Biomedical subjects

D A Kirschner

Publications and source records attributed to D A Kirschner.

At least 55 records · Page 3Linked to original sources

PubMed1993

Structure of beta-crystallite assemblies formed by Alzheimer beta-amyloid protein analogues: analysis by x-ray diffraction.

To elucidate the relation between amyloid fibril formation in Alzheimer disease and the primary structure of the beta/A4 protein, which is the major component of the amyloid, we have been investigating the ability of peptides sharing sequences with beta/A4 to form fibrils in vitro. In previous studies we focused on the macroscopic morphology of the assemblies formed by synthetic peptides corresponding in sequence to different regions of this protein. In the present study we analyze the x-ray diffraction patterns obtained from these assemblies. All specimens showed wide angle reflections that could be indexed by an orthogonal lattice of beta-crystallites having unit cell dimensions a = 9.4 A, b = 7 A, and c = 10 A, where a refers to hydrogen bonding direction, b to polypeptide chain direction, and c to intersheet direction. Given the amino acid sequence of beta/A4 as NH2-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT-COOH, we found that, based on their orientation and assembly, the analogues could be classified into three groups: Group A, residues 19-28, 13-28, 12-28, 11-28, 9-28, 1-28, 1-38, 1-40, 6-25, 11-25 and 34-42; Group B, residues 18-28, 17-28, and 15-28; and Group C, residues 22-35 and 26-33. For Groups A and C, the sharpest reflections were (h00), indicating that the assemblies were fibrillar, i.e., elongated in a single direction. Lateral alignment of the crystallites in Group A account for its cross-beta pattern, in which the hydrogen bonding (H-bonding) direction is the fiber (rotation) axis. By comparison, the beta-crystallites of Group C had no preferential orientation, thus giving circular scattering. For Group B, the sharpest reflections were (h0l) on the meridian, indicating that the assemblies were plate-like, i.e., extended in two directions. A series of equatorial Bragg reflections having a 40 A period indicated regular stacking of the plates, and the rotation axis was normal to the surface of the plates. Of the Group A peptides, the analogues 11-28 and 6-25 showed intensity maxima on the equator as well as on higher layer lines, indicating that the beta-crystallites are highly ordered relative to one another in the axial, H-bonding direction. This sampling of the layer lines by a larger period (60 A) suggests that the beta-crystallites are arrayed either in cylindrical or small restricted crystalline lattices. Consistent with its electron microscopic images, we modeled the structure as a tube with five or six f,-crystallites constituting the wall and with the individual crystallite, which either rotates freely or is restricted, made of five or fewer beta-pleated sheets. For the Group B peptides, the electron density projection along the b-axis was calculated from the observed intensities using phase combinations from fl-keratin.Amino acid side-chain positions were apparent and, when refined as 4-A-diameter spheres, led to a substantial decrease in the R-factors.For peptide 18-28 the electron density peaks, which are thought to correspond to side chains, were centered 3.3 A from the peptide backbone, whereas for peptides 17-28 and 15-28, these peaks were centered 1 A or more further from the backbone. Peaks having high electron density faced peaks having lower density, suggesting a favorable stereochemical arrangement of the residues. Thus, our analysis of the fiber x-ray patterns from beta/A4 peptides shows the organization of the beta-crystallites that form the wall of the amyloid fibrils as well as possible side-chain interactions.

Amino Acid Sequence↗

PubMed1993

Alpha 1-antichymotrypsin binding to Alzheimer A beta peptides is sequence specific and induces fibril disaggregation in vitro.

The serine protease inhibitor alpha 1-antichymotrypsin (ACT) consistently colocalizes with amyloid deposits of Alzheimer's disease (AD) and may contribute to the generation of amyloid proteins and/or physically affect fibril assembly. AD amyloid fibrils are composed primarily of A beta, which is a proteolytic fragment of the larger beta-amyloid precursor protein. Using negative-stain and immunochemical electron microscopy, we have investigated the binding of ACT to the fibrils formed by four synthetic A beta analogues corresponding to the wild-type human 1-40 sequence [Hwt(1-40)], a 1-40 peptide [HDu(1-40)] containing the Glu22-->Gln mutation found in hereditary cerebral hemorrhage with amyloidosis of the Dutch type, the N-terminal 1-28 residues [beta(1-28)], and an internal fragment of A beta containing residues 11-28 [beta(11-28)]. Each of these peptide analogues assembled into 70-90-A-diameter fibrils resembling native amyloid and, except for beta(11-28), bound ACT, as indicated by the appearance of 80-100-A globular particles that adhered to preformed fibrils and that could be decorated with anti-ACT antibodies. Under the conditions used, ACT binding destabilized the in vitro fibrils and produced a gradual dissolution of the macromolecular assemblies into constituent filaments and shorter fragments. The internal fragment (11-28) did not exhibit ACT binding or any structural changes. These results suggest that a specific sequence likely contained within the N-terminal 10 residues of A beta is responsible for the formation of the ACT-amyloid complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer Disease↗

PubMed1993

Myelin P0-glycoprotein: predicted structure and interactions of extracellular domain.

Protein zero (P0), a transmembrane glycoprotein, accounts for over 50% of the total protein in PNS myelin. The extracellular domain of P0 (P0-ED) is similar to the immunoglobulin variable domain, carrying one acceptor sequence for N-linked glycosylation. The x-ray diffraction analysis of PNS myelin has demonstrated reversible transitions that depend on pH and ionic strength, resulting in three distinct structures characterized by widths of about 36 A, 50 A (native), and 90 A between the extracellular surfaces of the membranes. In the current work, we considered the constraints imposed by these x-ray diffraction data on the orientation of P0-ED, and we propose how this immunoglobulin-like domain could be accommodated in the variable widths of the extracellular space between myelin membranes. The modeling made use of the finding that beta-strand predictions for P0-ED are virtually superimposable with those of the VH domain of the phosphocholine-binding immunoglobulin M603 of mouse, which has a similar number of residues as P0-ED and a structure that has been solved crystallographically. The dimensions of P0-ED from the space-filling model, developed using PC-based molecular modeling software, were found to be 44 A x 25 A x 23 A. On the assumption that neither the shape nor the orientation of P0-ED changes appreciably, then the different widths at the extracellular apposition would easily accommodate P0-ED from apposed membranes if the molecules were oriented so that the beta-strands were approximately perpendicular to the membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Fibril formation by primate, rodent, and Dutch-hemorrhagic analogues of Alzheimer amyloid beta-protein.

Deposition of extraneuronal fibrils that assemble from the 39-43 residue beta/A4 amyloid protein is one of the earliest histopathological features of Alzheimer's disease. We have used negative-stain electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and fiber X-ray diffraction to examine the structure and properties of synthetic peptides corresponding to residues 1-40 of the beta/A4 protein of primate [Pm(1-40); human and monkey], rodent [Ro(1-40); with Arg5-->Gly, Tyr10-->Phe, and His13-->Arg], and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) [Du(1-40); with Glu22-->Gln]. As controls, we examined a reverse primate sequence [Pm*(40-1)] and an extensively substituted primate peptide [C(1-40); with Glu3-->Arg, Arg5-->Glu, Asp7-->Val, His13-->Lys, Lys16-->His, Val18-->Asp, Phe19-->Ser, Phe20-->Tyr, Ser26-->Pro, Ala30-->Val, Ile31-->Ala, Met35-->norLeu, Gly38-->Ile, Val39-->Ala, and Val40-->Gly]. The assembly of these peptides was studied to understand the relationship between species-dependent amyloid formation and beta/A4 sequence and the effect of a naturally occurring point mutation of fibrillogenesis. The three N-terminal amino acid differences between Pm(1-40) and Ro(1-40) had virtually no effect on the morphology or organization of the fibrils formed by these peptides, indicating that the lack of amyloid deposits in rodent brain is not due directly to specific changes in its beta/A4 sequence. beta-Sheet and fibril formation, judged by FT-IR, was maximal within the pH range 5-8 for Pm(1-40), pH 5-10.5 for Du(1-40), and pH 2.5-8 for Ro(1-40).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Is myelin basic protein crystallizable?

Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used convention strategies in our search for a suitable composition of a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization - homogeneity of tertiary structure--is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized.

Ammonium Sulfate↗

PubMed1992

Phylogenetically conserved amino acids of MBP and P0 from amphibian myelin.

Myelin basic protein (MBP) and P0 glycoprotein are major structural proteins of myelin. In adult frog, MBP is found in both the central and peripheral nervous systems (CNS and PNS), while P0 is found exclusively in the PNS. To assess the phylogenetic conservation of these proteins, MBP and P0 were isolated from adult bull-frog. A cyanogen bromide cleavage peptide of MBP (8-26), and the amino-terminal region (1-20) and an endoproteinase Lys-C peptide (67-79) of P0 were sequenced and compared to those of other vertebrate species. Residues that were conserved among other vertebrate species were found also to be conserved in frog: MBP--Ala18, Ser19, Thr20, Asp22; P0--Ile1, Val3, Thr5, Val13, Gly14, Ser15, Val17, Leu19, Trp72, Val73, Gly74, Lys79. These residues are located within or adjacent to regions that have been postulated to form beta strands and to be essential to the folding and function of these proteins.

Amino Acid Sequence↗

PubMed1992

Amyloid-like properties of a synthetic peptide corresponding to the carboxy terminus of beta-amyloid protein precursor.

A synthetic peptide whose sequence corresponds to the 20 carboxy-terminal amino acids of beta-amyloid protein precursor (APP) was found to form fibrils in vitro. These fibrils showed birefringence in polarized light when stained with Congo red, fluoresced when bound with thioflavin S, were resistant to proteases, and had a cross-beta conformation. By contrast, peptides with other sequences from the intracellular domain of APP and a peptide corresponding to this entire domain did not exhibit the full range of beta-amyloid properties. These results suggest that a fragment from the C-terminus of the beta-amyloid protein precursor could bind to intraneuronal paired helical filaments and account for some of its amyloid-like properties.

Amino Acid Sequence↗

PubMed1992

Effects of sulfate ions on Alzheimer beta/A4 peptide assemblies: implications for amyloid fibril-proteoglycan interactions.

To model the possible involvement of sulfated proteoglycans in amyloidogenesis, we examined the influence of sulfate ions, heparan, and Congo red on the conformation and morphology of peptides derived from the Alzheimer beta/A4 amyloid protein. The peptides included residues 11-28, 13-28, 15-28, and 11-25 of beta/A4. Negative-stain electron microscopy revealed a sulfate-specific tendency of the preformed peptide fibrillar assemblies of beta(11-28), beta(13-28), and beta(11-25), but not beta(15-28), to undergo extensive lateral aggregation and axial growth into "macrofibers" that were approximately 0.1-0.2 micron wide by approximately 20-30 microns long. Such effects were observed at low sulfate concentrations (e.g., 5-50 mM) and could not be reproduced under comparable conditions with Na2HPO4, Na2SeO4, or NaCl. Macrofibers in NaCl were only observed at 1,000 mM. At physiological ionic strength of NaCl, fibril aggregation was observed only with addition of sulfate ions at 5-50 mM. Selenate ions, by contrast with sulfate ions, induced only axial and not substantial lateral aggregation of fibrils. X-ray diffraction indicated that the original cross-beta peptide conformation remained unchanged; however, sulfate binding did produce an intense approximately 65 A meridional reflection not recorded with control peptides. This new reflection probably arises from the periodic deposition of the electron-dense sulfate along the (long) axis of the fibril. The sulfate binding could provide sites for the binding of additional fibrils that generate the observed lateral and axial aggregation. The binding of heparan to beta(11-28) also produced extensive aggregation, suggesting that in vivo sulfated compounds can promote macrofibers. The amyloid-specific, sulfonated dye Congo red, even in the presence of sulfate ions, produced limited aggregation and reduced axial growth of the fibrils. Therefore, electrostatic interactions are important in the binding of exogenous compounds to amyloid fibrils. Our findings suggest that the sulfate moieties of certain molecules, such as glycosaminoglycans, may affect the aggregation and deposition of amyloid fibrils that are observed as extensive deposits in senile plaques and cerebrovascular amyloid.

Amyloid↗

PubMed1991

Folding and function of the myelin proteins from primary sequence data.

To explain how the myelin proteins are involved in the organization and function of the myelin sheath requires knowing their molecular structures. Except for P2 basic protein of PNS myelin, however, their structures are not yet known. As an aid to predicting their molecular folding and possible functions, we have developed a FORTRAN program to analyze the primary sequence data for proteins, and have applied this to the myelin proteins in particular. In this program, propensities for the secondary structure conformations as well as physical-chemical parameters are assigned to the amino acids and the pattern of these parameters is examined by calculating their average values, autocorrelation functions and Fourier transforms. To compare two proteins, their sequences are aligned using a unitary scoring matrix, and homologies are searched by plotting a two-dimensional map of the correlation coefficients. Comparison of the corresponding myelin basic proteins (MBP) and P0 glycoproteins (P0) for rodent and shark showed that the conserved residues included most of the amino acids which were predicted to form the alpha or beta conformations, while the altered residues were mainly in the hydrophilic and turn or coil regions. In both rodent and shark the putative extracellular domain of P0 glycoprotein displayed consecutive peaks of beta propensity similar to that for the immunoglobulins, while the cytoplasmic domain showed alpha-beta-alpha folding. To trace the immunoglobulin fold along the P0 sequence, we compared the beta propensity curve of P0 with that of the immunoglobulin M603, whose three-dimensional structure has been determined. We propose that the flat beta-sheets of P0 are orientated parallel to the membrane surface to facilitate their homotypic interaction in the extracellular space. An extra beta-fold in the extracellular domain of shark P0 compared with rodent P0 was found, and this may result in a greater attraction between the apposed extracellular surfaces and may account for a smaller extracellular space as measured by x-ray diffraction. A computer search of the myelin protein sequences for functional motifs revealed sites for N-glycosylation, phosphorylation, nucleotide binding, and certain enzyme activities. We note especially that there are potential nucleotide binding sites in proteolipid protein (PLP), MBP and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). This is consistent with the experimental observations that PLP acts like an ionophore or proton channel when reconstituted into planar lipid bilayers, MBP binds GTP, and CNP catalyzes in vitro the hydrolysis of 2',3'-nucleotides into corresponding 2'-nucleotides.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗

PubMed1991

Morphology and antibody recognition of synthetic beta-amyloid peptides.

To elucidate the relationship between amyloid fibril formation in Alzheimer disease (AD) and the primary structure of the beta-amyloid protein (beta-AP), we investigated the ability of peptides sharing sequences with beta-AP to form fibrils in vitro and to recognize anti-beta-amyloid antisera. The peptides, which were synthesized using a FMOC solid phase procedure and purified by HPLC, consisted of residues 6-25 from the putative aqueous domain, residues 22-35, which overlaps the putative aqueous and transmembrane domains, and residues 1-38 and 1-40 representing nearly the full length of beta-AP. Electron microscopy of negative-stained or thin-sectioned preparations revealed that the peptides assembled into fibrils having different morphologies, some of which resembled in situ AD amyloid. Peptide 6-25 fibrils had diameters of 50-80 A and occasionally showed a central groove suggestive of constituent filaments. Cross sections of the fibril showed a penta- or hexameric arrangement of globular subunits with diameters of 25-30 A. Peptide 22-35 fibrils were helical, with a pitch of 1,100 A and a width of 120 A at its greatest and 50-60 A at its narrowest. The fibrils formed by peptides 1-38 and 1-40 were 70-90 A in diameter. When the peptide assemblies were singly oriented by sedimentation or doubly oriented in a magnetic field, their X-ray diffraction patterns all showed reflections typical of a cross-beta pleated sheet conformation. The patterns differed mainly in their small-angle equatorial intensity, which arises from the packing of fibrils having different widths. Antiserum raised to either native amyloid or to synthetic peptide beta-(1-28) was highly reactive in an inhibition-ELISA assay to beta-(6-25) and beta-(1-38), but not to beta-(22-35), and immunostained beta-(1-40) on Western blots. These studies show that the beta-(6-25), beta-(1-38) and beta-(1-40) peptides can assemble into cross-beta fibrils that retain epitopes characteristic of AD amyloid.

Amino Acid Sequence↗

PubMed1991

pH-dependent structural transitions of Alzheimer amyloid peptides.

To understand the molecular interactions leading to the assembly of beta/44 protein into the hallmark fibrils of Alzheimer's disease (AD), we have examined the ability of synthetic peptides that correspond to the beta/A4 extracellular sequence to form fibrils over the range of pH 3-10. Peptides included the sequences 1-28, 19-28, 17-28, 15-28, 13-28, 11-28, and 9-28 of beta/A4. The model fibrils were compared with isolated amyloid with respect to morphology, conformation, tinctorial properties, and stability under denaturing conditions. Electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and x-ray diffraction revealed that the ionization states of the amino acid sidechains appeared to be a crucial feature in fibril formation. This was reflected by the ability of several peptides to undergo fibril assembly and disassembly as a function of pH. Comparisons between different beta/A4 sequences demonstrated that the fibrillar structure representative of AD amyloid was dependent upon electrostatic interactions, likely involving His-13 and Asp-23, and hydrophobic interactions between uncharged sidechains contained within residues 17-21. The results also indicated an exclusively beta-sheet conformation for the synthetic (and possibly AD fibrils) in contrast to certain other (e.g., systemic) amyloids.

Amino Acid Sequence↗

PubMed1991

Quantitative differences between homozygous 'USA' and 'Swiss' mld mutant mice.

Parallel developmental studies of central nervous system myelin proteins and morphology (postnatal days 15-118; P15-118) confirm qualitative similarities but substantial quantitative differences between homozygous mld mice with Billings-Gagliardi and Wolf's 'USA' versus Matthieu's 'Swiss' genetic backgrounds. The USA mld/mld have fewer convulsions and significantly longer life span. While whole-brain homogenates from both Swiss and USA mld/mld show increases in myelin basic protein (MBP) and in 2',3'-cyclic nucleotide 3'-phosphohydrolase specific activity with age, at P50 and older the levels of both proteins are approximately twice as high in the Swiss. The number of optic nerve axons myelinated is always greater in Swiss mld/mld, and they have approximately twice as many myelin sheaths showing any apposition of cytoplasmic membrane faces (the location of the major dense line in normal myelin), except at the youngest age. Evidence is presented which suggests that these quantitative differences between Swiss and USA mld stocks most likely reflect different regulatory genes influencing the expression of the same (mld) allele, rather than the presence of a different allele at the MBP locus.

Aging↗

PubMed1991

Shiverer jimpy double mutant mice. V. Correlation of genotype and myelin proteins.

We have reexamined the levels of myelin basic protein (MBP) and proteolipid protein (PLP) in the brains of mice bred to carry both the shi/shi and jp/Y hypomyelination defects. The genotype of each putative double mutant was confirmed by direct DNA analysis: shi/shi by Southern blot analysis, and jp/Y by restriction enzyme analysis of polymerase chain reaction-amplified fragments. MBP and PLP levels were assessed by immunoblotting. All putative double mutants were found to be shi/shi. However, examination of the PLP locus revealed both jp and wild-type genotypes, the latter produced by an expected crossover. Animals proven to be shi/shi*jp/Y had no detectable MBP or PLP; those proven to be shi/shi*+/Y (the crossover) had no MBP but had PLP. These results differ from an earlier report of both MBP and PLP in the brains of presumed shi*jp animals.

Animals↗

PubMed1990

Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides.

The amyloid beta protein is deposited in the brains of patients with Alzheimer's disease but its pathogenic role is unknown. In culture, the amyloid beta protein was neurotrophic to undifferentiated hippocampal neurons at low concentrations and neurotoxic to mature neurons at higher concentrations. In differentiated neurons, amyloid beta protein caused dendritic and axonal retraction followed by neuronal death. A portion of the amyloid beta protein (amino acids 25 to 35) mediated both the trophic and toxic effects and was homologous to the tachykinin neuropeptide family. The effects of the amyloid beta protein were mimicked by tachykinin antagonists and completely reversed by specific tachykinin agonists. Thus, the amyloid beta protein could function as a neurotrophic factor for differentiating neurons, but at high concentrations in mature neurons, as in Alzheimer's disease, could cause neuronal degeneration.

Amino Acid Sequence↗

PubMed1990

Molecular determinants of amyloid deposition in Alzheimer's disease: conformational studies of synthetic beta-protein fragments.

The amyloid beta-protein (1-42) is a major constituent of the abnormal extracellular amyloid plaque that characterizes the brains of victims of Alzheimer's disease. Two peptides, with sequences derived from the previously unexplored C-terminal region of the beta-protein, beta 26-33 (H2N-SNKGAIIG-CO2H) and beta 34-42 (H2N-LMVGGVVIA-CO2H), were synthesized and purified, and their solubility and conformational properties were analyzed. Peptide beta 26-33 was found to be freely soluble in water; however, peptide beta 34-42 was virtually insoluble in aqueous media, including 6 M guanidinium thiocyanate. The peptides formed assemblies having distinct fibrillar morphologies and different dimensions as observed by electron microscopy of negatively stained samples. X-ray diffraction revealed that the peptide conformation in the fibrils was cross-beta. A correlation between solubility and beta-structure formation was inferred from FTIR studies: beta 26-33, when dissolved in water, existed as a random coil, whereas the water-insoluble peptide beta 34-42 possessed antiparallel beta-sheet structure in the solid state. Solubilization of beta 34-42 in organic media resulted in the disappearance of beta-structure. These data suggest that the sequence 34-42, by virtue of its ability to form unusually stable beta-structure, is a major contributor to the insolubility of the beta-protein and may nucleate the formation of the fibrils that constitute amyloid plaque.

Alzheimer Disease↗

PubMed1990

Radial component of CNS myelin: junctional subunit structure and supramolecular assembly.

The radial component is a structural specialization within CNS myelin that is believed to stabilize the apposition of membranes in the internode. Previous observations on thin sections and freeze-fracture replicas show that this junctional complex consists of linear, particulate strands that run parallel to the nerve fibre axis and radially through the myelin sheath, but details on its molecular organization are lacking. The objective of our current study was to gain further insight into its arrangement and composition by examining its fine-structure and incidence in: myelin with known deficits in protein composition (e.g., shiverer, transgenic shiverer, myelin deficient and jimpy mutant mice); isolated CNS myelin, which has been shown by X-ray diffraction to be more stable than intact CNS myelin; and human white matter, in which this junctional complex has not yet been described. Our results confirm the localization and general appearance of the radial component as previously reported. In addition, we found that: (1) the radial component occurs abundantly in human CNS myelin where it has a complex subunit structure; (2) the constituent junctional unit of this structure is organized as a pair of globular domains (each approximately 40 A diameter) at the extracellular apposition which is linked by approximately 15 A diameter filaments extending through the bilayer to approximately 25 A globular domains in the adjacent cytoplasmic apposition; (3) the radial component is present with apparently normal structure in the sparse, compact myelin of murine mutants containing either different amounts of MBP or no PLP which indicates that neither of these proteins is necessary for junctional integrity; (4) the radial component is present in purified CNS myelin membranes which may account for the stability of these membranes; and (5) the radial component is structurally resistant to Triton, which suggests a method for its further biochemical characterization. Finally, from an analysis of images from tilted transverse and longitudinal sections, we have reconstructed a model of its three-dimensional, supramolecular organization.

Animals↗

PubMed1990

Quaking*jimpy double mutant mice: additional evidence for independence of primary deficits in jimpy.

Mice which have the genotype qk/qk*Tajp/Y, and therefore simultaneously express both the quaking (qk) and jimpy (jp) mutations, have CNS white matter morphology intermediate between qk and jp with respect to amount of myelin, myelin structure, and oligodendrocyte number. The level of myelin basic protein in the CNS is also intermediate; however, myelin proteolipid protein (PLP) is virtually absent. Thus in the qk/qk*Tajp/Y double mutant mouse the PLP deficit is as severe as in jp alone but the oligodendrocyte survival deficit (reflected in number and myelin production) of jp alone is rendered less severe. The observation that these two cardinal deficits of the jp mutation can be independently altered in double mutant combinations is consistent with our previous suggestion that the PLP genetic locus may encode at least two independently regulated primary gene functions: a structural protein and signal influencing oligodendrocyte behavior.

Animals↗

PubMed1989

X-ray diffraction analysis of myelin lipid/proteolipid protein multilayers.

To examine the proposal that myelin proteolipid protein underlies the adhesion of neighboring membranes in central nervous system (CNS) myelin, we carried out X-ray diffraction studies on the structure and interactions of model bilayers composed of total myelin lipids plus proteolipid apoprotein (PLP). Total myelin lipids were used because their heterogeneity was expected to provide an appropriate environment for the integral membrane protein to achieve its native conformation and establish appropriate contacts with the apposed bilayer. We found that incorporation of PLP into myelin lipid bilayers, whether organized into multilamellar vesicles or oriented multilayers, did not appreciably affect the lamellar period, which ranged from 65-71 A. In oriented multilayers, the wide-angle spacing at approximately 4.8 A, which arises from the lateral packing of lipid chains and is perpendicular to the lamellar diffraction, was less oriented and weaker in bilayers containing the protein. These results indicate that PLP was incorporated into the bilayers and had a disordering effect on the hydrocarbon chains but did not extend into the spaces between bilayers. Bilayer profiles calculated from the lamellar diffraction to about 15 A spacing did not show any major changes in the distribution of electron density, suggesting that to moderate resolution, the protein was distributed uniformly across the width of the lipid bilayer. Periodicities measured from osmotically stressed multilamellar vesicles did not depend on the presence of PLP, indicating that the protein did not form stabilizing contacts between bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗