Detergent-insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane

Glycosphingolipids and cholesterol form lateral assemblies, or lipid 'rafts', within biological membranes. Lipid rafts are routinely studied biochemically as low-density, detergent-insoluble complexes (in non-ionic detergents at 4 degrees C; DIGs, detergent-insoluble glycosphingolipid/cholesterol microdomains). Recent discrepancies recommended a re-evaluation of the conditions used for the biochemical analysis of lipid rafts. We have investigated the detergent insolubility of several known proteins present in the glycosphingolipid/cholesterol-rich myelin membrane, using four detergents representing different chemical classes (TX-100, CHAPS, Brij 96 and TX-102), under four conditions: detergent extraction of myelin either at (i) 4 degrees C or (ii) 37 degrees C, or at 4 degrees C after pre-extraction with (iii) saponin or (iv) methyl-beta-cyclodextrin (MbetaCD). Each detergent was different in its ability to solubilize myelin proteins and in the density of the DIGs produced. Brij 96 DIGs floated to a lower density than other detergents tested, possibly representing a subpopulation of DIGs in myelin. DIGs pre-extracted with saponin were denser than DIGs pre-extracted with MbetaCD. Furthermore, pre-extraction with MbetaCD solubilized proteolipid protein (known to associate with cholesterol), whereas pre-extraction with saponin did not, suggesting that saponin is less effective as a cholesterol-perturbing agent than is MbetaCD. These results demonstrate that DIGs isolated by different detergents are not necessarily comparable, and that these detergent-specific DIGs may represent distinct biochemical, and possibly physiological, entities based on the solubilities of specific lipids/proteins in each type of detergent.     

Solubilization of Myelin Membranes by Detergents

The major proteins of myelin have classically been extracted in organic solvents. Here we investigated some of the characteristics of brain myelin solubilization in aqueous detergent solutions. At comparable molar concentrations, two nonionic detergents, i.e., octyl glucoside and Lubrol PX, proved relatively better myelin solubilizers than the detergents related to the bile salts, i.e., cholate and CHAPS. The two former detergents solubilized more protein than lipid and the two latter ones more lipid than protein from myelin membranes. All four detergents solubilized the phospholipid more efficiently than the cholesterol component of myelin. The detergent concentrations required for myelin solubilization were reduced substantially if the temperature and the salt concentration of the media were increased. As much as 3 mg of lyophilized myelin (about 1 mg of protein) were solubilized readily per milliliter of a solution containing 30 mM octyl glucoside and 0.1 M sodium sulfate in 0.1 M sodium phosphate buffer, pH 6.7. Each of the detergents studied, including the above four, sodium dodecyl sulfate (SDS). Triton X-100, and Zwittergent 3-14, had its own advantages and drawbacks as myelin protein extractors. The nonionic amphiphiles and CHAPS left a small residue mainly composed of proteins of the Wolfgram fraction, as revealed by SDS-polyacrylamide gel electrophoresis. Octyl glucoside was preferred, given its versatility as solubilizer, ultraviolet transparency, and high critical micellar concentration. Observations on possible difficulties that may be encountered are also included.     

4-Methylumbelliferyl Lipase in Human and Mouse Brain: A Possible Localization in Myelin

4-Methylumbelliferyl (4-MU) lipase activity in human and mouse brain, measured with 4-MU palmitate, was characterized with respect to effects of pH and detergents, and subcellular and myelin localization. Purified myelin isolated by Norton's procedure [J. Neurochem. 21, 749-757 (1983)] contained higher specific activity of 4-MU lipase, particularly in alkaline pH. Myelin lipase activity was markedly affected by the addition of different types of detergents, the amount of detergents added, and substrate. The optimal pH in myelin was bimodal--pH 4.5 and up to 8.0, respectively. These data indicate that myelin possesses 4-MU lipase activity at alkaline pH, with lower levels at acidic pH.     

Enhanced resolution of glycosylphosphatidylinositol-anchored and transmembrane proteins from the lipid-rich myelin membrane by two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2-DE) has become a powerful and widely used technique for proteomic analyses. However, the limited ability of 2-DE to resolve transmembrane and glycosylphosphatidylinositol (GPI)-anchored proteins has slowed the identification of proteins from membrane-rich biological samples. Myelin is an unusually lipid-rich membrane with relatively few major proteins but many quantitatively minor proteins, most of which have an unknown identity and/or function. The goal of this study was to identify the optimal conditions of 2-DE for the separation of myelin proteins. We have identified two detergents, the nonionic n-dodecyl beta-D-maltoside and the zwitterionic amidosulfobetaine ASB-14, that are more effective in solubilizing myelin proteins than the commonly used zwitterionic detergent 3-[(3-cholamidopropyl)- dimethylammonio]-1-propanesulfonate (CHAPS). These detergents significantly enhance the solubility of both transmembrane (e.g., the highly hydrophobic and multiply acylated myelin proteolipid protein) and GPI-anchored (e.g., contactin and neuronal cell adhesion molecule) myelin proteins and enable their resolution by 2-DE. We conclude that these detergents are effective tools for the 2-DE analysis of myelin, and that they may be more generally useful for the analysis of membrane-rich biological samples.     

Solubilization of thyroid peroxidase by nonionic detergents.

We have examined the ability of nonionic detergents to solubilize thyroid peroxidase from a porcine thyroid particulate fraction, as measured by the release of peroxidase activity into the supernatant fraction after centrifugation at 105,000 X g for 1 hour and the retardation of the supernatant peroxidase of Sepharose 6B. The parameters of peroxidase solubilization by Triton X-100 have been investigated in detail. Under optimum conditions, 60 to 95% of the thryoid peroxidase and about 50% of the total protein is released into the 105,000 X g, 1-hour supernatant. Under the optimum conditions established with Triton X-100, a series of Brij detergents of different chemical structure were equally effective in releasing peroxidase and protein. The protein patterns of the supernatants obtained with these detergents were similar on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, suggesting that the detergents studied release similar membrane proteins. The Triton X-100 and Brij 58 supernatants were chromatographed separately on Sepharose 6B equilibrated with 0.1% Triton X-100 or Brij 58, respectively. In both cases, 75 to 80% of the peroxidase activity was retarded, thereby indicating that the nonionic detergents effect solubilization of the peroxidase rather than dispersal of nonsedimentable membrane fragments. These studies report the first successful solubilization of thyroid peroxidase by nonionic detergents. Together with previous evidence from our laboratory, these experiments indicate that thyroid peroxidase is an integral membrane protein.     

Susceptibility of the Wolfgram Proteins and Stability of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase of Rat Brain Myelin to Limited Proteolytic Digestion

The susceptibility of proteins in the myelin membrane to proteases was studied. Lyophilized rat brain myelin suspended in water was subjected to controlled proteolytic digestion with pure trypsin (N-tosyl-L-phenylalanine chloromethyl ketone treated, 5 units/mg of myelin), and proteins remaining in the pellet were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under these conditions, large basic protein (LBP) was completely hydrolyzed in 5-10 min, proteolipid proteins remained largely intact until 60 min, whereas Wolfgram protein (WP) was progressively degraded from 10 min onward with the simultaneous appearance of a new protein band with a molecular weight of 35K. A similar pattern was obtained on treatment with chymotrypsin or subtilisin. The 35K protein band was shown to be derived from WP by its immunological cross-reactivity with WP antibodies. Western blot analysis showed that 35K protein is the only major breakdown product of WP under these conditions. Treatment with higher concentrations of trypsin (greater than 20 units/mg of myelin) resulted in the degradation of all myelin proteins. Essentially all the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) activity was observed in the myelin pellet after controlled or drastic digestion with trypsin. It is concluded that the major fragment of WP (35K) is located in the hydrophobic milieu of the bilayer, relatively inaccessible to trypsin, whereas a portion (20K) of the WP is exposed to the cytoplasmic side (major dense line), like LBP, and that peptide fragments (less than 14K) that remained in the myelin membrane lipid bilayer after trypsin digestion could exhibit CNP activity.     

The reaction of myelin phospholipids with phospholipase C and D

Phospholipase C and D hydrolyze the membrane-bound phospholipids of isolated, untreated myelin. When the membrane is treated with detergents or solvents which disrupt the membrane structure, the activity of the enzymes against the membrane-bound lipids increases. Myelin in the central nervous system is derived from the cell membrane of the oligodendroglial cell. Because the phospholipids in erythrocyte cell membranes are strikingly resistent to phospholipase C and D hydrolysis the question is raised of whether myelin in situ, as opposed to isolated myelin, is susceptible to phospholipase hydrolysis.     

Towards crystallization of hydrophobic myelin glycoproteins: P0 and PASII/PMP22

The preparation of a pure and homogeneous protein sample at proper concentration is a prerequisite for success when attempting their crystallization for structural determination. The detergents suitable for solubilization particularly of membrane proteins are not always the best for crystallization. Myelin of the peripheral nervous system of vertebrates is the example of a membrane for which neutral or "gentle" detergents are not even strong enough to solubilize its proteins. In contrast, sodium- or lithium-dodecyl sulfate is very effective. We solubilized myelin membrane in 2%(w/v) sodium dodecyl sulfate, followed by chromatographic purification of the hydrophobic myelin glycoproteins P0 and PASII/PMP22, and finally, we have exchanged the sodium dodecyl sulfate bound to protein for other neutral detergents using ceramic hydroxyapatite column. Theoretically, we should easily exchange sodium dodecyl sulfate for any neutral detergent, but for some of them, the solubility of myelin glycoproteins is low. To monitor the potential variability in the secondary structure of glycoproteins, we have used circular dichroism. Sodium dodecyl sulfate seems to be the appropriate detergent for the purpose of purification of very hydrophobic glycoproteins, since it can be easily exchanged for another neutral detergent.     

Detergents enhance EspB secretion from Escherichia coli strains harboring the locus for the enterocyte effacement (LEE) gene

The effects of detergents (cholic acid, deoxycholic acid, Triton X-100, and Nonidet P-40) on the secretion of EspB from the locus for enterocyte effacement (LEE) gene-positive Escherichia coli strains were examined. Clinical isolates of eight EPEC strains and seven STEC strains were used to detect EspB after they had been cultivated in Luria-Bertani (LB) broth containing one of the detergents. When the bacteria were cultured in LB broth supplemented with one of the detergents, the amount of EspB produced was increased by 2-32-fold depending on the detergent and the strain used. EspB was detected in all strains when they were cultured in LB broth containing all of the detergents. The results obtained in this study can be applied to immunological diagnostic methods for detecting EspB and also to the production of EspB for research purposes.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : IV. Chemical and Cytological Characterization and Biosynthetic Capabilities of Fragments Obtained by Mild Procedures

Membrane-envelope fragments have been isolated from Escherichia coli by comparatively mild techniques. The use of DNAase, RNAase, detergents, sonication, lysozyme, and ethylenediaminetetraacetate were avoided in the belief that rather delicate, but metabolically important, associations may exist between the plasma membrane and various cytoplasmic components. The membrane-envelope fragments have been characterized in terms of their content of major chemical components as well as their electron microscope appearance. Fractions containing membrane-envelope fragments were found to possess appreciable DNA- and protein-synthesizing activities. The fragments were rich in membrane content as determined by reduced nicotinamide adenine dinucleotide (NADH) oxidase activity and deficient in soluble components as measured by NADH dehydrogenase activity. The particulate fraction obtained between 20,000 g and 105,000 g and usually considered a ribosomal fraction was rich in membrane content and had a relatively high capacity for DNA synthesis. Envelope fragments sedimenting at 20,000 g attained very high levels of incorporation of amino acids into protein.     

Thermal stability of bovine-brain myelin membrane

The thermal behaviour of bovine-brain myelin membrane has been studied by high-sensitivity differential scanning calorimetry, Fourier-transform infrared spectroscopy and thermal gel analysis. Spectroscopic results indicate that protein transitions take place between 60°C and 90°C, while thermal gel analysis has provided the thermal denaturation profiles of myelin proteolipid, DM-20 protein and the Wolfgram Fraction. An irreversible calorimetric transition centred at 80.3 ± 0.2°C with a specific enthalpy of 4.7 ± 0.6 J/g of total protein has been assigned to the thermal denaturation of myelin proteolipid and DM-20 protein. The effects of the myelin storage conditions, scan rate, ionic strength and pH on this calorimetric transition have also been investigated. The thermal transition of the proteolipid practically disappears after treatment of the myelin with different amounts of chloroform-methanol 2:1 (v/v), a treatment which is generally used in proteolipid purification. On the other hand, the addition of several detergents to myelin only causes minor modifications to this transition, which then occurs at about 70°C, with a specific enthalpy of between 2.5 and 3.6 J/g of total protein. These results appear to show that detergents preserve the native conformation of the proteolipid far more than do organic solvents. Hence the use of detergents would seem to be the appropriate method for proteolipid purification.Abbreviations DSC Differential scanning calorimetry - TGA Thermal gel analysis - FTIR Fourier-transform infrared spectroscopy - PLP Proteolipid protein - MBP Myelin basic protein - DM-20 Protein DM-20 - WF Wolfgram fraction - BSA Bovine serum albumine - SDS Sodium dodecyl sulfate - ANSA 4-amino-3-hydroxynaphthalene-1-sulphonic acid - OG -d-glucopyranoside - PAGE Polyacrylamide gel electrophoresis - Chaps 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CNS Central nervous system Correspondence to: P. L. Mateo     

A CHOLESTEROL-ESTERIFYING ENZYME IN RAT CENTRAL NERVOUS SYSTEM MYELIN1

Aontrary to our earlier finding (Eto & Suzuki , 1971), the myelin fraction purified from young adult rat brain consistently showed cholesterol-esterifying activity. The specific activity in myelin was the highest among subcellular fractions. Extensive washing wiih various aqueous salt solutions failed to remove the activity from myelin. The enzyme was evenly distributed among the arbitrarily defined light, medium and heavy myelin subfractions. The myelin-localized activity showed the pH optimum and heat stability identical to the microsome-bound activity. Although there were minor differences in the effect of detergents or exogenous lipids added to the reaction mixture, no firm evidence was obtained to indicate that the myelin-bound cholesterol-esterifying enzyme is different from that in other subcellular fractions. On the other hand, the distribution among the myelin subfractions and heat stability of the myelin-bound cholesterol-esterifying activity were different from those of the myelin-specific cholesterol ester hydrolase. Therefore, the esterification does not appear to be a mere reverse reaction catalyzed by the previously known myelin-specific hydrolase. The rat brain myelin, therefore, is capable of both synthesizing and hydrolyzing cholesterol esters.     

EFFECTS OF CATIONS ON ISOLATED BOVINE OPTIC NERVE MYELIN1

Abstract— Myelin fragments were isolated from bovine optic nerves and then exposed to solutions of NaCl, CaCl₂, LaCl₃ or to water. Measurements of the water content of myelin pellets and the hydrophobicity of myelin fragments indicated an apparent isoelectric point at about pH 4.0 which increased with increasing membrane counterion valence. The exposure of myelin to CaCl₂ and LaCl₃ solutions for 1 hr removed relatively more cholesterol and galactolipid than protein or phospholipid. The same changes were observed after 12 days of storage in all four solutions. Myelin ultrastructure was evaluated by electron microscopy after positive and negative staining. No pronounced changes in myelin ultra-structure were seen after exposure to any of these solutions although extensive beading of the lamellae was observed and the magnitude of the major period was greater than that reported for native myelin. While differences in the physical properties of myelin after exposure to Na⁺, Ca⁺⁺, or La⁺⁺⁺ ions could be explained by considering the fixed charge shielding capabilities of these cations, changes of state of the membrane infrastructure could not be ruled out. At pH values above 4.0 myelin fragments behaved like a cation exchange system.     

Effects of acyl chain length on the conformation of myelin basic protein bound to lysolipid micelles.

The interactions of myelin basic protein with micelles of lysophosphatidylcholine detergents of different acyl chain lengths were investigated by circular dichroism (CD), small-angle X-ray scattering, Fourier transform infrared spectroscopy (FT-IR), and 1H, 13C and 31P nuclear magnetic resonance spectroscopy (NMR). Circular dichroic, FT-IR, and 1H NMR measurements indicated that the conformational changes induced in the protein molecules by association with micelles depended on the acyl chain length of the detergents. Size is one of the physical properties of micelles which is a function of the length of the acyl chains. The radii of gyration of detergent micelles in complexes with the protein measured by small-angle X-ray scattering indicated that the average size of the micelles was a quadratic function of the acyl chain length. The dependence of the protein conformational changes on micelle size was used to ascertain the order in which different protein segments associate with the detergents. Several procedures were employed to change the fluidity of micelles formed with detergents of given acyl chain lengths. The conformational changes observed on the MBP molecule by varying the micelle properties without changing the length of the chain, suggested that the changes depended on the size and fluidity of the micelles.     

Acid endopeptidase activity of human myelin, elicited by using exogenous myelin basic protein as enzyme substrate

Purified human myelin was incubated with exogenous myelin basic protein (MBP) at pH 4.0 to see if there is acid proteinase activity associated with myelin. Following incubation for 12 h up to 70% of MBP was degraded. On electrophoresis peptide fragments of MBP between 15.8 and 9.4 kDa were consistent with an endopeptic cleavage of MBP. Unlike the exogenous substrate MBP associated with myelin was only slightly degraded under the experimental conditions used. The results show that proteinase activity associated with isolated myelin may be elicited and further evaluated by using MBP as enzyme substrate.     

Myelin basic protein inhibits histone-specific protein methylase I

Bovine brain myelin basic protein, free of associated proteolytic activity, was found to be a specific inhibitor of histone-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23) purified from bovine brain. 50% of the methyl group incorporation into the histone substrate catalyzed by the methylase I was inhibited by myelin basic protein at a concentration of 0.326 mM. However, neither of the peptide fragments (residues 1-116 and residues 117-170) generated by the chemical cleavage of myelin basic protein at the tryptophan residue retained the inhibitory activity for histone-specific protein methylase I. Proteins such as gamma-globulin, bovine serum albumin, bovine pancreatic ribonuclease and polyarginine did not exhibit significant inhibitory activity toward the enzyme. The Ki value for myelin basic protein was estimated to be 3.42 X 10(-5) M for histone-specific protein methylase I and the nature of the inhibition was uncompetitive toward histone substrate.     

Interactions of myelin basic protein with mixed dodecylphosphocholine/palmitoyllysophosphatidic acid micelles.

The interactions of myelin basic protein and peptides derived from it with detergent micelles of lysophosphatidylglycerol, lysophosphatidylserine, palmitoyllysophosphatidic acid, and sodium lauryl sulfate, and with mixed micelles of the neutral detergent dodecylphosphocholine and the negatively charged detergent palmitoyllysophosphatidic acid, were investigated by 1H NMR spectroscopy and circular dichroic spectropolarimetry. The results with single detergents suggested that there are discrete interaction sites in the protein molecule for neutral and anionic detergent micelles and that at least some of these sites are different for each type of detergent. The data on the binding of the protein and peptides to mixed detergent micelles suggested that intramolecular interactions in the intact protein and in one of the longer peptides limited the formation of helices and also that a balance between hydrophobic and ionic forces is achieved in the interactions of the peptides with the detergents. At high detergent/protein molar ratios, hydrophobic interactions appeared to be favored.     

On the binding of brain myelin basic protein to chromatographic resins

Myelin basic protein, only in association with certain detergents, is able to bind irreversibly to the usual gel filtration media. While this binding is greatly advantageous in purification of proteolipid (the other major myelin protein), the question arises of the uncommon, nonphysiological behaviour of the basic protein. A relationship between binding property and basic protein structure is suggested.     

Detergent activation and solubilization of 2'':3''-cyclic nucleotide 3''-phosphodiesterase from isolated myelin and c6 cells.

Several detergents were investigated for their ability to increase activity of 2':3'-cyclic nucleotide 3'-phosphodiesterase in isolated myelin. The ability of Triton X-100 and Sulfobetaine DLH to solubilize the enzyme was also examined. Solubilization with Triton X-100 was only effective in the presence of salt, for example with NaCl 51% of the activity was solubilized. A single extraction with Sulfobetaine DLH yielded slightly more solubilized enzyme and did not require added salt. Both activation and solubilization of 2':3'-cyclic nucleotide 3'-phosphodiesterase appeared to be similarly dependent on detergent concentration, suggesting a common action of the detergent in the two processes. Myelin basic protein was solubilized more readily than the enzyme. In contrast with the enzyme in myelin, 2':3'-cyclic nucleotide 3'-phosphodiesterase activity in C6 cells was not increased in the presence of Triton X-100, and was partially solubilized by either Triton X-100 or NaCl alone. No myelin basic protein could be detected in C6 cells by radioimmunoassay.