In vitro incorporation of (U-C14)-glucose and (1-C14)-sodium acetate in peripheral nerves of malnourished young rhesus monkeys

The effect of protein calorie malnutrition (PCM) on synthesis of lipids in peripheral nerves was studied by in vitro incorporation of (U-C14)-glucose and (1-C14)-sodium acetate. Ulnar and tibial nerves obtained from five young rhesus monkeys with PCM, five rehabilitated monkeys, and five control monkeys were incubated for 2 h with the radioactive precursors. Uptake of both radioactive precursors in whole peripheral nerves as well as myelin marker lipids was significantly decreased in animals with PCM. However, uptake returned to normal in rehabilitated monkeys.     

Effect of protein calorie malnutrition on in vitro incorporation of (U-C14)-glucose in brain of young rhesus monkeys

Twelve young rhesus monkeys weighing 1.5-2.0 kg were acclimatised for a month in the animal house of the Institute. After acclimatization 6 monkeys were placed on a protein calorie deficient diet and remaining 6 on normal diet. The deficiency state was indicated by reduction in body weight, significant decrease in serum albumin, lack of physical activity and shedding of fur. At this stage, animals of both groups were sacrificed and brains were removed. Cerebrum was separated, blotted on filter paper and weighed. In vitro incorporation of (U-C14)-glucose was done for 2 hr in approximately 300 mg cerebrum. Incorporation of this precursor was determined in cerebrum, lipids, its fractions and proteins in both groups. There was increased incorporation of (U-C14)-glucose into tissue, gangliosides, free fatty acids and esterified cholesterol but no significant change in myelin marker lipids and proteins in PCM group was noticed as compared to age matched controls.     

Postnatal change in lipid composition and nerve conduction of peripheral nerves of young rhesus monkeys.

Body weight, motor and sensory nerve conduction velocities of fore and hind limbs, and lipid composition were measured sequentially in peripheral nerves of 15 rhesus monkeys. Initially measurements were made with monkeys six to eight months of age. There were significant increases in body weight, motor, and sensory nerve conduction and myelin marker lipids over a five months period, but no change was observed in free fatty acids, triglycerides, and esterified cholesterol. These results indicate that myelination continues at least for 11 to 13 months of postnatal age in rhesus monkeys.     

Effects of Detergents, Proteins, and Lipids on 2'':3''-Cyclic-Nucleotide 3''-Phosphodiesterase Activity

The myelin marker 2':3'-cyclic-nucleotide 34'-phosphodiesterase (CNPase) was isolated to a lipid- and phosphate-free stage. The effects of exogenously added lipids were tested on this preparation and compared to the known stimulation of the enzyme by detergents and proteins. CNPase could be stimulated 2-3 fold by these various agents which appeared to be additive in their effect. Enzyme-protein and enzyme-lipid interactions and possible medical use of the improved assay conditions for CNPase employed in the study are discussed.     

Protein determinants of myelination in different regions of developing rat central nervous system.

Measurements of several different protein determinants correlated with the time and rate of myelination in five areas of the central nervous system are presented. The deposition of protein in the subcellular fraction corresponding to the density of adult myelin, the appearance of basic protein characteristic myelin, the change in proportions of the individual myelin proteins, the appearance and distribution of the myelin marker 2':3'-cyclic nucleotide3'-phosphohydrolase, and the results of morphological studies of purified myelin are compared. According to these various criteria, and in agreement with the morphological observations of others, myelin appears earliest in the spinal cord, then in the brain stem, and latest in the cerebral hemispheres. Multilamellar myelin was observed in the rat brain stem and spinal cord as early as 5 days of age. The relative proportion of the individual myelin proteins changed with myelin maturation in all areas, with the larger basic protein decreasing reciprocally with increase of the smaller basic protein. The proportion of Wolfgram protein also decreased with maturation. Larger proportions of the enzyme 2':3'-cyclic nucleotide 3'-phosphohydrolase were located in the microsomal fraction at early ages. During development the enzyme activity gradually became associated more with a fraction of a density corresponding to adult myelin, suggesting the presence of precursor membrane fragments in microsomal fractions in the early stages of myelination before compact myelin formation. A significant proportion of the total nucleotide phosphohydrolase activity of the homogenate could not be recovered in subcellular fraction at early ages, but the recovers of the enzyme increased with maturation and the activity was found more in the myelin fraction.     

The presence of 2'', 3''-cyclic AMP 3''-phosphohydrolase in glial cells in tissue culture

Abstract— T he E nzyme 2', 3'-cyclic AMP 3'-phosphohydrolase (CNP) is regarded as a marker for myelin (KURI- HARA and MANDEL, 1970) on the basis of its regional and subcellular distribution (K urihara and T sukada , 1967), its ontogenetic characteristics (KURIHARA and TSUKADA, 1968), and its behaviour in two strains of myelin-deficient mutant mice (K urihara , N ussbaum and M andel , 1969). However we have isolated highly-purified preparations of neuronal plasma membrane from rat brain synaptosomes which contain this enzyme activity (M organ , W olfe , M andel and G ombos , 1971). Two explanations of this finding are possible. The activity could be due to the presence of myelin, but this explanation is ruled out by electron microscopy and by the low level of cerebrosides in the synaptosomal plasma membrane preparations. Myelin is extremely rich in cerebrosides ( norton and A utilio , 1966). The second possibility is that the enzyme, 2', 3'-cyclic AMP 3'-phosphohyrolase, may also be found in the glial cells from which myelin is derived (B unge , B unge and P appas , 1962). To test our hypothesis that 2', 3'-cyclic AMP 3'-phosphohydrolase is not a specific marker for myelin, but is also found, in glial cells, we have examined a tumoral glial cell line maintained in myelin-free tissue culture.     

Morphological and Biochemical Changes in Myelin Subfractions of Developing Rats Fed Microbial Lipids

Morphological, biochemical, and physicochemical studies of myelin subfractions were undertaken on the progeny of Sprague-Dawley rats fed diets containing lipids either extracted from yeasts grown on n-alkanes or from margarine. Myelin subfractions obtained from pooled brain homogenates of littermates by sucrose density gradient centrifugation at 7, 14, and 21 days postnatally were subjected to electron microscopy, sodium dodecylsulfate polyacrylamide gel electrophoresis and assayed for 2', 3' cyclic nucleotide 3'-phosphohydrolase activity (CNPase; EC 3.1.4.37). Additionally, surface pressure measurements were made of lipid monolayers derived from myelin subfractions, which were subsequently injected with myelin basic proteins. The myelin subfractions of test animals, when compared with those of controls, show an earlier increase in the specific activity of CNPase, the earlier appearance of low-molecular-weight proteins, and an increase in the affinity of basic proteins for lipids derived from the myelin light fraction. This biochemistry suggests the presence of a more mature myelin between 7 and 14 days in the experimental group. The morphological studies, however, do not seem to concur with the biochemical data. The observed changes are discussed in relation to the influence of dietary lipids on myelinogenesis.     

Identification of 2':3'-cyclic nucleotide 3'-phosphodiesterase in the vertebrate retina

The enzyme, 2':3'-cyclic nucleotide 3'-phosphodiesterase (2':3'-cNMP-3'-ase) has been used as a marker in the nervous system for the presence of myelin membrane or myelin-producing glial cells. In this study, goldfish and bovine neural retinas are found to have high levels of such a diesterase activity. Analysis of retinal tissue incubated with 2':3'-cAMP shows only 2'-AMP as the reaction product, indicating the selective hydrolysis of the cyclic nucleotide. Microdissection of the goldfish retina demonstrates the highest 2':3'-cNMP-3'-ase activity in the region of the photoreceptors. A fraction enriched in bovine rod outer segments has about a 5-fold increase in specific enzyme activity when compared to whole retina preparations. These data suggest that 2':3'-cNMP-3'-ase is either closely associated with or is an intrinsic feature of vertebrate photoreceptor elements. The retina, which contains this enzyme, may serve as a model to investigate the influence of 2':3'-cyclic nucleotides on a function of the nervous system.     

2',3'-Cyclic nucleotide 3'-phosphohydrolase in the developing chick brain and spinal cord

Developmental changes of the 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in the chick brain and spinal cord are reported. The greater part of increase in enzyme activity occurred between 18 days of incubation and 3 days after hatching in the whole brain, and between 18 and 21 days of incubation in the spinal cord. These periods are those of active myelination in the chick brain and spinal cord, respectively. The possibility was emphasized that 2',3'-cyclic nucleotide 3'-phosphohydrolase can be used as a marker for the myelin sheaths in the developing central nervous system. Comparisons were also made among the developmental changes in the forebrain, midbrain, brain stem, cerebellum, and spinal cord.     

THE SYNTHESIS OF MYELIN IN DEVELOPING RAT BRAIN

Abstract— —The synthesis of myelin proteins has been studied in the grey and white matter slices of developing rat brain by measuring the incorporation of [³H]lysine and [¹⁴C]arginine into polypeptide. The incorporation was sensitive to cycloheximide and puromycin at 1 mM concentration. Developing rat optic nerve slices, free of retinal ganglion cells, were able to synthesize myelin basic and proteolipid proteins, but rat retinal preparation failed to synthesize myelin basic protein. Rabbit retinae were able to synthesize myelin basic and proteolipid proteins. Significant activity of the myelin marker enzyme 2',3'-cyclic nucleotide-2'-phosphodiesterase has been found in the rabbit retina but not in rat retina. The results presented in this communication suggest that myelin proteins in the rat CNS are synthesized by the oligodendroglial cells and that neurons probably do not participate.     

Detergent activation and solubilization of 2'':3''-cyclic nucleotide 3''-phosphodiesterase from isolated myelin and c6 cells.

Several detergents were investigated for their ability to increase activity of 2':3'-cyclic nucleotide 3'-phosphodiesterase in isolated myelin. The ability of Triton X-100 and Sulfobetaine DLH to solubilize the enzyme was also examined. Solubilization with Triton X-100 was only effective in the presence of salt, for example with NaCl 51% of the activity was solubilized. A single extraction with Sulfobetaine DLH yielded slightly more solubilized enzyme and did not require added salt. Both activation and solubilization of 2':3'-cyclic nucleotide 3'-phosphodiesterase appeared to be similarly dependent on detergent concentration, suggesting a common action of the detergent in the two processes. Myelin basic protein was solubilized more readily than the enzyme. In contrast with the enzyme in myelin, 2':3'-cyclic nucleotide 3'-phosphodiesterase activity in C6 cells was not increased in the presence of Triton X-100, and was partially solubilized by either Triton X-100 or NaCl alone. No myelin basic protein could be detected in C6 cells by radioimmunoassay.     

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.     

Separation and identification of 2',3'-cyclic nucleotide 3'-phosphodiesterase on isoelectric focusing gels

A method is presented for the separation and detection of the myelin marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase on isoelectric focusing gels and by immunoblotting. The gel staining procedure is a modification of a method used to demonstrate enzyme activity on blots after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis. The results show that immunologically active 2',3'-cyclic nucleotide 3'-phosphodiesterase can be separated under equilibrium conditions on isoelectric focusing gels with an expanded alkaline pH range after solubilization in a mixture of nonionic/zwitterionic detergents and urea. Enzymatically active 2',3'-cyclic nucleotide 3'-phosphodiesterase focused as two closely spaced bands at pIapp 8.1 and 8.8, respectively, while 2',3'-cyclic nucleotide 3'-phosphodiesterase immunoreactivity was detected as four distinct bands at pIapp 4.2, 7.4, 8.8, and 9.3 and a diffuse band at pIapp 7.9-8.2. By two-dimensional separation these five bands showed molecular weights of about 43-47 kDa, i.e., corresponding to reported values for immunologically active 2',3'-cyclic nucleotide 3'-phosphodiesterase. Since enzyme activity is associated with only two of the bands, nonspecific and artifactual banding due to, e.g., detergent micelle formation, is unlikely.     

Inhibition of bovine and human brain 2':3'-cyclic nucleotide 3'-phosphodiesterase by heparin and polyribonucleotides and evidence for an associated 5'-polynucleotide kinase activity

The present paper establishes a 5'-polynucleotide kinase activity associated with the bovine and human brain enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37) in addition to known extremely high hydrolysis rates against 2':3'-cyclic nucleotides. Modulation of the enzyme activity by the addition of polyadenylate (5') and polyuridylate (5'), histone F3, myelin basic protein (MBP), and other basic molecules suggest that RNA may be the natural substrate for both enzymes. These enzymes, isolated from brain and present in very high activities in oligodendrocytes and in isolated myelin, probably have complex functions.     

A biochemical comparison of Xenopus laevis and mammalian myelin from the central and peripheral nervous systems.

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P0 protein and a basic protein of slightly larger mol wt. than the P1 protein of rat myelin.     

Myelin formation in dissociated cell cultures of rat embryonic cerebral hemispheres

Developing cultures of dissociated cerebral hemispheres obtained from 18-day-old embryonic rats synthesized, or activated, a myelin-related enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). This increase in activity coincided with the onset of myelination. The presence of CNPase in oligodendrocytes and myelin was demonstrated using immunocytochemical staining techniques. Myelinated axons and myelin sheaths were clearly made visible by electron microscopy of 28-day-old cultures.     

EFFECT OF EXOGENOUS LIPIDS ON ACTIVITIES OF THE RAT BRAIN CHOLESTEROL ESTER HYDROLASE LOCALIZED IN THE MYELIN SHEATH1

Abstract— Activity of cholesterol ester hydrolase localized almost exclusively in the myelin sheath (Eto & Suzuki , 1973a) was greatly affected by exogenous lipids added to the assay mixture. With isolated myelin as the enzyme source, phosphatidylserine was most effective in stimulating the activity. Other phospholipids were less effective. Efhanolamine phospholipid was slightly inhibitory and lysolecithin was strongly inhibitory. Differences in the fatty acid composition did not appear to account for such different effects. Glucosylceramide, galactosylceramide and digalactosylceramide were stimulatory while sulfatide, ganglioside and its asialo-derivative were inhibitory. Saturated fatty acids were generally stimulatory while corresponding unsaturated acids were strongly inhibitory. In order for exogenous lipids to be effective they had to be added to the assay mixture as free dispersion. When heat-inactivated myelin was used as the lipid source, no effect was observed, while equivalent amounts of a whole white matter lipid mixture was effective. Although phosphatidylserine was the most effective activator among the lipids tested, it could not completely replace sodium taurocholate present in the standard assay system. When isolated myelin was stored frozen, the activity of the enzyme declined gradually in the standard system without additional lipids. The stimulating effect of phosphatidylserine was greater for such partially inactivated enzyme sources, although it did not completely restore the activity to that of fresh preparations. When myelin was fractionated into basic protein, proteolipid protein and the high molecular weight acidic protein (Wolfgram) fractions, the last fraction contained most of the recovered activity. However, Wolfgram protein was less active than the intact myelin when assayed without additional lipid. The addition of phosphatidylserine completely restored the activity of this partially delipidated preparation.     

Further Characterization of a Myelin-Associated Neuraminidase: Properties and Substrate Specificity

A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity.     

CNPase activity in myelin from adult rat brains after prolonged lead exposure in vivo

The present study investigated the sensitivity of rats cerebral myelin to prolonged toxicity of lead (Pb) that imitates environmental exposure to this metal. The results indicated that 90 days exposure of young adult rats to lead in drinking water affects the morphology of myelin sheaths, expressed in disintegration of its multilamellar structure. Both, the protein content and the activity of the myelin-specific enzyme CNPase (2',3'-cyclic nucleotide 3-phosphodiesterase), were lowered. The Michaelis-Menten kinetic for CNPase in myelin obtained from control and Pb-treated rats was different. Km increased and Vmax decreased when compared to controls. Observed disturbances in enzyme activity may be one of the potential reasons of the ultrastructural changes. It is thus tempting to speculate that Pb may be considered as a one of the factors contributing to demyelinating diseases.     

Specific localization of 2',3'-cyclic nucleotide 3'-phosphohydrolase, (Ca2+/Mg2+)-ATPase, and acetylcholinesterase in human erythrocyte membrane

A procedure was developed for the detection of 2',3'-cyclic nucleotide 3'-phosphohydrolase in myelin. This assay was sufficiently to detect the low levels of 2',3'-cyclic nucleotide 3'-phosphohydrolase in human erythrocytes. The 2',3'-cyclic nucleotide 3'-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghosts and resealed ghosts were assayed for 2',3'-cyclic nucleotide 3'-phosphohydrolase (Ca2+/Mg2+)-ATPase, and acetylcholinesterase activity, and the 2',3'-cyclic nucleotide 3'-phosphohydrolase profile is the same as that of the (Ca2+/Mg2+)-ATP, an established inner membrane marker.