhibit choline phosphotransferase) did not influence the incorporation of palmitic acid into the main surfactant lipid. Thus, a deacylation-acylation reaction involving 2-lysophosphatidylcholine appears to be an important pathway for the synthesis of surfactant lipid in alveolar type II cells; the control of acyl specificity at the 2-position is determined by the relative concentrations of the coparticipating substrates, l-palmitoyl-$\text{sn}$-glycero-3-phosphocholine and palmitoyl-CoA.     

Cross-linking of the sarcoplasmic reticulum ATPase protein.

Treatment of rabbit sarcoplasmic reticulum vesicles with the cross-linking agent, cupric phenanthroline, causes production of high-molecular weight bands on SDS-gel electrophoresis. A plot of log mol wt $\text{vs}$ mobility indicates that the main band produced from the ATPase (mol wt = 10⁵) has a mol wt of 4 × 10⁵ and thus suggests formation of a tetramer. Notably, bands corresponding to dimers, trimers, pentamers, etc., are absent. The bands attributable to calsequestrin and calcium binding protein are unchanged by cupric phenanthroline. With extended treatment, the tetramer itself is polymerized (mol wt>10⁶). Partial disruption of the membranes with deoxycholate or Triton X-100 before cross-linking favors tetramer formation; the presence of sodium dodecyl sulfate, on the other hand, prevents intermolecular cross-linking. Our results suggest that the ATPase is at least partially associated within the membrane as a tetramer.     

The mitogenic principle of Escherichia coli lipoprotein: B-lymphocyte mitogenicity of N-palmitoyl-cysteine and N-palmitoyl-glutamic acid α-methyl esters

N-Palmitoyl-cysteine methyl ester and N-palmitoyl-glutamic acid α-methyl ester, which are analogues to the lipophilic N-terminal part of the lipoprotein from the outer membrane of $\text{Escherichia coli}$, were synthesized and tested for biological activity in an $\text{in}\text{vitro}$ lymphocyte culture system: in spleen cells from several inbred mouse strains the fatty acid derivatives exhibited mitogenic activity towards B-lymphocytes comparable to the effect of lipoprotein, as measured by ³H-thymidine incorporation and by hemolytic plaque assays. These results confirm former investigations, which have shown that the mitogenic principle of the lipoprotein molecule resides in its N-terminal fatty acid-containing part. The proper dispersion of the water-insoluble substances was critical for their mitogenic activity. Optimal mitogenicity was obtained by sonicating the substances at concentrations of 0.1 mg/ml in culture medium.