METABOLISM OF HIGHLY BASIC PROTEINS OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— (1) The encephalitogenic basic protein obtained from adult rat brain by treatment with 0·03 N-HCl was demonstrable in the brain on the 10th day after birth. It showed a marked increase in quantity during the phase of active myelination.
(2) The proteins extracted under similar conditions from 5-day old rat brain contained several highly basic proteins other than the encephalitogenic basic protein. These basic proteins, which were electrophoretically similar to highly basic proteins extracted similarly from adult rat liver, are histones.
(3) For metabolic studies the entire group of highly basic proteins in the acid extract was obtained after one-step adsorption of other proteins on DEAE-cellulose equilibrated at pH 9·8
(4) After injection of [¹⁴C]lysine the fractions containing highly basic proteins, water soluble non-basic proteins and other tissue proteins of the brain showed higher relative specific radioactivities during the period 1–10 days after birth than during later stages of postnatal development. The fraction containing proteolipid protein, another myelin protein, showed a low relative specific radioactivity throughout the whole period of postnatal development. The relative specific radioactivity of proteolipid protein was somewhat higher in young than in adult rat brain.     

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.     

ELECTROPHORETIC ANALYSIS OF THE HIGHLY BASIC PROTEINS OF THE RAT BRAIN FRACTION WHICH INDUCES EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS

Abstract— A purified basic protein fraction of adult rat brain when injected into guinea pigs induced experimental allergic encephalomyelitis (EAE). The freeze-dried preparations were subjected to electrophoresis on 5% polyacrylamide gel at pH 10.6 in the presence of 8 m -urea to obtain one-step separation of highly basic proteins from other proteins. Under these conditions the highly basic proteins whose isoelectric point exceeded pH 10.0 gave seven distinct components. After staining these protein bands with naphthalene black 10B they were scanned densitometrically: the area under each peak was computed and used for calculation of the amounts of individual basic proteins. The acid extracts of rat brain contained 2.61–3.95 mg highly basic proteins/g fresh tissue.
A comparison of the electrophoretic patterns of acid extracts of rat brain and liver showed that two of the highly basic proteins (components 1 and 2) were present only in the brain and not in the liver. These two components in the brain were attributed to proteins of the myelin sheath.     

ELECTROPHORETIC CHARACTERIZATION OF BASIC PROTEINS IN ACID EXTRACTS OF CENTRAL NERVOUS SYSTEM TISSUE

A technique has been outlined for identification of myelin basic proteins in mixtures of CNS proteins. Myelin basic proteins can be recognized easily by high cathodic mobility at low pH, a unique electrophoretic pattern exhibited at high pH and a characteristic colour when complexed with Amido black. The major protein extracted at pH 3·0 from either brain or spinal cord is myelin basic protein. In the low pH electrophoretic pattern of these extracts it is the most conspicuous component and the component migrating farthest cathodically; it does not appear in comparable electrophoretic patterns of liver extracts. Guinea pig myelin basic protein appears as a single dense blue-green band in low pH electrophoretic patterns, in contrast to the other proteins which are stained greyish-blue or greyish-purple by Amido black. The pattern of rat myelin basic protein is similar except that it consists of a pair of dense blue-green bands. A third characteristic which facilitates the identification of myelin basic proteins in mixtures is a considerable cathodic mobility and electrophoretic heterogeneity at pH 10·6. Most other basic CNS proteins barely penetrate the gel at this pH. We have also examined in detail the behaviour of two other components of pH 3·0 extracts which migrate close to myelin basic protein at low pH. Both are present in pH 3·0 extracts of liver and brain but not of spinal cord, and both stain grey instead of blue-green, a characteristic which readily distinguishes them from myelin basic protein. Neither of these components affects the characteristic pattern of microheterogeneity observed in high pH electrophoretograms of myelin basic proteins. One of these components has been purified and tentatively identified as lysine-rich histone F1.     

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.     

ISOLATION OF PURIFIED BASIC PROTEIN FROM HUMAN BRAIN

—A simplified method for the isolation of an encephalitogenic basic protein from myelin is described. The basic polypeptide is easily extracted from the interfacial protein which separates when chloroform-methanol extracts of myelin are treated with a synthetic upper phase containing citrate. Evidence is presented for the purity and identity of the protein. The disc polyacrylamide gel electrophoretic pattern of this protein was consistent with a high degree of homogeneity.     

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.     

COMPARISON OF THE FATTY ACIDS OF LIPIDS OF SUBCELLULAR BRAIN FRACTIONS

Abstract— Rat brain grey and white matter were fractionated to yield myelin, nerve terminal, synaptic vesicle, nerve terminal 'ghost', and microsomal fractions of white and grey matter. Ester-type glycolipids were found in all fractions except myelin, while cerebrosides occurred in significant concentrations only in myelin and white microsomes. Comparison of the fatty acid profile of the ethanolamine- and serine-containing phospholipids showed marked differences between myelin and the particles from grey matter, while the microsomes of white matter were of intermediate composition. Docosahexaenoic acid, a minor acid in myelin, was a major fatty acid in microsomes of grey and white matter. The fatty acid composition of sphingomyelin was distinctly different in the fractions derived from grey and white matter, clustering about stearate and nervonate in the latter, but only about stearate in the grey. Marked differences in the positional distribution of fatty acids were seen within phosphatidyl choline from myelin and nerve terminals. Ribonucleic acid was found in nerve terminal and synaptic vesicle fractions. The sphingosine found in the ganglioside from microsomes of both grey and white matter was similar with respect to distribution of the C₁₈ and C₂₀ homologues.
The possibility is discussed that microsomes furnish characteristic lipids for the synthesis or renewal of specific membranes, and that these lipids are accumulated somewhat before being released.     

Chemical compositions of brain and myelin in two patients with multiple sulphatase deficiency (a variant form of metachromatic leukodystrophy)

The lipid composition of the brain, including myelin, was studied in detail in two cases with a variant form of metachromatic leukodystrophy (multiple sulphatase deficiency type). In the white matter, the sulphatide concentration was 3-4 times higher than the normal level in both cases. There was a significant accumulation of cholesterol sulphate in the brain, liver and kidney of both cases. The ganglioside pattern in the grey and white matter was abnormal, with a higher proportion of GM3, GM2 and GD3-gangliosides. Non-lipid hexosamine contents were increased 1.5-2 times in brain, 8-10 times in liver and 2-3 times in kidney. Increased amounts of glucocerobroside, ceramide lactoside and ceramide trihexoside were present in grey and white matter of both cases. Recovery of purified myelin from two patients' brains was much less than from control (1-2% in case 1 and 20-30% in case 2). The lipid composition of myelin was almost normal except for a higher proportion of sulphatide, with a decreased amount of cerebroside. The fatty acid compositions of myelin sulphatide and sphingomyelin were almost normal, while non-hydroxy fatty acids of cerebroside contained less long-chain fatty acids, as characterized by a significant increase of C16:0 and C18:0 fatty acids. The myelin polypeptide pattern by SDS-disc gel electrophoresis showed a relative decrease of basic protein and of proteolipid protein. A possible mechanism of myelin loss in MSD is discussed.     

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.     

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.     

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.     

NONHISTONE NUCLEAR PROTEINS OF RAT BRAIN

Abstract— The rat brain was dissected into cerebral cortex, cerebellum and the remaining regions. From the nuclei, isolated from these three brain sections, were extracted two fractions of nuclear sap proteins (proteins soluble in 014 M NaCl and proteins soluble in 01 M Tris-HCl buffer pH 7-6) and two fractions of nonhistone chromosomal proteins (one soluble in 0-35 M NaCl and one which is not soluble at this salt concentration). Each of these four fractions of the nonhistone nuclear proteins was further separated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the studied fractions of nuclear proteins are qualitatively identical regardless of the brain section from which the analysed protein fraction was isolated. In addition, there arc no qualitative differences in the electrophoretic patterns of nonhistone chromosomal proteins which are and which are not soluble in 0-35 M NaCl. In contrast to the qualitative similarity of the electrophoretic patterns of proteins from different sections of the brain, the amount of the nonhistone nuclear proteins is characteristic for each studied brain section. The ratio of the total nonhistone nuclear proteins to DNA is highest in the brain cortex and lowest in the cerebellum. The most expressed difference between the nuclei is in the ratio of the nonhistone chromosomal proteins soluble in 0-35 M NaCl to DNA. This ratio is 0-52 in the cortex. 0-38 in the mixture of noncortical and noncerebel-lar regions and only 0-18 in the cerebellum. The amount of the three fractions of nonhistone nuclear proteins in the nuclei of individual brain sections is proportional to the activity of the genome in these nuclei. The only exception are the nonhistone chromosomal proteins which are not soluble in 0-35 M NaCl. These proteins and the histones are present in the same amounts in nuclei isolated from all three studied sections of the brain. The results support a proposal that the nonhistone nuclear proteins are involved in the expression of the genetic activity of the cell, without the majority of the proteins in any of the four fractions being the specific regulatory molecules.     

IMMUNOCHEMICAL STUDIES ON THE BRAIN SPECIFIC PROTEIN

Abstract— In the soluble brain proteins of various species-man, ox, cat, rabbit, rat, mouse, hen, snake, frog and fish–there is a protein group which migrates more slowly than Moore's S-100 protein and faster than the albumin fraction on disc electrophoresis. The protein group is absent from any organs other than brain, and has a different pattern and number of fractions in different species. Immunochemically, the protein fraction group of the mammalian brains shows some common and identical distinctive antigenic determinants compared with the brain protein of the other animals-hen, snake, frog and fish. The protein group was designated the 'SPR' proteins, which were separated to 'P_II,', 'P_III', 'P_IV' and 'P_v' fractions. Common antigenic determinants are found in these fractions. The protein group is found in human brain in larger amounts in grey matter than in white matter and in small amounts in the cellular nuclei of human and bovine brain.     

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.     

THE HYALURONIDASE OF BRAIN

Abstract— Hyaluronidase (hyaluronate glycanohydrolase, EC 3.2.1.35), with a pH optimum of 3.7, was detected in rat and bovine brain. It degraded hyaluronic acid and, at a slower rate, chondroitin sulphate to a mixture of higher oligosaccharides with N-acetylhexosamine at the reducing end. The enzyme was enriched 5- and 6-fold in a crude lysosomal fraction of rat brain or bovine cerebral cortex, and was further purified to a total enrichment of 9-fold by ammonium sulphate fractionation. The enzyme activity in grey matter was more than twice that found in white matter, and there was no significant change in enzyme activity as a function of increasing age from the neonatal to the adult rat brain. The level of hyaluronidase activity in rat brain is considerably greaterthan that required to account for the rate of catabolism of hyaluronic acid and chondroitin sulphate measured in vivo.     

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.     

THE OCCURRENCE OF TWO MYELIN BASIC PROTEINS IN THE CENTRAL NERVOUS SYSTEM OF RODENTS IN THE SUBORDERS MYOMORPHA AND SCIUROMORPHA

Extracts containing myelin basic proteins have been prepared from CNS tissue of representatives of the three suborders of Rodentia—Myomorpha, Hystricomorpha and Sciuro-morpha. Analyses of the extracts by electrophoresis at low pH showed that one type (L) of myelin basic protein is present in the CNS of all of the rodents examined (rat, mouse, hamster, guinea pig, chinchilla, prairie dog, woodchuck and squirrel). This protein is comparable in molecular size and charge to the CNS myelin basic proteins found in several other mammalian orders. In the CNS of the myomorphs (rat, mouse, hamster) and sciuro-morphs (prairie dog, woodchuck, squirrel) there is an additional type (S) of myelin basic protein of higher cathodic mobility and smaller molecular size. This additional protein is absent from the CNS of the hystricomorphs (guinea pig, chinchilla). These findings indicate that the presence of two myelin basic proteins originally reported in the CNS of the inbred rat is not an anomaly of inbreeding. These data further suggest that the presence of a single L-type CNS myelin basic protein might be a general characteristic of hystricomorphs, while the presence of both L- and S-type CNS myelin basic proteins might be a general characteristic of the myomorphs and sciuromorphs. Analyses by electrophoresis at low pH failed to reveal differences in mobility among either the L-type or the S-type CNS myelin basic proteins of the different species. In contrast, when electrophoresis was carried out cathodically at high pH, species-related differences in mobility were observed among the L- and S-type CNS myelin basic proteins.     

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.     

White Matter Proteins in Multiple Sclerosis

Abstract: The SDS-soluble membrane proteins of plaques and of macroscopically normal white matter from multiple sclerosis brain were investigated by gradient polyacrylamide gel electrophoresis (PAGE). Eleven protein bands were analyzed in detail. The extensive loss of myelin proteins in plaque samples was accompanied by changes in at least three other non-myelin proteins, besides glial fibrillary acidic protein (GFAP), which probably reflect gliosis. Densitometric analysis of the PAGE patterns of membrane fractions from MS and control white matter revealed significant quantitative differences in a number of protein bands. A reduction in myelin basic protein (BP) was associated with an equally significant increase in a high-molecular-weight peptide fragment which may prove to be a breakdown product of BP. Small but highly significant differences in the Wolfgram protein and in one non-myelin protein were also a consistent feature of the normal-appearing white matter samples. The problem of defining normal white matter in multiple sclerosis brain is discussed in relation to the results of the present study, which suggest that one of the early events in the pathogenesis of the disease prior to frank demyelination is an alteration in the protein components of the myelin sheath and possibly of glial cells.