脊髓和周围神经可溶性酸性蛋白质的研究

 利用微型双向电泳、SDS电泳、免疫印迹法、DEAE-Sephadex色谱、高效液相色谱及氨基酸分析等方法,对牛脊髓(中枢神经)和马尾神经(周围神经)的可溶性酸性蛋白质进行了研究。结果表明在牛脊髓和马尾神经中有钙调素(CaM)、S-100蛋白和神经元特异烯醇化酶(NSE)等可溶性酸性蛋白质存在;脊髓中这些酸性蛋白质的含量远较马尾神经为高。     

IMMUNOCHEMICAL AND BIOCHEMICAL STUDIES DEMONSTRATING THE IDENTITY OF A BOVINE SPINAL CORD PROTEIN (SCP) AND A BASIC PROTEIN OF BOVINE PERIPHERAL MYELIN (BF)

A protein extracted from bovine peripheral myelin (BF) and a protein extracted from bovine spinal cord (SCP) have been shown to be identical: the proteins cross-react immunochemicaliy with each other but not with highly purified CNS myelin basic protein. Neither BF nor SCP have anti-encephalitogenic activity. Their electrophoretic behavior is the same at three different pH values. Their apparent molecular weight by sodium dodecyl sulfate-gel electrophoresis is 13,800 ± 550. The amino acid compositions of the proteins are essentially identical. BF and SCP each contain 2 cysteine residues and have valine at the C terminus. The 23 major tryptic peptides are identical on peptide maps. Circular dichroic analyses yield essentially identical curves, which, when computed by best-fit curve analysis, indicate that each has 0%α helix and a large percentage of β structure.     

DISTRIBUTION AND OPTICAL ACTIVITY OF THE BASIC PROTEIN IN BOVINE PERIPHERAL NERVE MYELIN

Abstract— Antiserum to BF protein isolated from bovine spinal roots has been used to study the distribution of the protein in other species and tissues.
Significant amounts of protein could be demonstrated in bovine, pig and rabbit peripheral nerve myelin. It was, however, scarcely detectable in guinea pig peripheral nerve myelin. There was BF protein in rabbit spinal cord as well as in peripheral nerve, but little or no BF protein in the liver, kidney, muscle or brain. BF protein in bovine spinal cord was localized in the myelin. The ratio of the BF protein to the encephalitogenic protein in the spinal cord myelin was around 0.15:1.0. BF protein was extractable from peripheral nerve myelin by saline as well as by acid solutions.
The circular dichroism spectrum of the BF protein in aqueous solution suggested that this protein contained a very large amount of β-structure. This structure was not considered to be the result of acid denaturation because the protein purified from the saline extract of peripheral nerve also showed a similar spectrum.     

HISTOCHEMISTRY OF MYELIN XIV PERIPHERAL NERVE MYELIN PROTEINS: ELECTROPHORETIC AND HISTOCHEMICAL CORRELATIONS

Abstract— Myelin from the peripheral nervous system has been shown to contain two basic protein components and an electrophoretically slower-moving major protein, the 'J' band. The 'J' band protein cannot be selectively removed by aqueous or organic solvents and does not correspond to proteolipid or acidic protein. Histochemical stains applied to peripheral nervous systems myelin proteins separated by polyacrylamide electrophoresis indicate that 'J' band protein is analogous with the neurokeratin of the nerve sheath. Trypanophilia observed histochemically in unfixed myelin is principally due to basic proteins. With prolonged tryptic digestion 'J' band protein is degraded. Thus, previous classifications of myelin proteins based on trypsin sensitivity have been modified. All peripheral nervous system myelin proteins should be regarded as trypsin-sensitive, the basic protein being relatively more and the 'J' band protein relatively less susceptible.     

ISOLATION AND CHARACTERIZATION OF GLYCOSAMINOGLYCANS IN PERIPHERAL NERVE AND SPINAL CORD OF MONKEY

—The isolation of uronic acid-containing glycosaminoglycans from peripheral nerve and spinal cord of monkey was done by combining the cetyl pyridinium procedure and DEAE-Sephadex column chromatography. The constituent analyses of the isolated GAG-fractions indicated that hyaluronic acid, chondroitin-4-sulphate, chondroitin-6-sulphate, heparan sulphate and a testicular hyaluronidase-resistant galactosamine-containing GAG were present in both tissues. Hyaluronic acid was the predominant GAG (63 per cent) in both tissues and its level was much higher than in brain. Chondroitin-4-sulphate constituted 16 per cent in both tissues. The levels of heparan sulphate and hyaluronidase-resistant galactosamine-containing GAG in these tissues were much lower than in brain. The results indicate that the patterns of GAGs in peripheral nerve and spinal cord of monkey are similar but differ from that of brain.     

RADIOAUTOGRAPHIC STUDIES OF CHOLINE INCORPORATION INTO PERIPHERAL NERVE MYELIN

This radioautographic study was designed to localize the cytological sites involved in the incorporation of a lipid precursor into the myelin and the myelin-related cell of the peripheral nervous system. Both myelinating and fully myelinated cultures of rat dorsal root ganglia were exposed to a 30-min pulse of tritiated choline and either fixed immediately or allowed 6 or 48 hr of chase incubation before fixation. After Epon embedding, light and electron microscopic radioautograms were prepared with Ilford L-4 emulsion. Analysis of the pattern of choline incorporation into myelinating cultures indicated that radioactivity appeared all along the length of the internode, without there being a preferential site of initial incorporation. Light microscopic radioautograms of cultures at varying states of maturity were compared in order to determine the relative degree of myelin labeling. This analysis indicated that the myelin-Schwann cell unit in the fully myelinated cultures incorporated choline as actively as did this unit in the myelinating cultures. Because of technical difficulties, it was not possible to determine the precise localization of the incorporated radioactivity within the compact myelin. These data are related to recent biochemical studies indicating that the mature myelin of the central nervous system does incorporate a significant amount of lipid precursor under the appropriate experimental conditions. These observations support the concept that a significant amount of myelin-related metabolic activity occurs in mature tissue; this activity is considered part of an essential and continuous process of myelin maintenance and repair.     

GANGLIOSIDES OF PERIPHERAL NERVE MYELIN

—Gangliosides have been isolated from myelin obtained from three types of peripheral nerve: bovine spinal roots, bovine sciatic nerve and human sciatic nerve. Yields in most cases were 218–287 μg of lipid-bound sialic acid per g myelin, less than half that previously obtained from CNS myelin. Myelin accounted for approx 60% of total ganglioside present in whole spinal root. The human sample contained only N-acetylneuraminic acid but the two bovine preparations contained that as well as N-glycolylneuraminic acid; N-acetylglucosamine and N-acetylgalactosamine were both present in all three preparations. Sphingosine was the major long-chain base in each preparation while 4-eicosasphingenine (d20:1) comprised about 14% in the two bovine samples and 3% in the human sample. The major fatty acids in all preparations were 16:0, 18:0, 22:0, 24:0 and 24:1. Sialosylgalactosyl ceramide (G₇), a ganglioside characteristic of CNS myelin, was not detected in any of the PNS samples. The majority of gangliosides in bovine spinal root myelin were monosialo species, although the structures differed in some respects from those of CNS myelin. The molar concentration of lipid-bound sialic acid in PNS myelin is roughly equivalent to that of the P₁ basic protein.     

THE ROLE OF WATER IN THE STRUCTURE OF PERIPHERAL NERVE MYELIN

In the study of the drying kinetics of nerve fibres, at least five "phases" of water evaporation can be distinguished. A consideration of the accompanying changes in low-angle x-ray diffraction patterns permits a tentative identification of the "phases" and a quantitative interpretation of the data in terms of the water distribution in nerve fibres. These results suggest that the myelin sheath of frog sciatic nerve contains 40 to 50 per cent water, and it is suggested further that the greater part of this water is "organised" in relation to the hydrophilic groups of the lipide and protein components.     

THE BIOSYNTHESIS OF TRIPHOSPHOINOSITIDE BY PURIFIED MYELIN OF PERIPHERAL NERVE

The distribution of diphosphoinositide kinase activity in homogenates and myelin of rabbit nerve was determined by measuring the synthesis of labelled triphosphoinositide. Evidence is presented to show that the biosynthesis of triphosphoinositide in peripheral myelin involves a membrane-bound diphosphoinositide kinase.     

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 IONIC STRENGTH OF IMMERSION MEDIUM ON THE STRUCTURE OF PERIPHERAL NERVE MYELIN

A study of the effects of hypertonic solutions on the structure of peripheral nerve myelin reveals an expansion rather than a contraction of the layer spacing. This suggests the absence of "free" water between the myelin layers. Hypotonic solutions bring about a change in radial repeat period from 171 A to 250 to 270 A. These findings are of significance in relation to the structure of myelin.     

COMPARATIVE STUDIES OF GUINEA PIG AND BOVINE MYELIN BASIC PROTEINS. PARTIAL CHARACTERIZATION OF CHEMICALLY DERIVED FRAGMENTS AND THEIR ENCEPHALITOGENIC ACTIVITIES IN LEWIS RATS

Guinea pig and bovine myelin basic proteins were chemically cleaved at the carboxyl peptide bonds of methionyl and tryptophanyl residues to yield several fragments. Comparison of the bovine fragment consisting of the first 20 residues of the protein with the corresponding guinea pig fragment showed that the latter differs in containing histidine and glycine (one residue of each), an additional threonyl residue, and one fewer alanyl residues. Comparison of the bovine fragment consisting of the C-terminal 54 residues of the protein (residues 117-170) with the corresponding guinea pig fragment showed that the latter differs in containing one fewer histidyl and leucyl residues and an additional phenylalanyl residue. Tests of encephalitogenic activity in Lewis rats showed that these two fragments from both species were much less active, on a molar basis, than the uncleaved protein. On the other hand, examination of the bovine fragments consisting of residues 1-116 and 21-116 and the corresponding fragments obtained from the guinea pig protein revealed activity at least as high as that of the respective uncleaved proteins.     

THE ISOLATION AND PROPERTIES OF LARGE BASIC PEPTIDES FROM BOVINE SPINAL CORD

Abstract— Two basic peptides (B1 and B2) were derived from bovine spinal cord following in situ proteolysis at 37°C for 10–24 h. These peptides do not arise as degradation products from the A1 protein as shown by amino acid composition and end group analysis; rather they appear to originate from some larger basic protein in the spinal cord having similarities to the P2 protein, a basic protein found in peripheral nerve myelin. The peptides were purified following defatting, acid extraction, and ammonium sulphate fractionation, by chromatography on Amberlite IRC-50 resin using guanidinium chloride. The peptides, found generally in a 4:1 ratio of B1 to B2, appeared homogeneous on gel electrophoresis and immunodiffusion. Approximately 25–60 mg of peptides was obtained per 100 g wet spinal cord.
In contrast to the basic A1 protein from myelin, neither of these peptides nor their pepsin digests were encephalitogenic. They do not cross-react immunologically with the basic A1 protein, but cross-react with each other. These peptides further differ from the A1 protein in their tryptic peptide map, size (B1, 63 residues; B2, 54 residues), and composition particularly the high lysine: arginine ratio, and low histidine content. Like the A1 protein, however, they contain a tryptophan residue and a blocked NH₂-terminal amino acid; peptide Bl has COOH-terminal valine. It was concluded that the basic peptides represent a fragment of a hitherto unidentified protein(s) of the nervous system.     

HISTOCHEMISTRY OF MYELIN–XV. CHANGES IN THE MYELIN PROTEINS OF THE PERIPHERAL NERVE UNDERGOING WALLERIAN DEGENERATION–ELECTROPHORETIC AND MICRODENSITOMETRIC OBSERVATIONS

The chronological order of changes in rat peripheral nerve proteins during Wallerian degeneration has been investigated by microdensitometric and electrophoretic techniques. Both methods revealed an early loss of myelin proteins. The histochemical microdensitometric study showed a very substantial early loss of stainable protein basic groups and a somewhat slower progressive loss of the major protein component of peripheral nerve myelin (the J band). The electrophoretic study showed an early loss of both the J band protein and the slower-moving basic protein band. The histochemical study also suggested that some cerebroside may be lost in the early stage of Wallerian degeneration. It is concluded that degradation of myelin proteins is an initial event in the process of myelin breakdown.     

ELECTRON MICROSCOPE AND LOW-ANGLE X-RAY DIFFRACTION STUDIES OF THE NERVE MYELIN SHEATH

1. A close correlation has been obtained between high resolution electron microscopy and low-angle x-ray diffraction studies of the myelin sheath of frog and rat peripheral and central nerves. Extensive studies were performed by application of both techniques to the same specimens, prepared for examination by OsO₄ or KMnO₄ fixation, and embedding either in methacrylate or in gelatin employing a new procedure. Controlled physical and chemical modifications of the myelin sheath prior to fixation were also investigated. 2. A correspondence was established between the layer spacings observed in electron micrographs and the fundamental radial repeating unit indicated by the low-angle x-ray diffraction patterns. The variations in relative intensities of the low-angle x-ray reflections could be related to the radial density distributions seen in the electron micrographs. 3. An analysis of the preparation procedures revealed that OsO₄ fixation introduces a greater shrinkage of the layer spacings and more pronounced changes in the density distribution within the layers than KMnO₄ fixation. The effects of methacrylate and gelatin embedding are described, and their relative merits considered in relation to the preservation of myelin structure by OsO₄ fixation. 4. The experimental modifications introduced by freezing and thawing of fresh whole nerve are described, particularly the enhancement of the intermediate lines and the dissociation of the layer components in the myelin sheath. A characteristic collapsing of the radial period of the sheath is observed after subjecting fresh nerve trunks to prolonged and intense ultracentrifugation. 5. Controlled extraction of fresh nerve with acetone at 0°C., which preferentially removes cholesterol, produces characteristic, differentiated modifications of the myelin sheath structure. Electron microscopy reveals several types of modifications within a single preparation, including both expanded and collapsed layer systems, and internal rearrangements of the layer components. Alcohol extraction leads to a more extensive structural breakdown, but in certain areas collapsed layer systems can still be observed. The components of the lipide extracts could be identified by means of x-ray diffraction. These modifications emphasize the importance of cholesterol in the myelin structure, and disclose a resistance of the dense osmiophilic lines to lipide solvents. 6. The significance of these structures is discussed in relation to present concepts of the molecular organization of myelin. The available evidence is consistent with the suggestion that the primary site of osmium deposition is at the lipoprotein interfaces and that the light bands probably represent regions occupied by lipide chains. The electron microscope and x-ray diffraction data also indicate the possibility of a regular organization within the plane of the layers, probably involving units of 60 to 80 A. The myelin sheath is regarded as a favourable cell membrane model for detailed analysis by combined application of x-ray diffraction and electron microscopy.