Evidence for the Presence of CDP‐Ethanolamine: 1,2‐diacyl‐sn‐glycerol Ethanolaminephosphotransferase in Rat Central Nervous System Myelin

Abstract

Highly purified rat brain myelin isolated by two different procedures showed appreciable activity for CDP-ethanolamine: 1,2-diacyl-sn-glycerol ethanolaminephosphotransferase (EC 2.7.8.1). Specific activity was close to that of total homogenate and approximately 12-16% that of brain microsomes. Three other lipid-synthesizing enzymes, cerebroside sulfotransferase, lactosylceramide sialyltransferase, and serine phospholipid exchange enzyme, were found to have less than 0.5% the specific activity in myelin compared with microsomes. Washing the myelin with buffered salt or taurocholate did not remove the phosphotransferase, but activity was lost from both myelin and microsomes by treatment with Triton X-100. It resembled the microsomal enzyme in having a pH optimum of 8.5 and a requirement for Mn2+ and detergent, but differed in showing no enhancement with EGTA. The diolein Km was similar for the two membranes (2.5-4 x 10(-4) M), but the CDP-ethanolamine Km was lower for myelin (3-4 x 10(-5) M) than for microsomes (11 - 13 x 10(-5 M). Evidence is reviewed that this enzyme is able to utilize substrate from the axon in situ.

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

“…Myelin obtained by the modified Norton and Poduslo (1973) procedure, shown previously to possess very high purity (Wu and Ledeen, 1980;Haley et al, 1981), was found to possess phosphoethanolamine cytidylyltransferase, of specific activity amounting to some 27-32% of that observed in simultaneously isolated microsomes. The same myelin preparations contained virtually negligible levels of t h e microsomal marker NADPH-cytochrome c reductase or the cytosol marker lactate dehydrogenase (Table 1).…”

“…Recent evidence has indicated that the myelin membrane itself has a capacity for synthesis of specific membrane constituents-principally lipids-although such synthesis is not neces-sarily functionally related to the above-mentioned process of myelinogenesis. Evidence for such myelin-localized synthesis came indirectly from experiments demonstrating incorporation of axonally derived substrates into a variety of myelin lipids Ledeen, 1978, 1979;Dtoz et al, 1978Dtoz et al, , 1981Brunetti et al, 1981;Ledeen and Haley, 1983) and more directly from reports of specific lipid-synthesizing enzymes in purified myelin (Costantino-Ceccarini and Suzuki, 1975;Deshmukh et al, 1978;Choi and Suzuki, 1978;Ledeen and Wu, 1979;Wu and Ledeen, 1980;Kunishita and Ledeen, 1982).…”

“…Recent work in this laboratory (Wu and Ledeen, 1980) demonstrated the presence of CDP-ethanolamine: 1,2-diacyl-sn-glycerol ethanolaminephosphotransferase, the enzyme which completes the synthesis of ethanolamine phosphoglycerides in rat CNS myelin; a preliminary report on the presence of the analogous cholinephosphotransferase, which completes the synthesis of choline phosphoglycerides, has also been presented (Ledeen and Wu, 1979). We now report the presence in purified rat brain myelin of CTP:phosphoethanolamine cytidylyltransferase (EC 2.7.7.14), the enzyme catalyzing the penultimate step giving rise to CDP-ethanolamine formation: CTP + phosphoethanolamine + A preliminary account of this work has been presented (Kunishita and Ledeen, 1982).…”

Fractionation of Isolated Rat CNS Myelinated Axons by Sucrose Density Gradient Centrifugation in a Zonal Rotor

“…Our results indicate that CDP-choline : 1,2-diglyceride choline phosphotransferase, Na+ ,K+-ATPase, and 5'-nucleotidase may be myelin-associated, since they all demonstrate RSAs greater than 1.0 in the region of the sucrose gradient (fractions 7-lo), where the paranodal loop regions of myelin may be found. Several laboratories have reported that 5'-nucleotidase (Cammer et al, 1980), Na+,K+-ATPase (Reiss et al, 1981), and CDP-choline : 1 ,Zdiglyceride choline phosphotransferase ( Wu and Ledeen, 1980) are intrinsic CNS myelin constituents. 5'-Nucleotidase has been localized to the outer regions of the myelin sheath (Scott, 1965), but our results indicate that even the least dense multilamellar myelin-rich region of the density gradient has significant 5'-nucleotidase activity (RSA = 1.1).…”

“…After thorough mixing by vortexing, the upper phase was clarified by centrifugation (1000 x g for 5 min), and a 1-ml aliquot was counted in 10 ml of Aquasol (Beckman Instruments, Fullerton, CA) in an Intertechnique SL-30 scintillation counter. CDPcholine: 1,2-diglyceride choline phosphotransferase was assayed using the incubation system described by Wu and Ledeen (1980). The radioactive product was extracted by the procedure of Hajra and Radin (1978), and a 0.5-ml aliquot of the radioactive product was evaporated dry and then counted in 5 ml of Aquasol by liquid scintillation counting.…”