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.     

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.     

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.     

PROTEIN KINASES IN MYELIN OF RAT BRAIN: SOLUBILIZATION AND CHARACTERIZATION

Myclin from rat brain contained adenosine 3′, 5′-monophosphate (cyclic AMP)-dependent protein kinase activity, which was solubilized by 0.2% Triton X-100 and required exogenous protein substrate for its activity. Also present was a protein kinase which catalysed the phosphorylation of the endogenous substrate and which was neither solubilized by Triton X-100 nor stimulated by cyclic AMP. Sodium fluoride was required to maintain the activity of the endogenous phosphorylation, probably by inhibiting ATPase activity, but had no effect on the phosphorylation of histone by the solubilized enzyme. Protamine and myelin basic protein served as well as histone as a substrate for the solubilized enzyme. A protein kinase modulator had no effect on the endogenous phosphorylation, but inhibited histone phosphorylation by the solubilized enzyme. Cyclic AMP-binding activity was observed in both the solubilized and non-solubilized preparations. The concentration of cyclic AMP required to give half-maximal binding activity of the preparations was about 2.5 nM. The results indicate that the cyclic AMP-binding site of the protein kinase in myelin may partially be accessible, whereas the catalytic site may be integrated into the membrane structure of myelin.     

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.     

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.     

CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS

—Myelin preparations from the whole brains of 16-day-old rats and from cortical regions and brainstem, respectively, of 40-day-old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16-day-old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16-day-old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one-third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16-day-old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16-day-old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40-day-old rats was 4-fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16-day-old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin-related membranes, although the heavy fraction from 16-day-old rats also may be substantially contaminated with membranes which are unrelated to myelin.     

THE SYNTHESIS OF MYELIN IN DEVELOPING RAT BRAIN

Abstract— —The synthesis of myelin proteins has been studied in the grey and white matter slices of developing rat brain by measuring the incorporation of [³H]lysine and [¹⁴C]arginine into polypeptide. The incorporation was sensitive to cycloheximide and puromycin at 1 mM concentration. Developing rat optic nerve slices, free of retinal ganglion cells, were able to synthesize myelin basic and proteolipid proteins, but rat retinal preparation failed to synthesize myelin basic protein. Rabbit retinae were able to synthesize myelin basic and proteolipid proteins. Significant activity of the myelin marker enzyme 2',3'-cyclic nucleotide-2'-phosphodiesterase has been found in the rabbit retina but not in rat retina. The results presented in this communication suggest that myelin proteins in the rat CNS are synthesized by the oligodendroglial cells and that neurons probably do not participate.     

PHOSPHORYLATION OF ENDOGENOUS PROTEINS IN MYELIN OF RAT BRAIN

Abstract— The phosphorylation of endogenous proteins occurring in the myelin of rat brain was examined using the method of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Two myelin basic proteins and at least five more proteins were phosphorylated after incubation of myelin fraction in the presence of ATP + Mg²⁺. The apparent molecular weights of the proteins other than the myelin basic proteins were 120,000, 76,000, 60,000, 41,000 and 38,000, respectively. The proteins of mol wt 60,000. 41,000 and 38,000 were extracted by treatment with hydrochloric acid, whereas those of mol wt 120,000 and 76,000 were insoluble in hydrochloric acid and chloroform-methanol. Folch-Lees proteolipid protein was not found to be phosphorylated under the conditions studied. The endogenous phosphorylation of the proteins was not stimulated by adenosine 3',5'-monophosphate.     

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.     

MULTIPLE FORMS OF PROTEIN KINASES IN MYELIN AND MICROSOMAL FRACTIONS OF BOVINE BRAIN

Abstract— The activity profiles of the solubilized protein kinases from the microsomal and myelin fractions of bovine brain were examined by column chromatography and sucrose density gradient centrifugation. The main peak of adenosine 3',5'-monophosphate (cyclic AMP)-dependent activity with histone as substrate for each membrane enzyme was eluted with about 0.2 m -NaCl on a DEAE-cellulose column. A peak of activity stimulated with cyclic AMP was also eluted with about 0.1 m -NaCl for the microsomal enzyme. A peak with protamine and casein as substrate for the microsomal or myelin enzyme, respectively, was larger than that with histone as substrate for each enzyme. The first peak with histone as substrate on a DEAE–cellulose column appeared as two peaks on the Sepharose 6B column. The second peak with histone as substrate on DEAE–cellulose column was shown to be a holoenzyme consisting of regulatory and catalytic subunits. The holoenzyme and subunits were eluted at similar positions to each other between both membrane enzymes on Sepharose 6B column. The holoenzyme sedimented as two peaks of activity on sucrose density gradient centrifugation, both of which were stimulated with cyclic AMP. The preincubation of the holoenzyme with cyclic AMP resulted in shifting to a position of a smaller molecular size.
The results indicate the occurrence of multiple forms of protein kinases in membrane fractions of brain with respect to substrate specificity and physical property.     

ACTIVITY OF 2'',3''-CYCLIC-NUCLEOTIDE 3''-PHOSPHODIESTERASE IN REGIONS OF RAT BRAIN DURING DEVELOPMENT: QUANTITATIVE RELATIONSHIP TO MYELIN BASIC PROTEIN

2′,3′-Cyclic-nucleotide-3′-phosphodiesterase activity was examined in several regions of rat brain during development, namely optic nerve, olfactory bulb, cerebrum, cerebellum, midbrain, brain stem, and spinal cord. From 4 to 120 days the total activity increased in all regions, although the specific activity approached a constant value in adults. The developmental profile of the enzyme appeared to correlate with the onset of myelination and with the levels of myelin basic protein as well as the appearance of galactocerebroside sulfotransferase. A correlation coefficient of 0.91 was found between total basic protein, expressed as the per cent of the adult (120 day) value, and total enzyme activity over 12–42 days of age (P < 0.001) from six different brain regions as well as for whole brain. By increasing the sensitivity of the assay with the use of [³H-8]adenosine 2′,3′-cyclic monophosphate, we were able to detect activity at birth in both whole brain and spinal cord.     

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.     

ELECTROSHOCK-INDUCED SEIZURES AND THE TURNOVER OF BRAIN PROTEIN IN THE RAT

Abstract— A total of ten electroshock seizures (two seizures per day) were induced in rats beginning 3 days after an injection of [U-¹⁴C]glucose. Despite the intense stimulation, the labelling of the protein and nucleic acid fractions in the brains of convulsed animals decreased only slightly and not significantly. During the first 2 days after administration of [¹⁴C]glucose to untreated animals, there was a slight decrease in the specific activity of protein-bound glutamic acid relative to that of aspartic acid and the total protein fraction, suggesting the presence of a protein with a high content of glutamic acid and a rapid turnover.     

MYELIN SUBFRACTIONS IN DEVELOPING RAT BRAIN: CHARACTERIZATION AND SULPHATIDE METABOLISM

Centrifugation of isolated myelin on discontinuous sucrose gradients resulted in a separation into three bands and a pellet. The three bands were morphologically identical to myelin, whereas the pellet consisted primarily of vesicular membranes. These four fractions differed from one another in their lipid-to-protein ratios and in molar ratios of cholesterol:phospholipid:galactolipid. All of the fractions contained proteins typical of myelin, although the proportions of the proteins varied, with the pellet being the lowest in basic protein and proteolipid protein. High activity of 2′,3′-cyclic nucleotidase and low activity of cerebroside sulphotransferase further distinguished these fractions from the microsomal fraction. Distribution of radioactive sulphatide in the subfractions at 15 min after intracranial injection of radioactive sulphate indicated that newly-labelled sulphatide first appeared in the lipid-poor fractions, followed by the lipid-rich fractions; results of pulse-chase experiments also suggested this relationship. Several days or weeks after the injection of radioactive sulphate, most of the radioactive sulphatide was in the lipid-rich fractions.     

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.     

CHANGES IN THE PROTEIN COMPOSITION OF MOUSE BRAIN MYELIN DURING DEVELOPMENT

Abstract— Myelin was isolated from the brains of mice at various ages by a procedure involving a final purification on a continuous CsCl gradient. Myelin protein accumulated throughout development, increasing from 0.25 mg of protein/brain at 8 days of postnatal age to 3.5 mg of protein/brain at 300 days, although the rate of accumulation was greatest at about 21 days of age. Quantitative studies of the protein composition of these samples were carried out, utilizing discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Mouse brain myelin, contained (in order of increasing molecular weight) two basic proteins, an uncharacterized doublet, proteolipid protein, and a group of high molecular weight proteins. There were marked changes in the quantitative distribution of these proteins with increasing postnatal age. The basic protein fraction of total myelin protein increased from about 18 per cent at 8 days to 30 per cent at 300 days of age. Proteolipid protein increased even more dramatically, from 7 to 27 per cent in the same time interval. These chemical studies were correlated with ultrastructural investigations, both of the developing myelin sheath in situ and the isolated myelin obtained from mice of various ages. A hypothesis, relating the observed changes in protein composition of myelin during development to its mode of formation, is developed. Another subcellular fraction, separated from myelin, by virtue of its greater density in a CsCl gradient, was also studied. It was a vesicular, membranous fraction present at a level of 0.35 mg of protein/brain at all ages and was related to myelin in terms of protein composition.     

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.     

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.