THE INTRINSIC FLUORESCENCE CHARACTERISTICS OF THE MYELIN BASIC PROTEIN

—The encephalitogenic basic protein has been isolated from the myelin sheath of ox brain white matter and the purity and amino acid composition have been verified. The intrinsic fluorescence characteristics of the purified basic protein have been determined and the results interpreted in terms of current ideas on the structure of the protein. Fluorescence data obtained from the basic protein in aqueous solution indicate that the tyrosine and tryptophan residues are largely exposed to the solvent and that resonance energy transfer from tyrosine to tryptophan is very inefficient. Denaturing conditions in 8 m -urea have little effect on the fluorescence properties of the protein. The ionic detergent, sodium dodecyl sulphate, interacts with the basic protein and alters the fluorescence properties in a manner which indicates that the tryptophan residue is in the hydrocarbon chain region of the detergent while the local positive charge around the tyrosine residues is neutralized by the negatively charged sulphate head-groups. The fluorescence results suggest that the basic protein can be used as a natural, non-perturbing probe which will report on its environment after it has reacted with other membrane components.     

THE ENCEPHALITOGENIC ACTIVITY AND MYELIN BASIC PROTEIN CONTENT OF ISOLATED OLIGODENDROGLIA

Abstract— Oligodendroglia isolated from frozen human brain induced EAE in the guinea pig when injected with Freund's complete adjuvant. An acid extract of the isolated cells was highly encephalitogenic. The disease was clinically and histologically similar to EAE induced with CNS white matter or myelin basic protein(MBP). The isolated cells were essentially myelin-free but contained a large amount of myelin basic protein as determined by polyacrylamide gel electrophoresis. It was shown that the cells can absorb ¹²⁵I-MBP during isolation and the possibility that contamination from extra cellular MBP is responsible for the encephalitogenic activity is considered.     

METHYLATION OF MYELIN BASIC PROTEIN BY ENZYMES FROM RAT BRAIN

Abstract— In rat brain Methylase l activity ( S -adenosyl- l -methionine: protein-arginine methyl-transferase) is found predominantly in the cytoplasmic fraction, and it appears that several enzymes contribute to this activity. No evidence for the existence of two enzymes specific for the methylation of histone and myelin basic protein was found. The specific activity of Methylase I did not increase at the period of rapid synthesis of myelin basic proteins. Methylase I activity was strongly inhibited by S -adenosyl- l -homocysteine.     

ALTERATION OF MYELIN BIOSYNTHESIS IN SLICES OF RABBIT SPINAL CORD BY ANTISERUM TO MYELIN BASIC PROTEIN AND BY PUROMYCIN 1

Abstract— Slices of rabbit spinal cord were incubated with [³H]tyrosine and [³⁵SO₄] in the presence of either 5% antiserum to myelin basic protein or 0.21 mM-puromycin. The degree of incorporation of the precursors into the basic protein (BP), the proteolipid protein (PLP) and into sulphatides, as a representative lipid, in isolated myelin was investigated. Anti-BP serum inhibited the incorporation of [³H]tyrosine into BP and PLP from 22 to 46% as compared to controls, whereas puromycin nearly completely inhibited incorporation. The incorporation of [³⁵SO₄] into sulphatides was inhibited by anti-BP serum from 20 to 34% and by puromycin from 33 to 65% as compared to controls. These alterations were myelin-specific as shown by the equal or even increased incorporation of the precursors into the homogenates of spinal cord. The results are discussed in relation to the interaction of lipids and proteins in membrane assembly.     

MICROHETEROGENEITY AND PHOSPHOAMINO ACIDS IN THE CARBOXY-TERMINAL HALF OF MYELIN BASIC PROTEIN

—Three chromatographically distinct peptic peptides (F80-1, F80-2 and F80-3) derived from the C-terminal half of the bovine and guinea-pig myelin basic proteins were characterized. The three peptides of each animal species had the same N-terminal residue (phenylalanine) and essentially the same amino acid composition, but they differed in electrophoretic mobility at alkaline pH. The least basic peptide (F80-3) differed from the others in showing a deficit of C-terminal arginine residues and in containing phosphorus, 0·37 and 0·46 g-atom/mol of bovine and guinea-pig peptides, respectively. Other peptic peptides. derived from the N-terminal half of the basic protein, were essentially phosphorus-frcc. Analyses of partial acid hydrolyzates of peptide F80-3 by high voltage electrophoresis showed the presence of both phosphoserine and phosphothreonine. After incubation with E. coli alkaline phosphatase (EC 3.1.3.1). 34 and 40% of the bovine and guinea-pig F80-3 peptides. respectively, were converted to peptide F80-1. This reaction involved the loss of 2 net negative charges, and its extent corresponded to loss of essentially all of the phosphate originally present in the peptide. This result indicated that the phosphorylated species of peptide F80-3 contained one phosphate group per molecule.     

THE PRESENCE OF CYSTEINE IN FROG MYELIN BASIC PROTEIN

—Frog myelin basic protein, when subjected to ion-exchange chromatography at alkaline pH, underwent conversion to a higher molecular weight form. Treatment of the latter with 2-mercaptoethanol regenerated the monomeric basic protein. Amino acid analysis of the monomer and the higher molecular weight species after performic acid oxidation demonstrated the presence of approximately 1 mol of cysteic acid per mol of protein of molecular weight 19,700. Treatment of the monomer with the mild oxidizing agent azodicarboxylic acid bis dimethyl amide resulted in its partial conversion to the higher molecular weight form. These studies demonstrate that the frog myelin basic protein, unlike those of all other species hitherto examined, contains a single cysteinyl residue in its polypeptide chain.     

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.     

METHYLASES OF MYELIN BASIC PROTEIN AND HISTONE IN RAT BRAIN

—The two enzymes methylating myelin basic protein and histone were purified 170- and 250-fold respectively from the cell sap fraction of rat brain. These enzymes methylated only arginine residues of the two proteins. The enzyme activities were present in all organs tested. Testis has the highest, brain a moderate and liver the lowest activity. Most of the activities were present in the cell sap fraction in brain, liver and testis. Methylation of myelin basic protein and histone was examined in both the cell sap and solubilized nuclear fraction of rat brain during life span after birth. The myelin basic protein methylating activity in the cell sap fraction increased during myelination. Histone methylase from the nuclear fraction was highest at birth and dropped rapidly thereafter. The other activities remained unchanged. The natural occurrence of N^G-mono- and N^G,N^G-dimethylarginine residues in histones prepared from rabbit liver was demonstrated.     

MYELIN BASIC PROTEIN MICROHETEROGENEITY IN SUBFRACTIONS OF RAT BRAIN MYELIN

Abstract— Myelin subfractions were prepared from adult rat brain by discontinuous sucrose gradient ultracentrifugation. Gel electrophoretic studies at pH 10.6 in the presence of urea revealed differences in basic protein microheterogeneity among subfractions. With increasing myelin density there was a decrease in the most positively charged components of both large BP and small BP. Since these components are the least modified by deamidation and phosphorylation, it seems likely that the heavier myelin subfractions are enriched in the more modified components of the microheterogeneous population of BP. These observed differences may be related to the regulatory processes controlling biosynthesis, organization, and catabolism of BP in CNS myelin.     

LOCALIZATION OF A BASIC PROTEIN IN THE MYELIN OF VARIOUS SPECIES WITH THE AID OF FLUORESCENCE AND ELECTRON MICROSCOPY

In this study, alanine was shown to be the N-terminal amino acid of a basic protein of low molecular weight that was isolated from either human or guinea pig brain. Antibodies prepared against the guinea pig protein were labeled with either fluorescein or ferritin. Studies with the labeled antibodies showed that an immunohistochemically similar protein is found in the myelin sheaths of central and peripheral nervous tissues of chicken and frog and a variety of mammalian species. Loss of integrity of the myelin during processing was shown to enhance markedly the antigen-antibody reaction.     

A RADIOIMMUNOASSAY FOR MYELIN BASIC PROTEIN AND ITS USE FOR QUANTITATIVE MEASUREMENTS

—A specific radioimmunoassay (RIA) for myelin basic protein is described which is sensitive to 10⁻⁹ g of basic protein. The amount of basic protein detected in isolated myelin by the RIA and by SDS-gel electrophoresis and spectrophotometric quantitation agree to within experimental error. In contrast to isolated myelin, the major portion of the basic protein in fresh tissue is not accessible to its antibody. It is shielded from its antibody in a complex which is disrupted by heat, organic solvents, and various detergents. Maximum antibody binding was obtained with tissue heated to 100°C for 10 min. It is possible to calculate that the RIA quantitatively detects basic protein in boiled tissue. Boiled adult rat brain contains approximately 2·5 μg of basic protein/mg wet wt of cerebral cortex. The antibody to basic protein has no capacity to bind non-neural tissues.     

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.     

SUBCELLULAR DISTRIBUTION AND STRUCTURAL POLYMORPHISM OF MYELIN BASIC PROTEIN IN NORMAL AND JIMPY MOUSE BRAIN

Abstract— Brains from 20 day old normal and 20 day old Jimpy mice were fractionated by a modification of the procedure described by Eichberg et al. (1964). Each of the fractions obtained was subjected to radioimmunoassay (RIA) for myelin basic protein (MBP). From both the normal brain and the Jimpy brain MBP was recovered in three separate membrane fractions designated P1A. P2A. and P3A. which differed in their sedimentation properties but which had similar densities (less than 1.08 g'ml). In the Jimpy brain compared to normal brain the amounts of P1A and P2A were greatly reduced but the amount of P3A was increased. During development in the normal brain the amount of MBP in the PIA fraction increased in parallel with the accumulation of myelin. The amount of MBP in P2A increased gradually during active myelination and decreased slightly in the adult. The amount of MBP in P3A increased sharply during the period of most active myelination and decreased approx 10-fold as the rate of myelination in the brain declined. Electron microscopic examination revealed that the P1A and P2A fractions from normal brain contained myelin fragments while the P1A and P2A fractions from Jimpy brain contained numerous vesicular membranous structures with little if any identifiable myelin. The P3A fraction from both normal and Jimpy brain contained small vesicles of uniform size, some with polyribosomes attached. Each of the fractions was analyzed by a technique combining sodium dodecyl sulfate polyacrylamide gel electrophoresis with RIA for MBP in order to identify and quantitate the four different forms of MBP with molecular weights of 21.5 K. 18.5 K. 17 K and 14 K dalton. The proportions of the four MBPs were characteristic for each fraction. The relative proportions of the four proteins were 14 K > 18.5 K > 17 K > 21.5 K daltons in all the fractions except P1A Jimpy in which 21.5 K dalton protein was the predominant form of MBP present. The cellular origin of the MBP containing fractions from normal and Jimpy brain is discussed.     

THE EFFECT OF TRIETHYLLEAD ON TOTAL AND MYELIN PROTEIN SYNTHESIS IN RAT FOREBRAIN SLICES

Rats (20-day-old) were acutely intoxicated with triethyllead and their forebrains were studied during the following 14 days. All the lead in the tissue was found in the form of triethyllead, proving that the toxin per se was responsible for the pathological changes observed in the organ. The incorporation of [¹⁴C]leucine into the acid-insoluble protein was suppressed in the forebrain slices prepared from the intoxicated animals as well as in the slices, to which PbEt₃ was added in vitro. In both systems the synthesis of myelin protein was inhibited more than the total protein synthesis. The results suggest a specificity of triethyllead toward processes involved in the furnishing of the myelin membrane proteins.     

MYELIN BASIC PROTEIN PHOSPHATASE ACTIVITY IN RAT BRAIN

Abstract— Previous work from this and other laboratories has demonstrated phosphorylation of myelin BP in vivo and in vitro. The rapid turnover of BP phosphate has suggested the presence of a phosphatase. The present studies have identified two BP phosphatases. One is present in the cytosol of rat brain homogenate. It has the highest specific activity (37 pmol/min/mg) and total activity of BP phosphatase present in any subcellular fraction. The partially purified cytosol enzyme can readily dephosphorylate soluble ³²P-labelled BP but is only half as effective in dephosphorylating membrane-bound BP. Conversely, the phosphatase which remains associated with highly purified myelin is 2.3 times as effective on BP in the membrane (7.2 pmol/min/mg) as on soluble BP (3.2 pmol/min/mg). The myelin phosphatase is tightly bound to the membrane and cannot be removed with concentrated salt solutions. During development the specific activity of the cytosol phosphatase remains constant. The specific activity of the myelin phosphatase, however, is twice as high during the period of maximum myelin formation (6.8 pmol/min/mg at 18 days) as it is in adult myelin (3.2 pmol/min/mg at 12 weeks).
In order to compare enzyme effectiveness under the various conditions employed in these studies, we have assumed that both soluble and particulate substrates are phosphorylated at equivalent sites on the polypeptide. We have further assumed that soluble and/or particulate substrates are dephosphorylated at equivalent sites on the polypeptide chain and that the various particulate and soluble enzymes have comparable access to the substrate. Within the limitations of these assumptions, our data suggest myelin phosphatase may play a significant role in phosphate turnover of BP.     

THE PROTEIN COMPOSITION OF ISOLATED MYELIN1

—Three fractions, each containing markedly different proteins, was obtained from myelin: (1) The first fraction was obtained as an insoluble residue when myelin was extracted with neutral chloroform-methanol (CM, 2:1, v/v). It was digestible with trypsin and had an amino acid composition similar to that of the acidic proteolipid protein of Wolfgkam (1966). (2) The second fraction was obtained as a precipitate by the addition of various electrolytes (KCl, NaCl, CaCl₂, MgCl₂ or HCl) to the CM (2:1 v/v) extract. This fraction consisted mainly of a basic protein which exhibited an electrophoretic mobility and amino acid composition indistinguishable from those of the basic protein obtained from white matter (Martensson and LeBaron, 1966). This procedure provided for a simple and rapid isolation of the basic protein from myelin. Depending on the conditions of precipitation, this fraction was either free of lipid or contained tri- and diphosphoinositide. The effects of different ions at differing concentrations and the yield and nature of the precipitate have been studied. (3) A third fraction remained in solution in CM (2:1, v/v) after the addition of the electrolyte. It comprised the bulk of the myelin lipids and a protein fraction which was resistant to digestion with trypsin and had an amino acid composition similar to the classical proteolipid protein of Folch-Pi and Lees (1951). The possibility of a salt-type bonding between the basic protein and the polyphosphoinositides is discussed, and values for tri- and diphosphoinositide in bovine myelin are given.     

COMPARATIVE STUDIES OF HIGHLY BASIC PROTEINS OF OX BRAIN AND RAT BRAIN. MICROHETEROGENEITY OF BASIC ENCEPHALITOGENIC (MYELIN) PROTEIN

(1) The total amount of highly basic proteins in acid extracts of whole ox brain, ox white matter and ox grey matter was determined quantitatively after electrophoresis on 5% polyacrylamide gels at pH 10-6 in the presence of 8 M-urea. (2) Ox white matter gave 13 mg and ox grey matter 2 mg of highly basic proteins per g fresh tissue on treatment with 0-03 n -HCl. The yield of total basic proteins of ox white matter increased to 17-6 mg/g fresh brain on stepwise extraction at pH 3-0, 2-0 and 1-0; the extract at pH 3.0 accounted for 90 per cent of the total basic proteins. (3) The high encephalitogenic activity of the fraction of highly basic proteins extracted at pH 3.0 from ox white matter indicated that these basic proteins were derived from myelin. It is suggested that the amount of basic proteins in a sample of brain extracted under these conditions is proportional to the amount of white matter in the sample. (4) The encephalitogenic (myelin) basic protein fraction was homogeneous with respect to molecular size but could be resolved into at least six components by electrophoresis at high pH. (5) The myelin basic proteins extracted from ox white matter had lower electrophoretic mobilities at high pH than did those of two basic proteins of rat brain apparently derived from myelin.     

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.     

PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.