Evidence for the Presence of CDP-Ethanolamine: 1,2-diacyl-sn-glycerol Ethanolaminephosphotransferase in Rat Central Nervous System Myelin (original) (raw)
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Journal of Neurochemistry, 1984
Highly purified myelin from rat brain was previously shown to contain the ethanolaminephosphotransferase which completes the synthesis of phosphatidyl ethanolamine. We have now obtained evidence for the presence in myelin of CTP:phosphoethanolamine cytidylyltransferase, the enzyme catalyzing formation of CDP-ethanolamine. Myelin was isolated by two different procedures, one based on the Norton-Poduslo method and the other involving repetitive gradients with osmotic shocking deferred to the end. The fact that activity remained constant through all but the earliest steps suggested that the enzyme is intrinsic to myelin. Comparison of subcellular fractions revealed that approximately half the total activity was in the supernatant, the remainder being distributed among the particulate fractions. Relative specific activity of myelin was 27-31% that of microsomes, thus eliminating the possibility of appreciable contamination by the latter. The possibility of adsorption of the soluble enzyme by myelin was rendered unlikely by retention of activity after washing the myelin with buffered sodium chloride or sodium taurocholate. Furthermore, relative specific activity of the cytidylyltransferase was 10-fold higher than that of lactate dehydrogenase (a cytosolic marker) in myelin. The apparent K, for CTP was approximately the same for myelin and microsomes, but that for phosphoethanolamine was significantly higher for myelin. Key Words: Myelin enzymes-Phosphoethanolamine cytidylyltransferase-Myelin-CDP-ethanolamine synthesis. Kunishita T. and Ledeen R. W. Phospholipid biosynthesis in myelin: Presence of CTP:phosphoethanolamine cytidylyltransferase in purified myelin of rat brain. J. Neurochem. 42, 326-333 ( 1984). I This concentration of CTP was employed in several runs before it was realized that it is somewhat inhibitory (cf. . The optimal concentration of 0.5 mM would give rates 20-30% higher.
Ethanolamine Kinase Activity in Purified Myelin of Rat Brain
Journal of Neurochemistry, 1987
Highly purified rat brain myelin showed a significant level of ethanolamine kinase, amounting to 17% of the specific activity of whole brain homogenate. This kinase level in myelin was an order of magnitude higher than that of lactate dehydrogenase, a marker for cytosol. Subcellular distribution studies revealed that in addition to myelin, this kinase was present in the P1, P2, P3, and cytosolic fractions with highest relative specific activity in the latter. The possibility that myelin activity resulted from adsorption of the soluble enzyme was unlikely since activity was retained in myelin that had been washed with buffered sodium chloride or taurocholate. Mixing experiments and repeated purification further indicated that the enzyme is intrinsic to myelin.
Fatty acid synthesizing enzymes intrinsic to myelin
Molecular Brain Research, 2003
A recent study showing incorporation of acetyl groups from neuronal N-acetylaspartate into myelin lipids suggested the presence of fatty acid synthesizing enzymes in myelin that utilize the acetyl groups liberated by myelin-associated aspartoacylase [J. Neurochem. 78 (2001) 736]. We report here detection of the fatty acid synthase (FAS) complex and acetyl-CoA carboxylase (ACC) in purified myelin. The activity of myelin FAS was approximately half that of cytosolic FAS and, unlike the latter, required detergent for activation. Intrinsic association of FAS with myelin was indicated by failure to remove the activity with NaCl or Na-taurocholate. Myelin-associated ACC was approximately 10% of cytosolic ACC in myelin isolated by gradient centrifugation, and this was reduced by half following osmotic shock; this suggested bimodal distribution of myelin ACC, some being loosely associated within inter-lamellar cytoplasmic spaces and the remainder more firmly associated in a manner that resists NaCl / Na-taurocholate treatments. These results, in combination with earlier findings, provide a possible mechanism for the observed incorporation of neuronal NAA acetyl groups into myelin lipids.
Phosphatidate phosphohydrolase in purified rat brain myelin
Journal of Neuroscience Research, 1989
Highly purified myelin from rat brain stem has been shown to contain phosphatidate phosphohydrolase, an enzyme which converts phosphatidate to diacylglycerol. The high levels relative to cytosol and microsomes (17% and 22%, respectively) tended to preclude contamination by these fractions as the source of activity. Additional evidence came from study of repeated purification, mixing experiments, and washing of the myelin with salt and detergent. We conclude that this enzyme, in addition to being widely distributed in other subcellular fractions, is intrinsic to the myelin membrane. Through its activity it generates a key substrate for the cytidine (Kennedy) pathway which was previously shown to occur in this membrane.
The turnover of the lipid components of myelin
Journal of the American Oil Chemists' Society, 1965
The rate of loss of radioactivity in the lipid components of rat myelin labeled with acetate‐1‐C14 was determined over a period of one year. Rats were injected with acetate‐1‐C14 at 15舑16 days of age and purified myelin was prepared by differential ultracentrifugation from brain and spinal cord of this group at 1 day, 2 weeks, 1 month, 2 months, 3 months, 6 months and 1 year after injection. Total lipid was extracted from the myelin preparations and the lipids were separated into their components by thin‐layer chromatography. Cholesterol, galactolipid, ethanolamine phosphatide, choline phosphatide, inositol phosphatide, serine phosphatide and sphingomyelin specific activities at each age were measured. Three of the myelin lipid components, serine phosphatide, inositol phosphatide, and choline phosphatide decreased in specific activity faster than cerebroside, cholesterol, sphingomyelin, and ethanolamine phosphatide. Acetate‐1‐C14 injected into adult animals, though incorporated into...
Neurochemical Research, 1982
The distribution of UDP-galactose:ceramide galactosyltransferase (CGalT) was studied in subcellular fractions of rat forebrain during development using zonal centrifugation on linear gradients, Specialized subfractions: SN 1, a microsomal fraction, SN 4, a myelin-related fraction, and purified myelin were also used for this study, For comparison, two microsomal lipid synthesizing enzymes, a myelinspecific enzyme, 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin proteins were measured in the same subfractions. UDP-glucose:cerarnide glucosyltransferase and cerebroside sulfotransferase were confined to microsomes. CGalT was localized in microsomes, but also in myelin and myelin-related fractions. The
Enzyme and protein composition of myelin isolated in the presence of EGTA
Neurochemistry International, 1981
The efficiency of ethyleneglycol-bis (fl-amino-ethyl ether) N,N'-tetra-acetic acid (EGTA) in removing possible contamination from myelin was tested. Myelin fractions were isolated in the presence or absence of EGTA. An axolemma-enriched fraction was also prepared. Gel electrophoresis showed no important alteration of the protein pattern of myelin treated with EGTA. Only a minor band of about 41,000 dalton8 was selectively removed when EGTA was used during the two density gradient and differential centrifugation steps. EGTA, when used in the final washes, did not remove this band. It was absent from axolemma-enriched fractions. Different hypotheses are considered to explain these findings. Recently, a preliminary report (Samuels et al, 1979) indicated that extensive washing of myelin with solutions containing the calcium-chelating agent ethyl
Journal of Neurochemistry, 2008
Myelination, during both normal development and with respect to disorders of myelination, is commonly studied by morphological and/or biochemical techniques that assay as their end-points the extent of myelination. The rate of myelination is potentially a more useful parameter, but it is difficult and time-consuming to establish, requiring a complete developmental study with labor-intensive methodology. We report herein development of methodology to assay the absolute rate of myelination at any desired time during development. This involves intraperitoneal injection of 3H2O to label body water pools, followed by determination of label in the myelin-specific lipid, cerebroside. The absolute amount of cerebroside synthesized can then be calculated from the specific radioactivity of body water and knowledge of the number of hydrogens from water incorporated into cerebroside. During development, the rate of cerebroside synthesis correlated well with the rate of accumulation of the myelin-specific components, myelin basic protein and cerebroside. For purposes of control, we also tested other putative, albeit less quantitative, indices of the rate of myelination. Levels of mRNA for ceramide galactosyltransferase (rate-limiting enzyme in cerebroside synthesis) and for myelin basic protein did not closely correlate with myelination at all times. Cholesterol synthesis closely matched the rate of cholesterol accumulation but did not track well with myelination. Synthesis of fatty acids did not correlate well with accumulation of either fatty acids (phospholipids) or myelin markers. We conclude that measurement of cerebroside synthesis rates provides a good measure of the rate of myelination. This approach may be useful as an additional parameter for examining the effects of environmental or genetic alterations on the rate of myelination.
Biosynthesis of Peripheral Nervous System Myelin Proteins In Vitro
Journal of Neurochemistry, 1980
Myelin prepared from spinal roots and sciatic nerves of young rats (24 days of age) after incubation with [3H] amino acids in glucose-Krebs Ringer bicarbonate contained a considerable amount of radioactivity incorporated into the proteins. When these were separated on SDS-polyacrylamide gels, peaks of radioactivity were found to correspond to the Po, 23K, P,, and P2 proteins. Although very little Fast-Green-stained protein was visible in the high molecular weight region of the gels (>27,000), much radioactivity was located in this area. Incorporation of [ 3H] amino acids into the myelin proteins continued up to 3 h and was dependent on glucose, but not on Oz-CO, replacement of air in the flask, whereas cycloheximide inhibited incorporation by 80%. Much less amino acid was incorporated into myelin protein of old rats. High molecular weight proteins appeared to be more metabolically active than other myelin proteins, as estimated by the percent of total counts incorporated/amount of Fast-Green staining, and their activity decreased less with age. The 23K and P, proteins incorporated [3H] amino acid in approximate proportion to their content. Po was less, and Pz somewhat more metabolically active than their contents would predict. [3H]fucose also was incorporated in vitro into Po and P, (probably 19K) proteins as well as into the high molecular weight proteins, while glucosamine served less well as a glycoprotein precursor. This preparation may be useful for examining further parameters of PNS myelin synthesis, assembly, and the effects of neurotoxic agents.