Synthesis of myelin basic proteins in the developing mouse brain.

Mice ranging in age from 14 to 39 days were injected intracerebrally with [³H]lysine and rates of incorporation of the isotope were measured into total trichloroacetic acid-precipitable protein and purified myelin basic proteins (MBPs). MBPs were isolated by O-(carboxymethyl)-cellulose chromatography of pH 3 extracts prepared from chloroform-methanol insoluble residues of whole brains. The MBPs prepared in this fashion were further separated by polyacrylamide gel electrophoresis. The gels were sliced and the radioactivity incorporated into each of the two proteins was determined. Analysis of the rates of synthesis of the two basic proteins (using a 2-h labeling period) as a function of age revealed that synthesis of both proteins appeared to peak at about 18 days of age in the mouse. These data suggest that the maximum rate of MBP synthesis coincides with the age of maximal myelin deposition in the mouse. Furthermore the relative rates of synthesis of L and S changed considerably over the developmental period examined. It was observed that the ratio of the rates of synthesis of the small:large basic protein (S/L) increased by approximately 50% between 2 and 4 weeks and declined thereafter. Throughout the developmental period examined, however, the small basic protein appeared to be synthesized at a greater rate than the large protein. The latter data are consistent with previous observations by us and other workers that mouse and rat myelin becomes increasingly enriched in the small relative to the large basic protein with maturation of the membrane.     

Phosphoprotein phosphatases for myelin basic protein in myelin and cytosol fractions of brain.

Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) activity for myelin basic protein was found to be present in the myelin fraction of rat brain. The enzyme activity was in a latent form and solubilized by 0.2% Triton X-100 treatment with about 50% increase of activity. The cytosol fraction from bovine brain also had phosphoprotein phosphatase activity for myelin basic protein, which was resolved into at least two peaks of activity on DEAE-cellulose column chromatography. Myelin basic protein was the best substrate for both the solubilized myelin fraction and the cytosol enzymes among the substrate proteins tested. The Km values of the solubilized myelin fraction were 4.2 muM for myelin basic protein, 7.4 muM for arginine-rich histone, 8.0 muM for histone mixture and 14.3 muM for protamine, respectively.     

Enzyme activity and composition of myelin and subcellular fractions in the developing rat brain

1. Subcellular fractions and myelin were isolated from developing and adult rat brain. 2. Measurements of chemical composition and enzyme activities indicate the presence of a second myelin-like fraction mainly in the brain of developing rats. 3. This membrane fraction has a different lipid composition from myelin, but resembles myelin in its content of phosphohydrolase and aminopeptidase activity. 4. It is suggested that the second myelin-like fraction may be a submicrosomal contaminant or it may be derived from oligodendroglial plasma membrane during myelinogenesis.     

Developmental regulation of myelin basic protein expression in mouse brain

Developmental regulation of myelin basic protein expression in mouse brain has been examined by comparing the myelin basic protein coding potential of mRNA in vitro with the accumulation of myelin basic protein-related polypeptides in vivo. In vitro translation of mRNA isolated from mouse brain generated eight myelin basic protein-related polypeptides with apparent molecular weights of 34K, 30K, 29K, 26K, 21.5K, 18.5K, 17K, and 14K. A similar set of eight myelin basic protein-related polypeptides with corresponding molecular weights was identified in vivo when total brain proteins were analyzed by immunoblotting. Each of the myelin basic protein-related polypeptides shows a characteristic developmental profile in terms of mRNA level and rate of accumulation implying a complex developmental program of myelin basic protein gene expression with regulation and modulation at several different biosynthetic levels.     

The renal degradation of myelin basic protein peptide 43-88 by two enzymes in different subcellular fractions.

Previous studies have demonstrated that the kidney is the major site for clearance and catabolism of a peptide (residues 43-88) of encephalitogenic or basic protein (BP) derived from central-nervous-system myelin. In the present investigation rat renal tissue was shown to be capable of degrading human BP peptide 43-88 over the pH range 4-11.5 with peaks of activity at pH5 and pH9. The enzymic activity at pH5 was localized mainly to the 5900 g pellet (crude mitochondrial fraction) and, on the basis of its sulphydryl features, was inferred to be cathepsin B. The enzyme activity at pH9 was greatly enriched in the 100 000 g pellet (microsomal brush-border fraction), and its sensitivity to inhibitors suggested that it was a metalloproteinase. The activity at alkaline pH in the 100 000 g pellet was stimulated 3-fold by non-ionic detergents and 20-fold by ATP and polyphosphates. Through a series of experiments the ATP stimulation of the alkaline proteinase activity was concluded to be the result of a reversal of inhibition imposed by the presence of another cationic protein, methylated bovine serum albumin. Inhibition by certain bivalent cations, the irregular effects of chelators and the effects of poly-L-lysine supported this conclusion. These studies indicate the availability of renal enzymes of different types and in different cellular compartments that are capable of degrading BP peptide 43-88. In particular, the relative amounts of bivalent cations, anions and charged proteins and peptides are likely to be major influences on the activity of the alkaline proteinase in vivo. The control of this degradation as well as the features of the smaller fragments of the peptide formed may determine biological and immune events subsequent to the release of this potentially autoantigenic material.     

Immunochemical measurement of myelin basic protein in developing rat brain: an index of myelin synthesis.

The initial time and rate of myelin basic protein synthesis in neural tissues of the rat have been measured from birth to 120 days. The protein was quantitated by a radioimmunoassay directly applied to unfractionated cerebrum, cerebellum, olfactory bulb, midbrain, brain stem, optic and trigeminal nerve, and areas of the spinal cord. Because the protein is a specific myelin constituent and its appearance correlates precisely with the synthesis of myelin lipids, the data in this report can be interpreted in terms of myelin synthesis and oligodendrocyte activity. The results show striking heterogeneity in the initial time and rate of myelin synthesis in neural tissue.     

Synthesis of the myelin proteolipid protein in the developing mouse brain

Mice ranging in age from 16 to 44 days were injected intracerebrally with ³H-leucine, and incorporation into total brain proteolipids and the myelin proteolipid protein was measured. All proteolipids were isolated from whole brain by ether precipitation and separated into their individual components by SDS polyacrylamide gel electrophoresis. Two major proteolipids with apparent molecular weights of 20,700 and 25,400 were observed in these preparations, and their proportion increased over the developmental period examined. A Ferguson plot analysis comparing these proteins with those of isolated myelin showed that the 25,400-dalton proteolipid component from whole brain was the myelin proteolipid protein. Rates of incorporation of ³H-leucine into total brain proteolipids peaked at 22 days of age. Synthesis of the myelin proteolipid protein increased rapidly to a maximum value at 22 days and decreased rather slowly until at 44 days it was about 83% of its maximum rate of synthesis. The data indicate that the developmental pattern of synthesis of the myelin proteolipid protein is unlike that of the myelin basic proteins. Synthesis of the major myelin proteins is developmentally asynchronous in that peak synthesis of the myelin proteolipid appears to occur several days later than the basic proteins. In addition, it maintains its maximum rate of synthesis over a longer period of time than do the basic proteins.     

Acylation of myelin proteolipid protein in subcellular fractions of rat brainstem

The acylation of myelin proteolipid protein (PLP) and intermediate protein (IP) was investigated in an in vitro system of tissue slices prepared from actively myelinating rat brainstem. The incorporation of [³H]palmitate into the proteins in nine subcellular fractions including myelin and other cellular membranes which are actively involved in the synthesis and intracellular transport of the proteins was measured. More than 80% of [³H]palmitate-labeled proteins were recovered in myelin. The incorporation was highest in the heavy myelin and lowest in the light myelin subfraction. Appreciable acylation was also detected in the myelin-like fraction. On the other hand, the remaining fractions comprising a variety of endo- and ectomembranes, which harbored over 90% of newly synthesized PLP and IP as seen from [³H]leucine labeling showed practically no [³H]palmitate incorporation. The results indicate that the acylation of PLP and IP is a late event in their posttranslational processing and occurs only at their entry into the myelin sheath.     

The mass distribution of the phosphatidylcholines in subcellular fractions of rat brain

Whole brains from 20-22-day-old rats were separated into the 15,000 g supernatant, myelin, nerve ending and mitochondrial fractions. Gas chromatography of the trimethylsilyl derivatives of 1,2-diglycerides obtained by hydrolysis with phospholipase C of the phosphatidylcholine from each fraction showed marked differences of carbon number distribution (i.e. the sum of the carbon atoms in the two fatty acids of the diglyceride) among the different membranous fractions. Further characterization of each diglyceride was obtained by preparative gas chromatography of the diglyceride-trimethylsilyl ethers and determination of the acyl moieties after collection, methanolysis and gas chromatography. The results indicate that at least three distinct populations of phosphatidylcholine exist in the brain. Nerve endings and the 15,000 g supernatant fraction exhibit a very similar diglyceride pattern with dipalmitoylglycerophosphorylcholine representing over 30 per cent of the species present. Myelin has a unique phosphatidylcholine composition with much less polyenoic species in the 36 and 38 carbon number peaks. Mitochondria contain phosphatidylcholines with relatively more long-chain polyunsaturated fatty acids. TLC of the phosphatidylcholines yielded partial separation into two spots, which differed in distribution of fatty acids. The faster migrating spot contained most of the polyenoic acids, whereas the slower migrating spot contained most of the palmitic, stearic and oleic acids.     

The distribution of free amino acids in subcellular fractions of guinea-pig brain

1. The free amino acids of homogenates of guinea-pig brain in 0.32m-sucrose and of subcellular fractions derived therefrom have been estimated by the method of Moore & Stein. 2. Seven amino acids together accounted for over 80% of the free amino compounds; these are, in decreasing order of abundance: glutamate, aspartate, gamma-aminobutyrate, glycine, serine, alanine and threonine. In addition, there are appreciable quantities of amide (presumably glutamine). 3. Control experiments showed that the pattern of free amino acid occurrence in sucrose homogenates was similar to that of brains of animals killed by freezing in liquid nitrogen and extracted immediately without thawing. 4. The subcellular distribution of the amino acids resembled that of soluble cytoplasmic markers; there was no specific localization in a fraction rich in isolated presynaptic nerve terminals of amino acids capable of exciting or depressing central neurones. 5. The significance of the results is discussed in relation to the possible role of centrally active amino acids as transmitters.     

The distribution of protein-bound N-acetylneuraminic acid in subcellular fractions of rat brain

Protein-bound N-acetylneuraminic acid and hexosamine, including the sialomucopolysaccharides, occur mainly in the least dense particles sedimented in the microsomal fraction from rat whole brain. Particles rich in protein-bound N-acetylneuraminic acid and hexosamine are also found in the subcellular fraction separated as a layer between 0.8m- and 1.2m-sucrose after centrifuging the crude mitochondrial preparation in a density gradient. This distribution is similar to that of the gangliosides and suggests an association of all of these substances in the same subcellular structures. It is postulated that the sialomucopolysaccharides, as well as the gangliosides, are components of cell membranes. Evidence is presented that indicates that there are quantitative differences between distribution of the gangliosides on the one hand, and protein-bound N-acetylneuraminic acid and hexosamine on the other. The ratio of protein-bound N-acetylneuraminic acid (and hexosamine) to gangliosidic N-acetylneuraminic acid (and hexosamine) present in individual subcellular fractions obtained by density-gradient centrifugation tends to increase with increasing particle density. Exposure of the crude mitochondrial fraction to osmotic ;shock' before density-gradient centrifugation causes a shift of the protein-bound N-acetylneuraminic acid and gangliosides to the less dense fractions. In some experiments, a selective shift of the protein-bound N-acetylneuraminic acid was observed.     

Plasmalogen distribution in the subcellular fractions of the vertebrate brain

Studies have been made on plasmalogen and diacylic forms of phospholipids, i. e. phosphatidylethanolamine and phosphatidylcholine (their relative content and ratio), in subcellular fractions isolated from the brain of the trout Salmo trideus, frog Rana temporaria, pigeon Columba livia, tortoise Testudo horsfieldi, and rabbit. Investigation was carried out on 5 subcellular fractions (myelin, nuclei, microsomes, mitochondria, synaptosomes) as well as on brain homogenates. In all the fractions, relative content of plasmalogens is the lowest in the trout, increasing in parallel with the increase in the complexity of the nervous system and reaching the highest values in pigeons and rabbits.     

Distribution of protein kinase activities in subcellular fractions of rat brain.

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.     

Effects of delta9-tetrahydrocannabinol on ATPases in mouse brain subcellular fractions.

The effect of (?)-Δ⁹-THC on the activities of Mg²⁺?, Na⁺?K⁺? and Mg²⁺Ca²⁺-ATPases were studied in mouse brain subcellular fractions. $\text{In vitro}$treatment with Δ⁹-THC produced a dose dependent stimulation of Mg²⁺ ATPase in the crude mitochondrial fraction and its subfractions and a dose-related inhibition of this activity in the microsomal fraction. Na⁺-K⁺- and Mg²⁺-Ca²⁺-ATPase activities were inhibited in a dose-related manner in all subcellular fractions studied.     

Purification of myelin basic protein from bovine brain

Myelin basic protein (MBP) is a commonly used substrate for in vitro determination of numerous protein kinase activities. Herein we describe a rapid method for isolating relatively large amounts of MBP from bovine brain with a purity greater than that currently available from commercial sources. Lipids were first extracted from the CNS tissue by homogenization in sec-butanol. Washes under neutral and mildly basic conditions were employed to remove neutral and acidic proteins from the defatted residue. MBP was subsequently extracted under acidic conditions and further purified by chromatography on CM Sephadex C-25. Potential contaminating enzyme activities were destroyed by heart treatment. This method typically yields a recovery of 1.0-1.5 mg MBP per gram of starting material with a purity of greater than 95%. The MBP prepared in this manner was suitable for determination of kinase activities by both solution and the "in gel" kinase assay systems.     

Metabolism of cerebrosides and sulfatides in subcellular fractions of developing rat brain

Previous studies on myelinating rat brain indicated that microsomes, Golgi-enriched and cytosol fractions may process galactolipids destined for myelin. To extend these findings we labeled brain galactolipids in vivo and determined the specific radioactivity of cerebrosides and sulfatides in several subcellular fractions. 17-day-old rats were treated by intracranial injection with [14C]galactose 60 min prior to and [3H]galactose 15 min prior to killing. Subcellular fractions were prepared from brain stem, and concentrations of cerebrosides and sulfatides were determined, their radioactivity measured and the 3H/14C ratio compared. Our results showed that the heavier Golgi-enriched fraction (designated Fraction 2) is unique in its low galactolipid content and high specific radioactivities of cerebrosides and sulfatides. The low ratio of the specific activity of cerebroside to that of sulfatide in Fraction 2 compared to other fractions indicates that it may be the site of most rapid conversion of newly synthesized cerebrosides to sulfatides. The specific radioactivities of cerebrosides and sulfatides in cytosol are intermediate between those in Golgi-enriched Fraction 2 and microsomes and those in myelin, consistent with the role postulated for cytoplasmic elements in the transport of cerebrosides and sulfatides to myelin.     

Accumulation of the four myelin basic proteins in mouse brain during development.

Myelin was isolated from the brains of mice 15, 20, 30, and 60 days after birth. The total amount of basic protein present in the isolated myelin was determined by radioimmunoassay. The 4 myelin basic proteins, with molecular weights of 21,500, 18,500, 17,000 and 14,000, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and their relative amounts were determined densitometrically. The absolute amount of each of the basic proteins was calculated from its relative amount on the gel and from the total amount of myelin basic protein in the sample as determined by radioimmunoassay. The results show that between 10 and 30 days after birth each protein accumulates at a characteristic rate so that the molar ratios among the 4 basic proteins are (in descending order according to their molecular weights) 1:5:2:10 during this period. Between 30 and 60 days after birth the 14 K and 18.5 K proteins continue to accumulate at reduced rates while the 21.5 K and 17 K proteins begin to disappear from the myelin membrane; 60 days after birth the molar ratios among the 4 basic proteins are 1:10:3.5:35. These developmental patterns of accumulation are discussed in relation to the possible role of each of the 4 myelin basic proteins in myelination.