Age-Related Changes of Myelin Proteins in the Rat Peripheral Nervous System

Age-related changes in amounts of myelin proteins from rat sciatic nerve or spinal root were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In the aged peripheral nerve myelin, the relative amounts of band 105K and proteins X and Y increased, whereas those of proteins P0 and P1 and band 190K decreased. Band 105K purified by preparative SDS-PAGE exhibited three bands of 105K, 28K, and 21K at the second electrophoresis. A repeated SDS-PAGE did not improve the purity of bank 105K, but increased the ratio of 21K to 28K. Compared with P0 protein, band 105K has a very similar peptide map pattern and amino acid composition, as well as the identical NH2 terminal residue, isoleucine. These findings suggest that band 105K is an aggregate form of P0 protein and its fragment, 21K. The 21K protein is a distinct entity from X protein. The quantitative and qualitative alterations in myelin proteins, as we report here, may reflect continuing demyelination and remyelination in aged peripheral nerves.     

A Monoclonal Antibody Raised to Corpus Callosum Extract Reacts with 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase

Monoclonal antibody against 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with delipidated white matter from rat corpus callosum. The antibody was characterized by solid-phase radioimmunoassay, immunoblot of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoprecipitation from C6 glioma cells, and indirect immunofluorescence staining of monolayer cultures containing oligodendrocytes. The monoclonal antibody bound specifically to an intracellular antigen of oligodendrocytes, but not to Schwann cells, astrocytes, neurons, or fibroblast cytoplasm. The immunoblot of SDS-PAGE of CNS myelin showed that the antibody identified two protein bands at 48,000 and 50,000 molecular weight. These proteins were not identified in peripheral nervous system myelin. The monoclonal antibody immunoprecipitated CNP enzyme activity from extracts of C6 glioma cells. This monoclonal antibody should prove useful in further study of this myelin-specific enzyme in CNS myelin and in cells responsible for myelin production.     

Purification of Neuropathy Target Esterase from Avian Brain After Prelabelling with [3H]Diisopropyl Phosphorofluoridate

Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The volatilizable counts assay and analytical SDS-PAGE were used to monitor the protein. The 150-kDa subunit polypeptide appears as a single band on analytical SDS-PAGE.     

Some properties and applications of Superose 6B

Applications of a new agarose-based medium for high-performance preparative gel filtration is described. The 30-micron bead size, high pore volume, and simple experimental technique to prepare highly efficient columns make Superose 6B very suitable for preparative purifications of protein mixtures in the molar mass range of 10(3) to 4 X 10(6). Sample load exceeds 100 mg/cm2, which indicates that gram quantities may be purified on K 50/60 columns. The scale-up procedure from analytical to preparative columns is demonstrated by the separation of serum samples and the industrial purification of an enzyme-antibody conjugate.     

高分子量麦谷蛋白１４和１５亚基的纯化、Ｎ—末端序列及部分生化特性研究

用ＳＤＳ－ＰＡＧＥ制备电泳技术结合一种新的凝胶中蛋白质显色方法,对普通小麦（Triticum aestivum)小偃六号的高分子量麦谷蛋白１４和１５亚基进行了有效的分离纯化,将其转印于ＰＶＤＦ膜上测定了Ｎ－端的氨基酸顺序,通过比较了发现它们与已知序列的其他的高分子是麦谷蛋白亚基高度同源。用两种双向电泳技术确定了它们的等电点（ＰＩ）属于碱性范围。     

Preparation of template plasmids for cell-free protein synthesis using a regenerated anion-exchange column

In this report, we describe how reactions of cell-free protein synthesis can be successfully conducted using plasmids prepared with regenerated anion-exchange columns. When washed, stripped, and equilibrated with appropriated buffers, regenerated columns were able to be used repeatedly to prepare plasmids with consistent yield and purity. The regenerated columns exhibited comparable performance to a fresh column with respect to the efficiency of protein synthesis using the plasmids prepared from them. Overall, we expect that the presented results will contribute significantly to economizing the technology of cell-free protein synthesis as a practical method for protein production in preparative scales.     

Recommended method for the analysis of amino acids in biological materials

Fifty-five ninhydrin-positive compounds in physiological fluids were determined with a Hitachi Model KLA-5 amino acid analyzer by a two-column chromatographic procedure. Both columns were packed with Hitachi Custom 2618 ion-exchange resin. The total analysis time was 9.5 h.In this procedure, particularly glucosamine, mannosamine and galactosamine were separated completely from normal “protein” amino acids, and N^G-monomethylarginine, N^G,N^G-dimethylarginine and N^G,N′^G-dimethylarginine, which were present in the myelin basic protein of several species and excreted in human urine, were separated from other basic amino acids. The method is useful for various applications with biological materials.     

p-Cresol sulfate is the dominant component of urinary myelin basic protein like material

Multiple sclerosis (MS) is clinically heterogeneous and has an uncertain natural history. A high priority for more effective treatment of MS is an objective and feasible laboratory test for predicting the disease's course and response to treatments. Urinary myelin basic protein (MBP)-like material (MBPLM), so designated because it is immunoreactive as a cryptic epitope in peptide 83-89 of the human MBP molecule of 170 amino acids, is present in normal adults, remains normal in relapsing-remitting, but increases in progressive MS. In the present investigation, MBPLM was purified from urine and characterized. p-Cresol sulfate is the major component of urinary MBPLM. This conclusion is based on the following: (1) MBPLM and p-cresol sulfate both have a mass of 187 on negative scans by electrospray ionization mass spectrometry, the same fragments on tandem mass spectrometry of 80 (SO(-)(3)) and 107 (methylphenol), and similar profiles on multiple reaction monitoring; (2) (1)H and (13)C nuclear magnetic resonance spectroscopy revealed identical spectra for MBPLM and p-cresol sulfate; (3) purified p-cresol sulfate reacted in parallel with MBP peptide 83-89 in the same radioimmunoassay for MBPLM; and (4) p-cresol sulfate has the same behavior on preparative HPLC columns as urinary MBPLM. The unexpected immunochemical degeneracy permitting a cross-reaction between p-cresol sulfate and a peptide of an encephalitogenic myelin protein is postulated to be based on shared conformational features. The mechanisms by which urinary p-cresol sulfate, possibly derived from tyrosine-SO(4), reflects progressive worsening that is disabling in MS are unknown.     

Protein kinase C in rat brain myelin

It has been suggested that phosphorylation of myelin basic protein (MBP) in CNS is catalyzed by protein kinase C (PKC). In order to demonstrate that PKC in the myelin phosphorylates MBP, PKC was partially purified from rat CNS myelin by solubilization with Triton X-100 followed by a DEAE-cellulose column. MBP and histone III-S were phosphorylated in the presence of Ca²⁺ and phospholipid by rat myelin PKC. High voltage electrophoresis revealed that the phosphoamino acids in MBP by this kinase was serine residue, which is known to be the amino acid phosphorylated by PKC. The activity of PKC extracted from myelin was inhibited by the addition of psychosine to the incubation mixture. To confirm the presence of PKC molecule and to identify the isoform of PKC in the myelin, the solubilized myelin fraction was applied on SDS-PAGE, transferred to a nitrocellulose sheet and stained with anti-PKC monoclonal antibodies. Rat CNS myelin contained the PKC of about 80 kDa (intact PKC), and no proteolytic fragments were observed. PKC isozymes in myelin were type II and III. A developmental study from 14 to 42 postnatal days showed that PKC activity in CNS myelin seemed to parallel the deposition of myelin protein.     

Purification and immunohistochemical localization of rat brain myelin proteolipid protein.

Abstract— A homogeneous preparation of proteolipid protein (PLP) from rat brain myelin was isolated by preparative gel electrophoresis in sodium dodecyl sulfate and chemically characterized. The results of amino acid and N-terminal amino acid analyses are reported. The same preparation of myelin PLP was used to produce specific precipitating antibodies. Rabbit and goat antisera to myelin PLP each gave a single precipitin line with purified PLP dissolved in Triton X-100. Under identical conditions, no precipitation was observed with antiserum to myelin basic protein or with control serum. Immunofluorescence localization employing antiserum to PLP demonstrated bright specific fluorescence restricted to the myelin sheaths of axons in all anatomical areas of the rat brain examined. Neuronal cell bodies and their dendrites were completely negative with respect to the presence of proteolipid protein. PLP could not be localized in the cell bodies or fibrous processes in any of the glial elements in the adult rat brain. However, myelin PLP was clearly visible in the cytoplasm and processes of actively myelinating oligodendrocytes in the corpus callosum in the brains of 10-day-old rats.     

Purification and characterization of a 36 kDa antigen of Mycobacterium leprae

A 36 kDa antigen of Mycobacterium leprae was purified by phenol biphasic partition followed by preparative SDS-PAGE. The purified antigen appeared as a single band in SDS-PAGE and eluted as a single peak in ion-exchange chromatography. The antigen comprised epitopes which were cross-reactive with M. tuberculosis, as well as a species-specific epitope (recognized by MAb F47-9). Different treatments of the 36 kDa antigen suggested it to be largely protein in nature; the amino acid composition of 81% of the antigen was determined. A majority of sera from leprosy patients contained antibodies recognizing the 36 kDa antigen.     

Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.     

Triton X-100 extractions of central nervous system myelin indicate a possible role for the minor myelin proteins in the stability of lamellae

Isolated CNS myelin membranes were extracted with Triton X-100 under conditions previously established for the isolation of cytoskeletal proteins. Treated myelin retained much of its characteristic lamellar structure despite the removal of most of the major myelin basic protein (18.5 kDa) and the proteolipid protein, which together normally constitute 60% of the total myelin protein. The SDS-PAGE profile of this extract residue demonstrated an enrichment in proteins of Mr 30 to 60 kilodaltons (the Wolfgram group). The major myelin proteins were identified by antibodies on Western immunoblots, as were the 23-cyclic nucleotide 3-phosphodiesterase (CNP), actin, tubulin, myelin-associated glycoprotein (MGP) and the 21.5 kDa MBP. The overall behavior of CNP, the 21.5 kDa MBP, MGP and tubulin towards Triton extraction is reminiscent of the behavior of other membrane-skeletal complexes, supporting the idea that these and other minor myelin proteins might be part of heteromolecular complexes with interactions spanning several lamellae of the myelin sheath.     

Myelin Structure and Composition in Zebrafish

To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were approximately 153 and approximately 162 Angstrom, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl(2)-containing Ringer's solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl(2) than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr and mbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave approximately 20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout--intermediate protein 1 (24 kDa) and intermediate protein 2 (28 kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of approximately 7 kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability.     

One-step affinity purification of amidase from mutant strains of Pseudomonas aeruginosa

Amidases (acylamide amidohydrolase EC 3.5.1.4) from mutant strains (i.e., B6, AI3, AIU1N, OUCH 4 and L10) of Pseudomonas aeruginosa were purified in one-step by ligand affinity chromatography using Epoxy-activated Sepharose 4B-acetamide. The yields of the purified enzymes were about 90% for all mutant strains with purification factors of about 10 and were apparently homogeneous when analysed by SDS-PAGE and native PAGE. The protein bands on native PAGE coincided with the stained band of enzyme activity for all amidase preparations. Affinity columns had a maximum binding capacity of 0.5 mg amidase protein/ml of sedimented gel and could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either semicarbazide or urea were also found useful for the isolation of amidase. The differences in substrate specificity of these amidases reported previously were also observed in the elution behaviour of these enzymes from the affinity columns.     

The in vivo synthesis of myelin proteins in rabbit sciatic nerve

Rabbits were injected into the sciatic nerves with either ³⁵S-methionine, or ³H-fucose. After times ranging from 45 min to 15 days the nerves were removed and the total particulate material from the nerves fractionated to give seven subfractions with densities between 0.2 and 1.2 M sucrose. The patterns of radio-labelled proteins were examined by SDS-PAGE and quantitative fluorography. The results showed that the P₂ basic protein was metabolically far more active than either the major P₀ glycoprotein, or the basic protein BP. The P₂ protein also entered the myelin fractions more rapidly than either P₀, or BP components. The net synthesis of P₀ was slower than P₂ and BP and this intrinsic membrane protein remained associated with the denser membrane fractions (>0.7 M sucrose) for longer than the basic proteins prior to entering myelin. Newly synthesized high molecular weight proteins remained concentrated in the denser membrane fractions and turned over faster than the myelin proteins.

A low density myelin fraction (B) was detected in which both the P₂ protein and certain high molecular weight proteins became more rapidly labelled than in compact myelin. In this fraction the specific activity remained higher than that of compact myelin for up to five days after the injection of ³⁵S-methionine into the nerve.

The results indicate that the major PNS myelin proteins are incorporated into and turn over in the various compartments of the Schwann cell plasma membrane—myelin continuum at very different rates.     

Colvalent linkage of phospholipid to myelin basic protein: identification of phosphatidylinositol bisphosphate as the attached phospholipid

Evidence presented demonstrates a covalent attachment of a phospholipid to bovine myelin basic protein. Partial characterization of the phospholipid moiety was performed on myelin basic protein obtained from 32P-phosphorylated whole myelin that was first delipidated by two ether/ethanol (3:2 v/v) extractions, ether extraction, and acetone extraction and then purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The myelin basic protein was precipitated with aqueous acetone and treated with proteases. Treatment with carboxypeptidase Y or trypsin for several hours released a lipophilic fragment, which was purified by reverse-phase high-performance liquid chromatography to yield two "lipopeptides". Such lipopeptides were obtained from both the major and minor myelin basic proteins of rat and bovine brain. Treatment with either mild base or phospholipase C removes the lipophilic character of the peptide fragment. The lipophilic fragment is a substrate for phospholipase D, but it does not comigrate on thin-layer chromatography with any 32P-labeled lipid obtained from myelin incubated with [gamma-32P]ATP. Polyphosphoinositides were shown to be released by mild acid treatment of myelin basic protein that had been extracted with organic solvent and then purified by SDS-polyacrylamide gel electrophoresis. Along with the fact that inositol monophosphate was identified in the partial acid hydrolysate of the lipopeptide, we have concluded that polyphosphoinositide (phosphatidylinositol 4-phosphate and/or phosphatidylinositol 4,5-bisphosphate) was the original phospholipid portion of the lipopeptide.     

Biochemical and immunohistochemical studies with specific polyclonal antibodies directed against bovine myelin/oligodendrocyte glycoprotein

Bovine myelin/oligodendrocyte glycoprotein (MOG) was purified from a Wolfgram protein fraction of brain myelin by molecular sieving and preparative gel electrophoresis. The N-terminal sequence of this wheat germ agglutinin reacting glycoprotein was determined. Antibodies against purified MOG and synthetic N-terminal octapeptide of MOG were produced in rabbits. Respective affinity purified antibody preparations gave identical results on Western blots. Treatment with specific glycosidases indicated that the oligosaccharide chains of MOG are only of N-chain type. This glycoprotein seems to be restricted to mammalian species since it was not detected in other animal species, ranging from fish up to reptiles. Immunohistochemical investigations on rat brain sections revealed that MOG is restricted to myelin sheaths and oligodendrocytes, thus corroborating previous results obtained with the MOG 8-18C5 monoclonal antibody. Decreased staining pattern in Jimpy brain further attested its specific localization in myelin-related structures. The octapeptide site-specific antibodies were not reactive on brain sections which may be attributed to the burying of this N-terminal sequence in the membrane. These MOG polyclonal antibodies appear to be valuable tools for further studies concerning this minor glycoprotein.Abbreviations BSA bovine serum albumin - CNS central nervous system - DM-20 minor myelin proteolipid protein - MAG Myelin-associated glycoprotein - MBP myelin basic proteins - MOG Myelin/oligodendrocyte glycoprotein - OMgp Oligodendrocyte/Myelin glycoprotein - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - PeptMOG n-terminal octapeptide of MOG - PLP major myelin proteolipid protein - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecylsulphate - TBS Tris buffered saline - WPF Wolfgram protein fraction - WGA Wheat germ agglutinin     

Separation of synthetic oligonucleotides on columns of microparticulate silica coated with crosslinked polyethylene imine

A rapid and effective method for separating oligonucleotides using high-performance ion-exchange chromatography is described. Columns were prepared by adsorbing a layer of polyethylene imine on 5-micron-diameter porous silica followed by crosslinking with a multifunctional oxirane. This weak anion-exchange matrix was found to be useful in the separation of mononucleotides and oligonucleosides containing up to 20 residues in either homo- or heteropolymers. Small analytical columns (4.2 X 150 mm) had a capacity ranging from less than a microgram in analytical separations to several milligrams in the preparative mode. The columns have proven to be especially useful in assessing the purity of precursor blocks, monitoring the chemical synthesis of oligonucleotides, and isolating reaction products after the synthesis.     

Activation of Complement by Myelin: Identification of C1-Binding Proteins of Human Myelin from Central Nervous Tissue

Myelin isolated from central nervous tissue activates the classic pathway of complement by directly activating C1. Activation of C1 can proceed to form membrane attack complex, C5b-9, in the myelin. Such an interaction between myelin and complement may be important in diseases involving myelin damage, in view of the role of complement in membrane attack and inflammation. To identify the C1-activating protein, myelin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The blots were incubated with C1 or with whole serum complement, followed by immunostaining for C1 or C3, respectively. A duplicate strip was stained with amido black or anti-myelin antibody to visualize the myelin proteins. The results showed that two major protein bands were capable of activating C1. An approximately 56-58-kilodalton band comigrated with the W2 protein and an approximately 45-47-kilodalton band migrated along with, but slightly behind, the W1 Wolfgram doublet.