Membrane structure in isolated and intact myelins.

The biochemical composition of myelin and the topology of its constituent lipids and proteins are typically studied using membranes that have been isolated from whole, intact tissue using procedures involving hypotonic shock and sucrose density gradient centrifugation. To what extent, however, are the structure and intermembrane interactions of isolated myelin similar to those of intact myelin? We have previously reported that intact and isolated myelins do not always show identical myelin periods, indicating a difference in membrane-membrane interactions. The present study addresses the possibility that this is due to altered membrane structure. Because x-ray scattering from isolated myelin sometimes consists of overlapping Bragg reflections or is continuous, we developed nonlinear least squares procedures for analyzing the total intensity distribution after film scaling, background subtraction, and Lorentz correction. We calculated electron density profiles of isolated myelin for comparison with membrane profiles from intact myelin. The change in the width of the extracellular space and the relative invariance of the cytoplasmic space as a function of pH and ionic strength that we previously found for intact nerve was largely paralleled by isolated myelin. There were two exceptions: isolated CNS myelin was resistant to swelling under all conditions, and isolated PNS myelin in hypotonic saline showed indefinite swelling at the extracellular apposition. However, electron density profiles of isolated myelins, calculated to 30 A resolution, did not show any major change in structure compared with intact myelin that could account for the differences in interactions.     

Structure determination of asymmetric membrane profiles using an iterative Fourier method.

An iterative Fourier method is applied to solving and refining the electron density profile projected into the line perpendicular to a membrane surface. Solutions to the continuous X-ray scattering pattern derived from swelling of multilayer systems or from membrane dispersions can be obtained by this technique. The method deals directly with the observed structure factors and does not rely on deconvolution of the Patterson function. We used this method previously to derive the electron density profile for acetylcholine receptor membranes (Ross et al., 1977). The present paper is an analysis of the theoretical basis for the procedure. In addition, the technique is tested on artificially generated continuous-scattering data, on the data for frog sciatic nerve myelin derived from swelling experiments by Worthington and McIntosh (1974), and on the data for purple membrane (Blaurock and Stoeckenius, 1971). Although the method applies to asymmetric membranes, the special case of centrosymmetric profiles is also shown to be solvable by the same technique. The limitations of the method and the boundary conditions that limit the degeneracy of the solution are analyzed.     

Preparation of membrane vesicles from isolated myelin. Studies on functional and structural properties

Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in ²²Na⁺ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myelin basic protein from 0–150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.     

Preparation of membrane vesicles from isolated myelin: studies on functional and structural properties.

Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in 22Na+ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myeline basic protein from 0--150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.     

Theoretical calculation of the circular dichroism of unordered polypeptide chains

A calculation has been done of the circular dichroism (CD) spectrum of an unordered polypeptide chain. This has been based on a Boltzmann averaging over a dipeptide conformational CD map. This is shown to be valid by comparing the CD spectra of 28-mer oligopeptides with those generated by summing dipeptide CD spectra. The calculated CD spectrum of an unordered polypeptide chain is found to agree with the assignment proposed by Tiffany and Krimm from experimental studies.     

X-ray diffraction study of the kinetics of myelin lattice swelling. Effect of divalent cations.

The time-course of myelin lattice swelling and its reversal in dissected peripheral nerves was determined by small-angle x-ray diffraction using a position-sensitive proportional detector. The process of swelling can take place either in several hours or in less than 1 h depending on pretreatment of the nerves. The reversal of swelling was always completed within 1 h. The rapid structural transitions involved the disordering of membrane pairs as indicated by the transient appearance of a continuous intensity distribution similar to the membrane pair transform for myelin. The slow transitions involved the gradual replacement of the discrete reflections from the native structure by the reflections from the swollen lattice. Myelin membrane arrays reformed in normal Ringer's solution were much more stable to subsequent swelling than arrays reformed in Ca+2 and Mg+2-free Ringer's. These results suggest that these ions participate in stabilizing the interactions between the external surfaces of adjacent membrane pairs.     

The location of redox centers in the profile structure of a reconstituted membrane containing a photosynthetic reaction center-cytochrome c complex by resonance X-ray diffraction

The technique of resonance X-ray diffraction (Blasie, J.K. and Stamatoff, J. (1981) Annu. Rev. Biophys. Bioeng. 10, 451–452) utilizing synchrotron radiation was used to determine the locations of the cytochrome c heme iron atom and the photosynthetic reaction center iron atom within the profile of a reconstituted membrane. The accuracy of these determinations was better than ±2 ?. The cytochrome c heme iron atom → reaction center iron atom vector was determined to have a magnitude of approx. 44 ? projected onto the membrane profile and to span most of the lipid hydrocarbon core of the membrane profile. Since the reaction center iron atom interacts magnetically with the primary quinone electron acceptor Q_I over a distance of less than 10 ?, the primary light-induced electron-transfer reactions for this system generate the electric charge separation between oxidized cytochrome c⁺ and Fe-Q^?_I across most (approx. $\text{2}\text{3}$) of the membrane profile including most or all of the lipid hydrocarbon core of the membrane.     

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca²⁺ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.     

Membrane Interactions Are Altered in Myelin Isolated from Central and Peripheral Nervous System Tissues

Isolated myelin has been used for determinations of membrane surface charge density and topographical mapping of components in the membrane. To determine how similar such myelin is to myelin of intact tissue, we have used x-ray diffraction to compare their intermembrane interactions. The interactions were monitored by measuring the myelin period in samples treated with distilled water, buffered saline at pH 4-9 and ionic strength 0.06-0.18, and saline containing HgCl2 or triethyl tin sulfate. Myelin was isolated from whole brains and sciatic nerves of mice by conventional methods involving sucrose gradient centrifugation and osmotic shock. Consistent with previous findings, electron microscopy showed that the multilamellar morphology, staining, and repeat periods of isolated myelin were essentially like those of intact myelin; however, the membrane stacks were less extensive than those in whole tissue. X-ray diffraction revealed that isolated CNS myelin was like intact myelin in showing reversible compaction in acidic media and in distilled water. However, unlike the myelin in whole tissue, isolated CNS myelin did not swell in hypotonic or alkaline media, or in the presence of HgCl2-saline or triethyl tin. The altered membrane interactions could result from an increase in adhesiveness of the apposed membrane surfaces. Reorganization of proteolipid protein and/or a reduction of surface charge could account for the change in surface properties of isolated CNS myelin. Isolated PNS myelin, like the membranes in whole tissue, showed both compaction and swelling; however, the membrane pairs were disordered in the swollen structure. This irregular membrane swelling could result from charge variation in the extracellular surfaces.     

Ferrochelatase activity in wild-type and mutant strains of Spirillum itersonii. Solubilization with chaotropic reagents

New low-angle X-ray diffraction data have been obtained from nerve myelin after rehydration. The X-ray patterns show the first six orders of diffraction of a lamellar repeat unit of about 100 Å. Direct methods of structure analysis have been used to determine uniquely the phases of the first three orders of diffraction. The electron density profile of rehydrated nerve myelin has been obtained on an absolute electron density scale and is compared with the electron density profile of normal nerve myelin at the same resolution of 16–17 Å. Possible electron-density profiles of rehydrated nerve myelin at a resolution of 8 Å are shown.     

Shiverer and Normal Peripheral Myelin Compared: Basic Protein Localization, Membrane Interactions, and Lipid Composition

We have correlated membrane structure and interactions in shiverer sciatic nerve myelin with its biochemical composition. Analysis of x-ray diffraction data from shiverer myelin swollen in water substantiates our previous localization of an electron density deficit in the cytoplasmic half of the membrane. The density loss correlates with the absence of the major myelin basic proteins and indicates that in normal myelin, the basic protein is localized to the cytoplasmic apposition. As in normal peripheral myelin, hypotonic swelling in the shiverer membrane arrays occurs in the extracellular space between membranes; the cytoplasmic surfaces remain closely apposed notwithstanding the absence of basic protein from this region. Surprisingly, we found that the interaction at the extracellular apposition of shiverer membranes is altered. The extracellular space swells to a greater extent than normal when nerves are incubated in distilled water, treated at a reduced ionic strength of 0.06 in the range of pH 4-9, or treated at constant pH (4 or 7) in the range of ionic strengths 0.02-0.20. To examine the biochemical basis of this difference in swelling, we compared the lipid composition of shiverer and normal myelin. We find that sulfatides, hydroxycerebroside, and phosphatidylcholine are 20-30% higher than normal; nonhydroxycerebroside and sphingomyelin are 15-20% lower than normal; and ethanolamine phosphatides, phosphatidylserine, and cholesterol show little or no change. A higher concentration of negatively charged sulfatides at the extracellular surface likely contributes to an increased electrostatic repulsion and greater swelling in shiverer. The cytoplasmic surfaces of the apposed membranes of normal and shiverer myelins did not swell apart appreciably in the pH and ionic strength ranges expected to produce electrostatic repulsion. This stability, then, clearly does not depend on basic protein. We propose that P0 glycoprotein molecules form the stable link between apposed cytoplasmic membrane surfaces in peripheral myelin.     

X-ray diffraction study of myelin structure in immature and mutant mice

X-ray diffraction patterns were obtained from freshly dissected central and peripheral nerves of quaking, myelin synthesis deficiency ($\text{msd}$), and trembler mutants, as well as immature and adult normal mice. The patterns were compared with respect to strength of myelin diffraction, background scatter level, repeat period, and intensity and linewidth of Bragg reflections. The deficiency of myelin in optic nerves was found to be (in decreasing severity): quaking > immature > trembler ? normal adult; and in sciatic nerves: $\text{trembler > immature > quaking}\text{msd ?}\text{normal}$ adult. Repeat periods about 3 Å less than that for normal adult sciatic myelin were detected in corresponding nerves from immature, quaking, and trembler mice. In some trembler sciatic nerves a second phase having a 190–200 Å period and accounting for about 60% of the total ordered myelin was also evident. Comparison of electron density profiles of membrane units calculated from the repeat periods and diffracted intensities for sciatic myelins indicate structural differences at the molecular level. The main findings are: (1) quaking myelin shows a significant elevation of density in the external protein-water layer between membrane bilayers; (2) the membrane bilayer of immature myelin is ≈ 2 Å thinner than that for normal adult; (3) the membrane bilayer of the more compact phase in trembler myelin is ≈ 5 Å thinner than for normal; and (4) the difference in repeat periods for the two phases present in some of the trembler nerves can be accounted for predominantly by distinct membrane bilayer separations at the external boundary.     

An X ray diffraction study of frog sciatic nerve myelin in dimethyl sulfoxide.

Reversible structure modification of frog sciatic nerve myelin bathed in Ringer's solution containing dimethyl sulfoxide (DMSO) at a concentration of 33% has been studied by low-angle X-ray diffraction using a linear position-sensitive counter. Fourier images of native myelin layers, derived using low-order reflections measured at various stages of the DMSO treatment, reveal that the bilayer profile of native myelin membrane undergoes a specific asymmetric change prior to the phase transformation: The high-density peak on the extracellular side of the central lipid hydrocarbon layer decreases reversibly as the nerve is permeated by DMSO, while the internal peak and the central layer remain virtually unaltered. The dynamic process by which the contracted phase of myelin is derived from native myelin is speculated on the basis of the observed profile change.     

Isolation of Membrane Attack Complex of Complement from Myelin Membranes Treated with Serum Complement

Abstract: The interaction between complement and myelin membranes and its possible role in myelin damage and in the disposal of damaged myelin in vivo is of interest because activation of complement generates both opsonin(s) and membrane attack complex of complement. In our studies on the role of complement in demyelin-ation, we have shown that isolated myelin activates serum complement in the absence of myelin-specific antibody and that membrane attack complex of complement is the required factor in antibody-mediated demyelination of mouse cerebellar expiant cultures. In the present study, we examined whether activation of serum complement by myelin is associated with the formation of membrane attack complex of complement in myelin membranes. Extracts of myelin-associated proteins following incubation of myelin with fresh serum were studied by ultracentrifugation on a sucrose density gradient for detection of C5b-9 neoantigen. The subunit structure of C5b-9 was determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, electroblotting, and immunostaining. Results indicate that the macromolecular complex consisting of late-acting complement components, C5-C9, was assembled in the target myelin membranes.     

Modelling the swelling assay for aquaporin expression

The standard method of assaying the water transporting capability of a putative aquaporin-like entity is to express that entity in a cell of normally low water permeability and to measure the enhancement of swelling when the cell is subjected to hypo-osmotic shock. Because of the heterogeneous nature of cytoplasm, the interplay of advection and diffusion, and the coupling of internal and external media via a semipermeable elastic membrane, even simplified mathematical models can be difficult to resolve. This class of diffusion problem seems to have been but little studied. In this paper, the cell and its surround are at first modelled as perfectly-mixed phases separated by an ideal semipermeable membrane with vanishingly small elastic modulus; and the time course of swelling is evaluated analytically. This time course was found to be non-exponential, but such unexpected behavior should not seriously affect the traditional interpretation of experimental results because its short time limit is linear as in the traditional model; and normally only short time data are available. Next, the simplifications of diffusive equilibrium and of vanishing elastic modulus are examined. It is shown that diffusive equilibrium will be true only when diffusive movement of osmolyte is rather faster than the swelling and that this will probably not be the case for many assays. On the other hand, it should often be possible to neglect the elastic modulus. Finally, a more comprehensive model is formulated for a spherical cell in a hypotonic medium and the swelling behavior described in terms as a moving boundary problem (This type of moving boundary problem is often called a Stefan problem [ http://en.wikipedia.org/wiki/Stefan_problem ]) in which two phases containing diffusive osmolyte are separated by a weakly-elastic ideally-semipermeable membrane, the water flux across which is linear in the osmolality difference across it. This type of behavior was evaluated numerically by finite-difference time-domain techniques and found to be qualitatively similar to that of the perfect-mixing simplification.     

The location of redox centers in biological membranes determined by resonance x-ray diffraction. II. Analysis of the resonance diffraction data

In the preceding paper (Stamatoff, J., Eisenberger, P., Blasie, J.K., Pachence, J.M., Tavormina, A., Erecinska, M., Dutton P.L. and Brown, G. (1982) Biochim. Biophys. Acta 679, 177-187), we described the observation of resonance X-ray scattering effects from intrinsic metal atoms associated with redox centers in membrane proteins on the lamellar X-ray diffraction from oriented multilayers of reconstituted membranes. In this paper, we discuss the possible methods of analysis of such data and present the results of our model refinement analysis concerning (a) the location of the cytochrome c heme iron atom in the profile structure of a reconstituted membrane containing a photosynthetic reaction center-cytochrome c complex and (b) the location of the heme a and a3 iron atoms in the profile structure of a reconstituted membrane containing cytochrome oxidase. The former results are of special importance because they provide a test of the validity of the resonance diffraction data and the methods of analysis, since the location of cytochrome c in the reaction center-cytochrome c membrane profile is known independently of the resonance diffraction experiments.     

PROTEIN AND GLYCOPROTEIN COMPOSITION OF MYELIN AND MYELIN SUBFRACTIONS FROM BRAINS OF ''QUAKING'' MICE

Abstract— Quaking mutants in mice are known to be affected by an arrest of myelinogenesis and to have a purified myelin which is more dense than that of controls. Their myelin has been shown to demonstrate a striking decrease in proteolipid protein, a lesser decrease in the small myelin basic protein and changes in glycoproteins comprising reduction in the major peak and shift of this peak towards a higher apparent molecular weight. The possibility that these findings might reflect merely contamination of myelin with other membranes was tested by subfractionation. Light myelin (floats on 0.62 m -sucrose) is generally accepted as more compact and mature than the heavier subfraction (floating on 0.85 m -sucrose). The changes previously found were present in both subfractions and even more marked in the light myelin. These results indicate that the anomalies of myelin proteins and glycoproteins were not caused by contaminants and are present in compact myelin as well as in membranes which are transitional between the glial plasma membrane and the myelin sheath. Therefore, we suggest that the Quaking mutation results in dysmyelination rather than hypomyelination.     

Effect of solute permeability in determination of elastic modulus using the vesicular swelling method.

The modulus of elasticity of artificial and biological membranes can be determined in membrane vesicles by monitoring the limitation of vesicular swelling during a slow decrease in medium tonicity. The higher the elastic modulus of the membrane, the more effectively the vesicles will resist swelling. This method assumes that the solutes in the system are impermeant, so that the final volume of the vesicles is determined solely by a balance of osmotic and hydrostatic forces. In this paper, we present the results of computer simulation of vesicular swelling in which the solute permeability of the membrane was varied. We find that even a small permeability will lead to a loss of solute from the vesicle that will retard the increase in vesicular volume during dilution of the medium, and thereby cause the apparent modulus of elasticity to be much greater than the true value. For example, if one takes the mannitol permeability in brush border membrane vesicles from small intestine to be 0.004 micron/s (a reasonable estimate), one finds that a vesicular swelling study using mannitol as the principal solute will show the apparent elastic modulus of the vesicles to be greater than 10 times larger than the true value. With higher permeabilities, the effect is even more dramatic. We conclude that determination of impermeance of solutes is a critical prerequisite for making valid determinations of membrane elastic modulus using the vesicular swelling method.     

Adsorption of ions at charged sites and phase boundary potentials

The Poisson equation is used to determine for the equilibrium case the space profile of the electric potential at the boundary of two phases, one of which contains anionic sites able to adsorb selectively available cations. The solution has given an expression for the effective width of the space profile of the phase boundary potential in terms of the concentrations and the adsorption characteristics of the ions. It is shown that the sign of one (lumped) parameter determines the sign of the potential. It is also shown that a value of 66 Å can be obtained for the effective width of the space profile of the phase boundary potential. The implications concerning the nature of the “membrane” are briefly discussed.     

Repetitive propagation of action potentials destabilizes the structure of the myelin sheath. A dynamic x-ray diffraction study

Time courses of myelin lattice swelling in toad sciatic nerves preexposed to different treatments were determined by x-ray diffraction using a one-dimensional position-sensitive detector. In the nerves supramaximally stimulated for 1 h at 200 Hz, the subsequent process of myelin swelling occurred 45.0 +/- 7.3 min (n = 24) sooner than in resting controls. Sciatic nerves incubated for 1 h in a Ringer's solution deprived of divalent cations (Ca++ and Mg++) exhibited a kinetics of swelling similar to that shown by the stimulated nerves, that is, 52.5 +/- 14.2 min (n = 6) sooner than controls preincubated for the same time in normal Ringer's solution (with divalent cations). The fact that both pretreatments supramaximal stimulation and removal of divalent cations from the perfusion solution produced a similar effect; namely, a decrease of the myelin lattice stability against swelling in distilled water, suggests that the repetitive propagation of action potentials could modify the ionic composition at either the intraperiod channel or the paranodal axoglial junction complexes.