Phospholipid Biosynthesis in Myelin: Presence of CTP: Phosphoethanolamine Cytidylyltransferase in Purified Myelin of Rat Brain

Abstract

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 Km for CTP was approximately the same for myelin and microsomes, but that for phosphoethanolamine was significantly higher for myelin.

Ethanolamine Kinase Activity in Purified Myelin of Rat Brain

“…The cytidine pathway for phosphatidylethanolamine synthesis, discovered by Kennedy and Weiss (1956), is generally considered to be localized in the endoplasmic reticulum. Two of these enzymes were previously shown by us to occur in purified rat brain myelin as well: CDP-ethanolamine: 1 ,Zdiacyl-snglycerol ethanolaminephosphotransferase (Wu and Ledeen, 1980) and CTPphosphoethanolamine cytidylyltransferase (Kunishita and Ledeen, 1984). With the present demonstration ofethanolamine kinase activity, myelin would appear to have all the enzymes needed for the Kennedy pathway conversion of diacylglycerol to phosphatidylethanolamine.…”

“…The subcellular distribution of this enzyme was similar to that of CTPphosphoethanolamine cytidylyltransferase (EC 2.7.7.14), which also had highest activity in cytosol along with appreciable levels in all the membranous subfractions (Kunishita and Ledeen, 1984). In rat liver, ethanolamine kinase was reported to occur exclusively in the cytosol (Weinhold and Rethy, 1972).…”

“…The significant relative specific activity (RSA) value (0.17) and high activity compared to microsomes (23%) tended to exclude the latter as a likely source of contamination. We previously reported (Kunishita and Ledeen, 1984) that myelin prepared in this manner had only 1-2% as much NAD-PH-cytochrome c reductase as microsomes, for which this enzyme is a marker. Occluded cytosol seemed an unlikely source of contamination due to the low level of lactate dehydrogenase detected in the myelin.…”

“…By comparison, lactate dehydrogenase activity in myelin was only 1.4% that of homogenate. Previous assay of NADPH-cytochrome c reductase in myelin prepared in identical fashion gave a value 1-2% of microsomes (Kunishita and Ledeen, 1984). It may be noted that isolated myelin accounted for a small f i xtion (< 1 %) of total kinase activity in starting homogenate, a phenomenon observed for various other nonspecific enzymes detected in this membrane (for review, see Ledeen, 1984;Norton and Cammer, 1984).…”

“…The existence of such enzymes in myelin had been suggested by several studies (Droz et al, 1978, 198 1;Haley and Ledeen, 1979;Toews and Morell, 1981;Alberghina et al, 1982;Gould et al, 1982;Ledeen and Haley, 1983) indicating transfer of various substrates from the axon to myelin, these substances then becoming enzymatically incorporated into myelin lipids. We have previously shown myelin to possess CDP-ethanolamine: 1,2-diacyl-sn-glycerol ethanolaminephosphotransferase (EC 2.7.8.1) (Wu and Ledeen, 1980) and CTP:phosphoethanolamine cytidylyltransferase (EC 2.7.7.14) (Kunishita and Ledeen, 1984), the two enzymes that complete the synthesis of phosphatidylethanolamine. Prelimary reports indicated the presence of the corresponding enzymes for phosphatidylcholine synthesis as well (Ledeen and Wu, 1979;Kunishita and Ledeen, 1982).…”