A CHOLESTEROL-ESTERIFYING ENZYME IN RAT CENTRAL NERVOUS SYSTEM MYELIN1

Aontrary to our earlier finding (Eto & Suzuki , 1971), the myelin fraction purified from young adult rat brain consistently showed cholesterol-esterifying activity. The specific activity in myelin was the highest among subcellular fractions. Extensive washing wiih various aqueous salt solutions failed to remove the activity from myelin. The enzyme was evenly distributed among the arbitrarily defined light, medium and heavy myelin subfractions. The myelin-localized activity showed the pH optimum and heat stability identical to the microsome-bound activity. Although there were minor differences in the effect of detergents or exogenous lipids added to the reaction mixture, no firm evidence was obtained to indicate that the myelin-bound cholesterol-esterifying enzyme is different from that in other subcellular fractions. On the other hand, the distribution among the myelin subfractions and heat stability of the myelin-bound cholesterol-esterifying activity were different from those of the myelin-specific cholesterol ester hydrolase. Therefore, the esterification does not appear to be a mere reverse reaction catalyzed by the previously known myelin-specific hydrolase. The rat brain myelin, therefore, is capable of both synthesizing and hydrolyzing cholesterol esters.     

Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Microsomal synthesis of cholesterol from squalene, Lanosterol, and desmosterol. Evidence for the presence of two noncatalytic activator proteins in the 105,000 g supernatant fraction from brain, heart, and kidney

The biosynthesis of C₂₇ sterols (used as a generic term for 3 β-hydroxysterols containing 27 carbon atoms) from squalene and lanosterol, of cholesterol from desmosterol, and of lanosterol from squalene by microsomal fractions from adult rat heart, kidney, and brain was investigated. These conversions required the presence of 105,000g supernatant fraction. Heat treatment of the supernatant fractions resulted in a significant loss of their capacity to stimulate the conversion of squalene to sterols, but the capacity to stimulate conversion of lanosterol to C₂₇ sterols and desmosterol to cholesterol was unaffected. The stimulatory activity (for the conversion of all three substrates) of both the heated and unheated supernatant fractions was lost on treatment with trypsin. Thus the soluble fraction appears to contribute at least two essential protein components for the overall conversion of squalene to cholesterol; one a heat labile protein, which functions in the squalene to lanosterol sequence, and the other a heat-stable protein, which is operative in the pathway between lanosterol and cholesterol. Hepatic supernatant factors required for cholesterol synthesis by liver microsomal enzymes function with heart, kidney, and brain microsomal enzymes in stimulating sterol synthesis from squalene and sterol precursors. Moreover, heart, kidney, and brain supernatant fractions prepared in 100 mm phosphate buffer stimulated cholesterol synthesis from squalene and other sterol precursors by liver microsomes. The supernatant fractions of the extrahepatic tissues prepared in 20 mm phosphate buffer lacked the ability to stimulate the biosynthesis of lanosterol from squalene by liver microsomes but were able to stimulate the conversion of lanosterol to C₂₇ sterols or conversion of desmosterol to cholesterol. These findings indicate that the heat-stable protein factor present in the supernatant fractions from extrahepatic tissues is perhaps identical to that in liver, but that the heat-labile factor in extrahepatic tissues, which catalyzes the cyclization of squalene to lanosterol, differs in some respect from that in liver.     

Cholesterol ester hydrolase(s) in mammalian brain: Is there a myelin-specific cholesterol ester hydrolase?

The present study compared the properties of cholesterol ester hydrolase(s) in myelin and microsomes from rat, mouse and human brain. The results indicated that the enzyme activity in both myelin and microsomes from rat, mouse and human brain was optimal at pH 6.5 and required Triton X-100 for optimal activity. The enzyme activity in myelin was 3- to 4-fold higher in the presence of Trition X-100 than taurocholate. Addition of phosphatidyl serine enhanced (2 to 4 fold) the hydrolase activity in both myelin and microsomes. The properties of the enzyme in solubilized preparation of myelin were also similar to the properties of the enzyme in partially delipidated and solubilized preparations of microsomes. The activity was again optimal at pH 6.5, required Triton X-100 for optimal activity and was stimulated by phosphatidyl serine. These results indicate that the properties of cholesterol ester hydrolase in myelin are similar to those of the microsomal enzyme and that this is true for the fractions from both human and rodent brain. The data thus lead us to believe that the hydrolase activity in mammalian brain myelin and microsomes may reflect the distribution of a single enzyme in the two fractions rather than two distinct enzymes, one being specific to each fraction.     

THE DESMOSTEROL REDUCTASE ACTIVITY OF RAT BRAIN DURING DEVELOPMENT1

Abstract— The reduction of desmosterol by cell-free preparations from developing rat brain was established and the age-dependent alterations in reductase activity were correlated with levels of desmosterol in brain. An increase in enzymic activity followed closely the sharp increase in levels of desmosterol that was observed at about 5 days of age and that reached a maximum at 8-11 days of postnatal age. Furthermore, the abrupt decrease in the desmosterol content of brain at 13-15 days of age was associated with a decrease in enzymic activity. We suggest that the enzyme may be substrate-induced and that this attribute may be of significance with respect to the ontogenesis of myelin. Cerebral desmosterol reductase exhibited a specific requirement for NADPH and was primarily a particulate enzyme.     

Synthesis of sterols by chick brain and liver during embryonic development

The evolution throughout embryonic development of the rate at which acetate was converted into sterols was studied in chick brain and liver. Acetate incorporation (nmol/h/g tissue) was clearly higher in brain than in liver and sharply decreased with the age of embryo. Cholesterol and desmosterol were the major sterols formed from acetate by chick embryo brain, followed by lanosterol and squalene. No desmosterol was found in chick embryo liver, organ where cholesterol was the major sterol synthesized. In brain, the relative percentage of cholesterol increased throughout embryonic development reaching more than 50% at hatching, while the percentage of desmosterol decreased during the same period and represented at hatching only about 10–15% of the total nonsaponifiable fraction. The relative percentages of lanosterol and squalene did not change significantly throughout the period assayed. In liver, the percentage of cholesterol increased until 19 days but sharply decreased at hatching.     

Cholesterol esters in developing rat brain: enzymes of cholesterol ester metabolism

Abstract— Three enzymes of cholesterol ester metabolism, a cholesterol-esterifying enzyme which incorporates free fatty acids into cholesterol esters without participation of CoA, and two cholesterol ester hydrolases with differing pH optima, all showed distinct changes in developing rat brains. The specific activity of the esterifying enzyme was approx. 20 percent of the adult level at birth, increased gradually to the adult level by 20 days of age and remained constant thereafter. The pH 4.2 hydrolase at birth also had a specific activity of about 20 per cent of the adult level but it increased rapidly to reach a peak at 13 days, by which time the activity had increased eight-fold. The activity declined somewhat thereafter to reach the adult level by 23–30 days. In contrast, there already was 60 per cent of the adult specific activity of the pH 6.6 cholesterol ester hydrolase at birth. The activity remained constant until 12 days and then doubled during the next two weeks, reaching a broad peak, then declining slightly to reach the adult activity by 50 days. Therefore, the developmental changes of both of the hydrolases appeared to be related to the process of myelination. The period of active myelination (10–30 days) was characterized by the sharp rise in the activity of pH 6.6 cholesterol ester hydrolase and by the rapid decrease of pH 4.2 cholesterol ester hydrolase.     

Developmental changes of cholesterol ester hydrolases localized in myelin and microsomes of rat brain

We Previously demonstrated two distinct cholesterol ester hydrolases in rat brain (Eto and Suzuki , 1971). One of the two hydrolases had a pH optimum of 6·6 and showed a bimodal subcellular distribution, in microsomes and myelin. A substantial activity of this enzyme was present in newborn rat brain. The activity remained relatively unchanged during the first 12 days and then increased sharply, concomitant with the period of active myelination (Eto and Suzuki , 1972a). The more recent investigation, however, clearly demonstrated that this pH 6·6 cholesterol ester hydrolase actually consists of two distinct cholesterol ester hydrolases, one primarily localized in microsomes and the other almost exclusively localized in the myelin sheath (Eto and Suzuki , 1972b, 1973). The microsomal hydrolase had a pH optimum of 6·0 and was activated by sodium taurocholate and Triton X-100, particularly by the latter. The myelin enzyme had a pH optimum of 7·2. It was activated by sodium taurocholate but slightly inhibited by Triton X-100. These new findings suggested that the previously reported developmental curve of the pH 6·6 cholesterol ester hydrolase was probably a composite of developmental changes of these two distinct cholesterol ester hydrolases. We report here the findings which confirm the above prediction and update the information regarding the developmental changes of the enzymes involved in cholesterol ester metabolism in rat brain.     

Sterol content and squaline-2(3)-epoxide-lanosterol cyclase activity in human foetal brain during early and mid-gestation

—Cholesterol, desmostcrol and squalene-2(3)-epoxide-lanostcrol cyclase were assayed in various regions of human foetal brain from 10 to 22 weeks gestation. The cerebellum, thalamus and hypothalamus, and the ‘remainder’ of the brain were analysed. The proportion of desmosterol to cholesterol was highest in the thalamus and hypothalamus and lowest in the cerebellum. It is suggested that the ratio of desmosterol to cholesterol gives an indication of the state of maturity of a given region, particularly with respect to myelination. Thus the higher levels of desmosterol detected in the thalamus and hypothalamus may be correlated with earlier myelination in this region compared to the cerebellum which myelinates much later in development. Squalene-2(3)-epoxide-lanosterol cyclase activity in whole brain showed three phases during the period studied; an initial low level up to 15 weeks, a slight increase in activity from 15 to 18 weeks (corresponding to neuroblast multiplication) and a sharp increase after 18 weeks (corresponding to glial multiplication prior to myelination). The enzyme was shown to be microsomal although freezing and thawing of whole brain tended to result in its solubilization. The apparent K_m was found to be 2.5 × 10^?6m and the effects of the inhibitors iminosqualene and di-iminosqualene were studied. A comparison was made between the brain enzyme and the liver enzyme at the same age and it was seen that at 22 weeks there was a large increase in activity in the brain, prior to myelination, which was not paralleled by an increase in the activity of the liver enzyme. In this study we have measured the levels of cholesterol, desmosterol and squa-lene-2(3)-epoxide-lanosterol cyclase in whole human foctal brain, thalamus and hypothalamus, and cerebellum during early and mid-gestation. We have also examined various properties of the cyclase enzyme and in particular the variations in its activity during early brain development.     

CHOLESTEROL ESTER METABOLIZING ENZYMES IN HUMAN BRAIN: PROPERTIES, SUBCELLULAR DISTRIBUTION AND RELATIVE LEVELS IN VARIOUS DISEASED CONDITIONS

We have in the present study examined the properties and subcellular distribution of cholesterol ester metabolizing enzymes in human brain, and compared the levels of these enzymes in brains from patients with phenylketonuria (PKU), metachromatic leucodystrophy (MLD), and Down's Syndrome (DS). Cholesterol esterification was optimal at pH 5.6, did not require ATP or CoA as cofactors and was inhibited by detergents (TWEEN-20 and Triton X-100) and bile acids (sodium taurocholate and sodium deoxycholate). The specific activity of the cholesterol esterifying enzyme was highest in the mitochondrial fraction. Cholesterol esterifying activity in brains from PKU, MLD, and DS patients was not significantly different. Cholesterol ester hydrolase activity in human brain peaked at two different pHs (4.5 and 6.5). The activity was optimal when the substrate was dispersed in Triton X-100 and sonicated. The specific activity of the pH 4.5 hydrolase was highest in the mitochondrial fraction, while that of the pH 6.5 hydrolase was highest in myelin. The sulfhydryl group reagent parachloromercuribenzoate (PCMB) inhibited the activity of the hydrolase(s) but diisopropylfluorophosphate (DFP), a typical serine reagent, had no effect on hydrolase(s) activity. Addition of either phosphatidyl serine or phosphatidyl inositol significantly enhanced the hydrolase activity at both pHs. The level of cholesterol ester hydrolase(s) in PKU brains was lower than in the brains from DS patients, and the level of these enzymes in the brains from two patients with metachromatic leucodystrophy was lower than in the brains from PKU patients. It is concluded that the properties and subcellular distribution of cholesterol esterifying enzyme in human brain is similar to that in rat brain (Ero & Suzuki , 1971) but that the hydrolases in human brain differ from that in rat brain in several respects, and that the low levels of hydrolase(s) activity in MLD and PKU brain may be related to reduced myelin content of those brains.     

In vivo acylation of proteolipid protein and DM-20 in myelin and myelin subfractions of developing rat brain: Immunoblot identification of acylated PLP and DM-20

The acylation of proteolipid protein (PLP) was examined in myelin and myelin subfractions from rat brain during the active period of myelination. Proteolipid protein and DM-20 in myelin and myelin subfractions were readily acylated in developing rat brain 22 hours after intracerebral injection of [3H]palmitic acid. No differences in the relative specific activity of PLP in myelin from 9-, 15-, and 30-day-old rat brains was observed; however, the relative specific activity of PLP in the heavy myelin subfraction tended to be higher than that in the light myelin subfraction. The acylation of PLP was confirmed by fluorography of immuno-stained cellulose nitrate sheets, clearly establishing that the acylated protein is in fact the oligodendroglial cell- and myelin-specific protein, PLP. Since PLP is acylated in the 9-day-old animal, when little compact myelin is present, it is possible that the acylation of PLP is a prerequisite for the incorporation of this protein into the myelin membrane.     

UDP-galactose-ceramide galactosyltransferase in rat brain myelin subfractions during development.

The localization and activity of the enzyme UDP-galactose-hydroxy fatty acid-containing ceramide galactosyltransferase is described in rat brain myelin subfractions during development. Other lipid-synthesizing enzymes, such as cerebroside sulphotransferase, UDP-glucose-ceramide glucosyltransferase and CDP-choline-1,2-diacylglycerol cholinephosphotransferase, were also studied for comparison in myelin subfractions and microsomal membranes. The purified myelin was subfractionated by isopycnic sucrose-density-gradient centrifugation. Four myelin subfractions, three floating respectively on 0.55 M- (light-myelin fraction), 0.75 M- (heavy-myelin fraction) and 0.85 M-sucrose (membrane fraction), and a pellet, were isolated and purified. At all ages, 70--75% of the total myelin proteins was found in the heavy-myelin fraction, whereas 2--5% of the protein was recovered in the light-myelin fraction, and about 7--12% in the membrane fraction. Most of the galactosyltransferase was associated with the heavy-myelin and membrane fractions. Other lipid-synthesizing enzymes studied appeared not to associate with purified myelin or myelin subfractions, but were enriched in the microsomal-membrane fraction. During development, the specific activity of the microsomal galactosyltransferase reached a maximum when the animals were about 20 days old and then declined. By contrast the specific activity of the galactosyltransferase in the heavy-myelin and membrane fractions was 3--4 times higher than that of the microsomal membranes in 16-day-old animals. The specific activity of the enzyme in the heavy-myelin fraction sharply declined with age. Chemical and enzymic analyses of the heavy-myelin and membrane myelin subfractions at various ages showed that the membrane fraction contained more proteins in relation to lipids than the heavy-myelin fraction. The membrane fraction was also enriched in phospholipids compared with cholesterol and contrined equivalent amounts of 2':3'-cyclic nucleotide 3'-phosphohydrolase compared with heavy- and light-myelin fractions. The membrane fraction was deficient in myelin basic protein and proteolipid protein and enriched in high-molecular-weight proteins. The specific localization of galactosyltransferase in heavy-myelin and membrane fractions at an early age when myelination is just beginning suggests that it may have some role in the myelination process.     

Carbonic Anhydrase, 5''-Nucleotidase, and 2'',3''-Cyclic Nucleotide-3''-Phosphodiesterase Activities in Oligodendrocytes, Astrocytes, and Neurons Isolated from the Brains of Developing Rats

The activities of three myelin-associated enzymes, carbonic anhydrase, 5'-nucleotidase, and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP), were measured in oligodendrocytes, neurons, and astrocytes isolated from the brain of rats 10, 20, 60, and 120 days old. The carbonic anhydrase specific activity in oligodendrocytes was three- to fivefold higher than that in brain homogenates at each age, and, at all the ages, low activities of this enzyme were measured in neurons and astrocytes. The oligodendrocytes and astrocytes from the brains of rats at all ages had higher activities of the membrane-bound enzyme 5'-nucleotidase than was observed in neurons. In oligodendrocytes from 10- and 20-day-old rats, the 5'-nucleotidase activity was two-to threefold the activity in the homogenates (i.e., relative specific activity = 2.0-3.0), and the relative specific activity of this enzyme in the oligodendrocytes declined to less than 1.0 at the later ages, concomitant with the accumulation of 5'-nucleotidase in myelin. The CNP activity was always higher in oligodendrocytes than in neurons, but not appreciably different from that in astrocytes from 20 days of age onward. The relative specific activity of CNP was highest in the oligodendrocytes from 10-day-old rats but was lower, at all ages, than we had observed in bovine oligodendrocytes. These enzyme activities in oligodendroglia are quite different in amount and developmental pattern from those reported previously for myelin.     

Alteration of 5'-Nucleotidase and Na+, K+-ATPase in Central and Peripheral Nervous Tissue from Dysmyelinating Mutants (jimpy, quaking, Trembler, shiverer, and mld). Comparison with CNPase in the Developing Sciatic Nerve from Trembler

Abstract: 5'Nucleotidase and Na⁺,K⁺-ATPase are very probably myelin-associated enzymes, although not specific for this membrane. Thus, it is important to determine their activity in dysmyelinating mutants in either CNS (quaking, jimpy, shiverer, and mld) or PNS (Trembler). CNS: The activity of 5'nucleotidase was lower in mouse than in rat (10.5 and 28.0 nmol/min/mg protein in brain, respectively). In mouse myelin, the activity was 30 nmol/min/mg protein (and 72 in rat myelin). In mutants, the brain activity was very close to normal. In contrast, ATPase, the activity of which was higher in myelin as compared with forebrain homogenate, presented a reduced activity in various 21-day-old and adult mutants, except Trembler. It was normal in 8-day-old quaking and in cerebella from mutants. PNS: ATPase was lower than in brain and reduced in most mutants, this being expected for Trembler and quaking but not for shiverer and mld. 5'-Nucleotidase activity was higher compared with that in brain homogenate (relatively stable between 10-day postnatal and adult). It was affected in the mutants; in Trembler it was nearly normal in young animals but increased during development. Thus in Trembler, two different myelin-related enzymes and a myelin-specific enzyme (CNPase) presented different developmental patterns: ATPase was always reduced, 5'-nucleotidase was normal, and CNPase was slightly below normal in young (68% of the control value); CNPase activity declined during development but 5'-nucleotidase increased (42% and 190% of the control in 60-day-old animals). It is necessary to consider these results in parallel with alterations in the PNS because of Schwann cell abnormalities. Thus, determination of these two enzymes will provide a useful tool to study myelination and myelin assembly under both normal and pathological conditions.     

Desmosterol may replace cholesterol in lipid membranes

Recently, knockout mice entirely lacking cholesterol have been described as showing only a mild phenotype. For these animals, synthesis of cholesterol was interrupted at the level of its immediate precursor, desmosterol. Since cholesterol is a major and essential constituent of mammalian cellular membranes, we asked whether cholesterol with its specific impact on membrane properties might be replaced by desmosterol. By employing various approaches of NMR, fluorescence, and EPR spectroscopy, we found that the properties of phospholipid membranes like lipid packing in the presence of cholesterol or desmosterol are very similar. However, for lanosterol, a more distant precursor of cholesterol synthesis, we found significant differences in comparison with cholesterol and desmosterol. Our results show that, from the point of view of membrane biophysics, cholesterol and desmosterol behave identically and, therefore, replacement of cholesterol by desmosterol may not impact organism homeostasis.     

Effect of sterol structure on ordered membrane domain (raft) stability in symmetric and asymmetric vesicles

Sterol structure influences liquid ordered domains in membranes, and the dependence of biological functions on sterol structure can help identify processes dependent on ordered domains. In this study we compared the effect of sterol structure on ordered domain formation in symmetric vesicles composed of mixtures of sphingomyelin, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, and in asymmetric vesicles in which sphingomyelin was introduced into the outer leaflet of vesicles composed of DOPC and cholesterol. In most cases, sterol behavior was similar in symmetric and asymmetric vesicles, with ordered domains most strongly stabilized by 7-dehydrocholesterol (7DHC) and cholesterol, stabilized to a moderate degree by lanosterol, epicholesterol and desmosterol, and very little if at all by 4-cholesten-3-one. However, in asymmetric vesicles desmosterol stabilized ordered domain almost as well as cholesterol, and to a much greater degree than epicholesterol, so that the ability to support ordered domains decreased in the order 7-DHC > cholesterol > desmosterol > lanosterol > epicholesterol > 4-cholesten-3-one. This contrasts with values for intermediate stabilizing sterols in symmetric vesicles in which the ranking was cholesterol > lanosterol ~ desmosterol ~ epicholesterol or prior studies in which the ranking was cholesterol ~ epicholesterol > lanosterol ~ desmosterol. The reasons for these differences are discussed. Based on these results, we re-evaluated our prior studies in cells and conclude that endocytosis levels and bacterial uptake are even more closely correlated with the ability of sterols to form ordered domains than previously thought, and do not necessarily require that a sterol have a 3β-OH group.     

Diffusion of cholesterol and its precursors in lipid membranes studied by 1H pulsed field gradient magic angle spinning NMR

Cholesterol content is critical for membrane functional properties. We studied the influence of cholesterol and its precursors desmosterol and lanosterol on lateral diffusion of phospholipids and sterols by1H pulsed field gradients (PFG) magic angle spinning (MAS) NMR spectroscopy. The high resolution of resonances afforded by MAS NMR permitted simultaneous diffusion measurements on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and sterols. The cholesterol diffusion mirrored the DPPC behavior, but rates were slightly higher at all cholesterol concentrations. DPPC and cholesterol diffusion rates decreased and became cholesterol concentration dependent with the onset of liquid-ordered phase formation. The activation energies of diffusion in the coexistence region of liquid-ordered/liquid-disordered phases are higher by about a factor of 2 compared to pure DPPC and to the pure liquid-ordered state formed at higher cholesterol concentrations. We assume that the higher activation energies are a reflection of lipid diffusion across domain boundaries. In lanosterol- and desmosterol-containing membranes, the DPPC and sterol diffusion coefficients are somewhat higher. Whereas the desmosterol rates are only slightly higher than those of DPPC, the lanosterol diffusion rates significantly exceed DPPC rates, indicating a weaker interaction between DPPC and lanosterol.     

Lipid composition of brain myelin from normal and hyperphenylalaninemic chick embryos

The influence of hyperphenylalaninemia on the lipid composition of brain myelin has been investigated in 19-day-old chick embryos. CNP-ase activity was used as myelin marker enzyme for myelin isolation. CNP-ase activity was significantly lower in hyperphenylalaninemic myelin when compared with control. No significant differences were observed after experimental treatment in the total lipid content of myelin as well as in the proportion of cholesterol:phospholipid:galactolipid. Nevertheless, a clear increase in the percentage of esterified cholesterol was found. No appreciable alterations were observed in the phospholipid composition of brain myelin from both control and hyperphenylalaninemic chick embryos. However, the ratio of unsaturated to saturated fatty acids in serine plasmalogen and sphingomyelin was considerably increased by this treatment. This ratio in choline and ethanolamine phosphatides from treated embryos did not differ from that of controls.     

Ethanolamine Kinase Activity in Purified Myelin of Rat Brain

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. Kinetic studies indicated similar Km values for ethanolamine in the microsomal, cytosolic, and myelin fractions but a significantly lower apparent Km for ATP in myelin. This and other differences suggested the possible existence of isozymes. Establishment of the presence of this kinase completes the list of phospholipid synthesizing enzymes needed to synthesize phosphatidylethanolamine from diacylglycerol within the myelin membrane.