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.     

REGULATION OF THE LEVEL OF ENDOGENOUS PHOSPHORYLATION OF SPECIFIC BRAIN PROTEINS BY DIPHENYLHYDANTOIN1

Diphenylhydantoin (DPH) in therapeutic concentrations caused a decrease in the net level of endogenous phosphorylation of two specific proteins from rat brain cerebri, while not significantly affecting the phosphorylation of other protein substrates. The apparent molecular weights of the DPH- specific substrate proteins were 60-63,000 and 49-52,000, and were designated proteins DPH-L and DPH-M, respectively. DPH decreased both the initial rate and the net level of [³²P] phosphate incorporation from [γ-³²P] ATP into proteins DPH-L and DPH-M. The concentrations of DPH required to produce a half maximal decrease in the levels of phosphorylation of proteins DPH-L and DPH-M was 3 × 10^-4 and 8 × 10 ^-4 M, respectively. The effects of DPH on the incorporation of [³²P] phosphate into these specific brain proteins were independent of the concentration of ATP over a wide range of ATP concentrations. The DPH-specific proteins were demonstrated to be present in synaptosomal preparations. The results are compatible with the hypothesis that some of the stabilizing actions of DPH on neuronal tissue and seizure discharge may be mediated by the effect of this anticonvulsant on the phosphorylation of specific brain protein substrates.     

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.     

OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA OF DEVELOPING RAT BRAIN

Abstract—

• 1 Oxygen uptake, ADP/O ratios and respiratory control ratios (RCR) were studied by oxygen electrode techniques in mitochondria prepared from developing rat brain.
• 2 Oxygen consumption, ADP/O ratios and RCR based on mitochondrial protein concentration increased with maturation. Of the substrates employed, succinate supported oxygen uptake best and malate poorest.
• 3 The addition of exogenous NAD to the mitochondrial preparation had no effect on rate of oxygen uptake.
• 4 Lack of change in ADP/O ratio in the presence of glucose, a tricarboxylic acid cycle intermediate (α-oxoglutarate), and ATP leads us to believe that there is no significant hexokinase activity in this preparation.

     

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 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.     

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.     

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.     

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.     

THE BINDING OF TRIETHYLTIN TO RAT BRAIN MYELIN

—A high affinity binding site for triethyltin was found in rat brain myelin with an affinity of approx 6·6 × 10⁵m ⁻¹ at pH 7·5. Competitive binding studies showed that triethyl-lcad had about the same affinity and trimethyltin 30 times lower affinity than triethyltin. Hexachlorophane and 3,5-diiodo-4′-chlorosalicylanilide did not prevent triethyltin binding to rat brain myelin. Since triethyltin, hexachlorophane and 3,5-diiodo-4′-chlorosalicylanilide all produce similar oedematous lesions in the brain of rats, whereas triethyl-lead and trimethyltin do not, it is concluded that the high affinity triethyltin binding site either is not involved or is not the only factor in oedema production.     

致癌大鼠肝细胞内蛋白磷酸化

本实验观察到二乙基亚硝胺(DENA)致癌大鼠肝细胞液及颗粒提取液中P21及P42磷酸化都比正常肝高,增高的程度似与肝细胞癌变相关。新生一天及四天大鼠肝中P21及P42磷酸化也有增高,但再生肝中不增高。磷酸化21的磷酸根接在丝氨酸羟基,PP21不含磷酸酪氨酸或磷酸苏氨酸,或含量极微。     

SOME IMMUNOCHEMICAL CHARACTERISTICS OF W1 AND W2 WOLFGRAM PROTEINS ISOLATED FROM RAT BRAIN MYELIN

Starting from a chloroform-methanol (2: 1 v/v) insoluble pellet of rat brain myelin, two pure proteins W1 and W2 were isolated by sodium dodecylsulphate preparative polyacrylamide gel electrophoresis. Their amino acid composition was compared. Antibodies against these proteins were prepared in rabbits. It was found that the two antigens have common antigenic similarities. The presence of one precipitin line of identity when myelin or isolated W1 and W2 from different animals were tested, led to the conclusion that there was no species specificity. The importance of the availability of such antisera is discussed.     

ASSOCIATION OF AXONALLY TRANSPORTED PROTEINS WITH GOLDFISH BRAIN MYELIN FRACTIONS

—The presence of rapidly transported axonal proteins in purified preparations of myelin has been investigated in the goldfish visual system. Fish were injected intraocularly with ³H proline and contralateral optic tecta were pooled 8–12 h later for purification of myelin. Three purification procedures were employed using continuous and discontinuous gradients of sucrose and continuous gradients of CsCl. All of the myelin preparations were found to have physical, chemical and enzymatic properties attributable to relatively pure preparations of myelin. The goldfish myelin differed from mammalian preparations in having a slightly lower density and in containing an additional major protein of approx. 45,000 mol. wt. All of the myelin preparations retained relatively high levels of axonally transported radioactivity with specific radioactivities which ranged from 70 to 80 per cent of that of the whole tectal homogenate. Acrylamide gel analysis showed the myelin-associated radioactivity to be confined to the higher molecular weight proteins with very little radioactivity associated with basic protein or proteolipid protein. Both the axonally transported radioactivity and the group of higher molecular weight proteins were found to be more concentrated in a myelin subfraction of relatively high density than in a subfraction of low density. The possible significance of the association of axonally transported proteins with myelin is discussed.     

PHOSPHORYLATION AND METHYLATION OF CHROMATIN PROTEINS FROM MOUSE BRAIN NUCLEI

Abstract— Chromatin protein kinase and histone methyltransferase are present in two nuclear populations, neuronal and oligodendroglial. At least 30% of the enzymes are tightly bound to chromatin. The specific activities of both enzymes are higher in neuronal populations than in oligodendroglial. Protein kinase from these nuclear populations phosphorylates endogenous protein; however, the methyltransferase requires exogenous histone as substrate. The methyltransferase from both nuclear populations preferentially methylates lysine-rich histone.     

THE CELL-FREE SYNTHESIS OF ALKALOID-BINDING PROTEINS IN IMMATURE RAT BRAIN1

Abstract— cell-free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [¹⁴C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble ¹⁴C-labeled proteins formed in the cell-free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of ¹⁴C-labeled amino acids. Criteria for the formation of vinblastine-binding, ¹⁴C-labeled proteins were: (1) aggregation of ¹⁴C-labeled soluble protein by one mm -vinblastine sulfate and (2) immunoprecipitation of ¹⁴C-labeled soluble protein by an antiserum against vinblastine sulfate-precipitable material. Criteria for the formation of [³H]colchicine-binding, ¹⁴C-labeled protein were based upon: (1) co-precipitation of the ³H-and ¹⁴C-labeled materials by vinblastine sulfate and (2) the coincidence of ³H- and ¹⁴C-labeled elution peaks from columns of Sephadex G-200, DEAE-Sephadex A-50 and isoelectric focusing. Both in the in vitro and in the in vivo system, ¹⁴C-labeled amino acids were incorporated into soluble proteins of the post-microsomal supernatant fraction. Proteins labeled with ¹⁴C-labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, ¹⁴C-labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G-200 which is identical to [³H]colchicine-binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine-binding and colchicine-binding proteins in the in vitro cell-free system.     

EVIDENCE FOR THE CLOSE ASSOCIATION OF A GLYCOPROTEIN WITH MYELIN IN RAT BRAIN

Abstract— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [³H]glucosamine or N -[³H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [³H]fucose and [¹⁴C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.     

FRACTIONATION OF GLYCOPROTEINS ASSOCIATED TO ADULT RAT BRAIN MYELIN FRACTIONS

Abstract— The chloroform-methanol insoluble residue of adult rat brain myelin fractions (My-CMI) contains only 20% of protein but all myelin-associated glycoproteins (Z anetta et al ., 1977a). After solubilization in sodium dodecyl sulphate, these glycoproteins were separated by sequential affinity chromatography on 4 immobilized lectins. Ten fractions (9 of which contained only glycoproteins) were obtained. Glycoproteins added up to at least 25% of My-CMI proteins. Many minor glycoproteins were detected in the different fractions. However most of them appeared not to be intrinsic to myelin. On the contrary a major glycoprotein electrophoretic band, component A, appeared to be intrinsic to myelin although its presence also on oligodendrocyte plasma membrane cannot be excluded. Component A was tentatively identified with the'major myelin associated glycoprotein'described by QUARLES (1972, 1973 a, b ). It accounted for less than 0.4% of proteins and 8% of glycoproteins of myelin fractions and consisted of at least two'glycopolypeptides'which, both, bind to concanavalin A and to the Ulex europeus lectin. The other major glycoprotein, component B, did not bind to any of the lectins used and, thus, must have N -acetyl neuraminic acid as only terminal sugar. The separation of myelin-associated glycoproteins according to their affinity for lectins allowed a tentative identification of the lectin binding sites of myelin sheath.     

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.     

吗啡及第二信使对鼠脑细胞膜蛋白质磷酸化的调节

 本文研究了几种蛋白激酶活化剂及吗啡对脑细胞膜蛋白质磷酸化的调节。cAMP刺激了一种68KDa蛋白质和几种60KDa相关的蛋白质的磷酸化作用,Ca~(++)刺激68KDa和50KDa蛋白质的磷酸化。μ吗啡受体的特异性兴奋剂D-脑啡肽(DAGO)增加68KDa蛋白质的磷酸化,而吗啡K受体的特异性兴奋剂,Bremazocyne抑制这一蛋白质的磷酸化。蛋白激酶c的特异性活化剂——磷脂酰丝氨酸(PS)和甘油二油酸酯(DO)不促进这一磷酸化。相反,却抑制cAMP、Ca~(++)、和DAGO所刺激的68KDa蛋白质的磷酸化。结果表明,在鼠脑细胞膜存在一种68KDa专一的蛋白激酶,其活性受吗啡及几种细胞内信使分子,如cAMP、Ca~(++)和DO的调节。     

ACIDIC PROTEINS IN RAT BRAIN NUCLEI: DISC ELECTROPHORESIS

—Disc electrophoretic patterns of soluble, acidic proteins in brain and liver nuclei and in the respective tissue homogenates were studied. In brain nuclei, a fast component moving with the electrophoretic front constitutes a relatively large amount of the acidic proteins which migrate toward the anode. Electrophoretic patterns obtained with fractionated brain nuclei (neuronal, astrocytic and glial) were similar to those obtained with total brain nuclei. The S-100 protein could not be detected in any of the nuclei examined. Protein patterns in brain and liver nuclei markedly differed both quantitatively and qualitatively.