Cerebroside Sulfotransferase Research Articles

Cerebroside and sulfatide biosynthesis in the brain of Snell dwarf mouse: effects of thyroxine and growth hormone in the early postnatal period.

Snell dwarf mice (dw/dw) and normal mice (+/?) were injected with thyroxine (T4) (1 microgram/animal, four injections) and growth hormone (GH) (20 micrograms/animal, four injections) from the 5th to the 15th day of life. In the untreated dw/dw mouse brain, the specific activities of UDP-galactose:ceramide galactosyltransferase (CGalT), PAPS:cerebroside sulfotransferase (CST), and 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNP) were decreased by 28, 25, and 37%, respectively, compared with the control untreated +/? mice. The major effect of T4 was an increase of the brain CNP in the +/? mice (+40%) and dw/dw mice (+111%). The treatment with T4 also brought to normal the level of CGalT in dw/dw brain; a somewhat less marked effect on CST was observed. The treatment with GH had a great stimulatory effect on CNP: the specific activity of this enzyme increased by 40 and 69% in +/? and dw/dw mouse brain, respectively. On the contrary, no effect of GH on the CGalT activity was observed in this study. Our results suggest that T4 and GH may have both independent and complementary actions on the myelin-associated enzymes during the early postnatal period of brain development.

Cerebroside sulfotransferase in Golgi-enriched fractions from rat brain.

Golgi-enriched fractions were prepared from brainstems of 17-day-old rats by first floating off myelin, then fractionating the remaining pellet by a series of differential and density gradient centrifugations in sucrose. Fractions enriched in Golgi membranes were recovered at 0.46/0.76 M and 0.76/0.87 M interfaces on the final sucrose gradient as indicated by morphology and the biochemical markers thiamine pyrophosphatase and [3H]fucose-labeled glycoprotein. Morphology of the two fractions indicated very little contamination with myelin lamellae; however, the presence of significant levels of 2',3'-cyclic nucleotidase in the lighter fraction suggested a contribution from oligodendroglial or myelin-related membranes. Cerebroside sulfotransferase was highly enriched in the lighter Golgi-enriched fraction relative to the denser fraction, the post-34,880 X g microsomes, and the myelin-like fraction. In contrast, ceramide galactosyl transferase was more evenly distributed among the fractions. Our results show a more highly localized distribution of sulfatide synthesis than of galactocerebroside synthesis, probably in Golgi membranes or oligodendroglia-related membranes with similar properties.

Distribution of lipid synthesizing enzymes, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin proteins in rat forebrain subfractions during development.

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 myelin-specific enzyme, 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin proteins were measured in the same subfractions. UDP-glucose:ceramide glucosyltransferase and cerebroside sulfotransferase were confined to microsomes. CGalT was localized in microsomes, but also in myelin and myelin-related fractions. The developmental change in distribution of CGalT in adult animals toward myelin containing fractions could indicate that the replacement of galactosylceramide in compact myelin could be carried out in close proximity to compact myelin (mesaxon, paranodal loops) rather than in the distant oligodendrocyte perikaryon.

Cerebroside sulfotransferase in rat gastric mucosa

1. 1. Cerebroside sulfotransferase activity was demonstrated in rat gastric mucosa. This enzyme preparation catalyzes the transfer of sulfate group from 3′-phosphoadenosine-5′-phosphosulfate to added cerebroside and lactosylceramide. 2. 2. Psychosine also serves as an acceptor for sulfate group. 3. 3. Both hydroxy-fatty acid and non-hydroxy-fatty acid containing cerebrosides are equally sulfated. However, this enzyme is inactive toward glucosylceramide. 4. 4. The optimum pH is around 6.0. The reaction is stimulated by the addition of 0.4–0.9% of Triton X-100. 5. 5. The presence of 2–4 mM ATP moderately stimulates the reaction, but is inhibitory when the concentration exceeds 6 mM. 6. 6. EDTA has no effect. 7. 7. Both Mg 2+ and Ca 2+ stimulate at lower concentrations (<16mM), but inhibit at higher concentrations (>20mM). Mn 2+, however, is a strong inhibitor even at 8mM concentration. 8. 8. The apparent K m for cerebroside is 0.04 mM, while that for 3′-phosphoadenosine-5′-phosphosulfate is 0.45 μM.

Cerebroside sulfotransferase: preparation of antibody and localization of antigen in kidney.

This immunohistochemical study describes the localization of the enzyme cerebroside sulfotransferase (phosphoadenosine phosphosulfate: galactosylceramide sulfotransferase, EC 2.8.2.11) in rat kidney. The enzyme was purified from kidney and the preparation was used to raise antibodies for immunocytochemical investigations. In the kidney, the antigen was present only on the brush border of the epithelial cells of the proximal tubules, suggesting that sulfation of glycolipids occurs in the cytoplasm and plasma membranes of these specific cells. Moreover, biochemical and immunocytochemical studies of cerebroside sulfotransferase during development indicate that catalytic activity is correlated with the appearance of enzyme protein.

Effect of glucose deprivation on sulfatide synthesis and oligodendrocytes in cultured brain cells of newborn mice.

Summary: The present work was designed to develop a model for studying the effect of substrates on brain cell metabolism. The synthesis of sulfatide, a myelin lipid, which takes place predominantly in oligodendrocytes was measured in vitro in dissociated cell cultures of newborn mouse brains at 13 days in culture. This corresponds to an active stage of in vivo myelination. The effect of increasing durations of severe glucose deprivation (below 1 mM in the medium) was assessed on morphology, cellular protein, DNA, sulfatide synthesis, cerebroside sulfotransferase activity, and mucopolysaccharide synthesis. Glucose was rapidly consumed by the cultured cells above a concentration of 0.4 mM in the medium. At high and intermediate glucose concentrations, between 43.8 and 63.6% of the glucose consumed was accumulated as lactate on a molar ratio, indicating that the pyruvate dehydrogenase complex was rate limiting. In severe glucose deprivation, lactate consumption of the cultures exceeded accumulation. With increasing duration of severe glucose deprivation (below 1 mM), the following major changes were observed. The number of phase dark cells identified by immunofluorescence as oligoden-drocytes progressively decreased. Immunologically identified astrocytes apparently were unaffected by glucose deprivation. Sulfatide synthesis progressively dropped to 3.8% of control, and cerebroside sulfotransferase dropped to 10.3% of control with prolonged glucose deprivation. Thus, oligodendrocytes and one of their specific functions were damaged by glucose deprivation. Lactate, in spite of its being consumed by the brain cell cultures, was unable to replace glucose as a substrate for sulfatide synthesis. Uninfluenced by glucose deprivation were total cellular phosphocreatinkinase, arylsulfatase A, as well as total mucopolysaccharide synthesis by the brain cell cultures. Speculation: The present study shows that sulfatide synthesis in cultured brain cells of newborn mice at a stage corresponding to active in vivo myelination is dependent upon availability of glucose. Lactate, in spite of its being consumed by the brain cells, is unable to replace glucose with respect to sulfatide synthesis. This system of cultivated brain cells may serve as a model for studying other potential fuels for the maintenance of oligodendrocyte function.

Quantitative measurement of in vivo sulfatide metabolism during development of the mouse brain: Evidence for a large rapidly degradable sulfatide pool

The in vivo metabolism of sulfatide was studied in the cerebellum and in the cerebrum of developing mice by intraperitoneal injection of [ 35 S]sulfate. After correction for the specific radioactivity of the sulfate, total sulfatide synthesis and degradation could be determined. The developmental patterns of synthesis and degradation of sulfatide in vivo were compared with the developmental activity patterns of the synthesizing enzyme cerebroside sulfotransferase (CST, EC 2.8.2.11), the degrading enzyme cerebroside sulfate sulfatase, measured as arylsulfatase A (ASA, EC 3.1.6.1), and the net sulfatide synthesis in both brain parts. During brain development, sulfatide synthesis per milligram of tissue was higher in the cerebellum than in the cerebrum. Its developmental pattern was similar but not identical to the CST activity in vitro . The in vivo pattern of sulfatide degradation followed that of sulfatide biosynthesis and was similar to the developmental activity pattern of ASA. During myelination 40–70% of the newly synthesized sulfatide was degraded within 24 hr, as measured in two ways: (1) by a 24-hr chase of the [ 25 S]sulfatide and (2) by calculating the difference between the total daily sulfatide synthesis and net daily accumulation of sulfatide. Both methods of determination gave similar results. The data presented show that the synthetic enzyme CST is fully active in vivo , producing more sulfatide than is necessary for incorporation into myelin. The resulting sulfatide excess is rapidly degraded in the lysosomes by the action of ASA. Thus net sulfatide synthesis is partly regulated by lysosomal degradation during myelination of the brain.

High cerebroside sulfotransferase activity in a hereditary demyelinating neuropathy

'Trembler', a spontaneous, dominantly-inherited murine mutation causes spastic paralysis and an action tremor in mice. Peripheral nerves of Trembler mice show recurrent demyelination, remyelination and onion bulb formation resembling findings reported for onion bulb neuropathies in man. The metabolism of sulfatide, a lipid enriched in myelin, was investigated in this mutation. The sulfatide content of the nerve was decreased in Trembler mice but reflected the low myelin content. The specific activity of cerebroside sulfotransferase (CST), the enzyme responsible for the transfer of sulfate to cerebroside to form sulfatide, was increased by 257 percent in 15-day-old mutants. No activator or inhibitor effect and no lack of product inhibition could explain these results. In mutant PNS, Km, Vmax and heat sensitivity values for CST differed from controls In contrast, no abnormality could be demonstrated in mutant brain. These results suggest the presence of an abnormal enzyme, with a decreased affinity for its substrate but present in larger amounts. In vivo incorporation of radioactive sulfate into sulfatide and nonlipidic material was increased. The lack of accumulation of sulfatides could be explained by the low levels of substrate available and high arylsulfatase A activities. High arylsulfatase A activities with normal CST activities were observed in demyelinating diseases.

Comparison of the inhibition by methylmercury and triethyllead of galactolipid accumulation in rat brain

Methylmercury chloride (MeHg) and triethyllead chloride (Et 3Pb) interfere with the myelin deposition in young rats. In order to establish how myelin synthesis and development are affected by these neurotoxins, we have investigated the synthesis of myelin galactolipids using two different systems: (1) slices of cerebellum from 2-week-old rats, (2) enzyme preparations from rat brain. MeHg (50 μ m) or Et 3Pb (5 μ m) gave maximum inhibition of the uptake of 35SO 4 by sulfatides (63%). The [ 3H]serine uptake by cerebrosides with normal unsubstituted fatty acids (NFA) was inhibited up to 45% at 70 and 7 μ m of MeHg and Et 3Pb, respectively, after incubation of the slices for 16 hr in an Eagle basal medium at 37°C. At the same concentrations the inhibition of [ 3H]serine uptake in cerebrosides with α-hydroxy fatty acids (HFA) was about 35%. MeHg (50–100 μ m) inhibited UDPgalactose:ceramide galactosyltransferase (CGalT) and phosphoadenosine-5′-phosphosulfate:cerebroside sulfotransferase (CST) activities up to 50%, while the inhibition with Et 3Pb was about 30% at 2.5 m m for the enzymes.

Abnormal metabolism of 35SO4-sulfatide in jimpy brains expressed in brain organotypic cultures.

Cerebroside-sulfotransferase (CST), creatine-phosphokinase (CPK), and 3-hydroxy-3-methylglutaroyl CoA (HMG CoA) reductase activity, protein, and DNA content were measured in an easy-to-perform organotypic culture system of newborn normal and jimpy brains. The defective sulfatide synthesis which has been shown in vivo in jimpy brains could also be demonstrated in organ cultures of jimpy mice in the form of lowered CST activity in the homogenate as well as reduced 35SO4 incorporation into 35SO4-sulfatide. HMG CoA reductase was reduced to 60% of that found in 16-day-old normal cultures, similar to the findings in vivo. DNA of jimpy cultures was significantly lower than that in normal cultures, suggesting the possibility of an arrest in the differentiation or increased cellular death of presumptive oligodendrocytes, as was found in vivo. Organ cultures of jimpy mouse brain can serve as an appropriate model for further study of the primary defect in this animal mutant.

Abnormal sulfate metabolism in a hereditary demyelinating neuropathy

Trembler mice are affected by dominantly inherited neuropathy. Total lipid content and sulfatides were decreased in peripheral nerves from 15-day-old mutants. The proportion of sulfatides in per cent of total lipids was similar in control and Trembler nerves. The specific activity of ceramide galactosyltrnsferase, the enzyme responsible for the synthesis of cerebrosides, was 36 and 13% of controls, in young and adult. Trembler nerves, respectively. In contrast, cerebroside sulfotransferase activities were increased by 257 and 172% in young and adult Trembler sciatic nerves, respectively. No activator or inhibitor effect could be demonstrated. In Trembler PNS, Km, Vmax and heat sensitivity of CST differed from controls. Low levels of substrate and high arylsulfatase A activity (218% of controls) could explain the lack of sulfatide accumulation. The increased in vivo sulfate and galactose incorporation into non-lipidic material couild reflect the overproduction of endoneurial and perineurial connective tissue, whereas the high turnover rate of sulfatides could be correlated with intense demyelination and remyelination observed in Trembler PNS.

Evidence for the presence of CDP-ethanolamine: 1,2-diacyl-sn-glycerol ethanolaminephosphotransferase in rat central nervous system myelin.

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.

Progressive somatofugal activation of cerebroside sulfotransferase in the developing chick optic fibers.

A myelination parameter of the chick optic pathway has been investigated: the activity of cerebroside sulfotransferase. Evidence is presented for a somatofugal progression of the enzyme activity peak along the optic nerve, chiasm, optic tract, tectum anterior, and tectum posterior. The enzyme activity appears on the fifteenth embryonic day and reaches a maximum in the nerve two days before hatching. One day after hatching, the peak is found in the chiasm, and three days after that in the optic tract. The peak is found in the tectum anterior on the tenth day. The results suggest a somatofugal progression of myelination.

Effect of glucose deprivation on sulfatide synthesis in dissociated brain cell cultures of newborn mice

Sulfatide synthesis (SFS) in brain cells is a marker for cell differentiation. SFS was measured by incorporation of 35S04 into sulfatide in cultured brain cells of newborn mice at 13 days in culture after exposure to various glucose concentrations for the preceding 72hrs. This age range corresponds to an in vivo developmental stage of rapid myelination. Glucose extraction from the medium during the first 24hrs was independent from the initial glucose level down to lmM. Thus the duration of severe glucose deficiency (below lmM) was related to the initial glucose supply. SFS was progressively reduced with increasing duration of severe glucose deprivation reaching 3.5% of controls after 48hrs of glucose deficiency. In the cell homogenates the sulfatide synthetizing enzyme cerebroside sulfotransferase was similarly reduced following glucose depletion keeping a residual activity of 10%. This indicates that a lack of precursors or substrates was not the only cause of SFS reduction. Replenishment of glucose stores in the medium after a starvation period of 14hrs only failed to recover SFS. Reexposure of glucose deprived cells to glucose even led to a further decrease of SFS suggesting a possible toxic effect of glucose on brain cells previously damaged by glucose depletion.

Cerebroside Sulfotransferase Activity in Human Lung Tissues&lt;subtitle&gt;An Elevated Level in Lung Adenocarcinoma&lt;/subtitle&gt;

Cerebroside sulfotransferase activity was demonstrated in particulate fractions from human lung and its carcinoma tissues. The activity in human lung adenocarcinoma was significantly higher than those in a different histological type of carcinoma (squamous cell carcinoma) and in the normal tissue from which each carcinoma was derived.

Chronic lead intoxication: effects on developing optic nerve.

The effects of chronic lead intoxication were studied by examining the optic nerve of mice given lead-containing mother's milk from day 1 to day 21 of life. Biochemical assays for myelin basic protein, 2',3'-cyclic nucleotide phosphodieterase, and cerebroside sulfotransferase showed that the total amount of myelin produced by the lead-exposed animals was decreased. Lead exposure did not alter the number of oligodendroglia or the relationship between axons and myelin sheaths. The hypomyelination was paralleled by a reduction in size of optic axons in the lead-exposed animals. The data suggest that chronic exposure to lead in developing mice results in a primary effect on neurons and that hypomyelination is secondary to reduction in axon size.

Influence of reduced cholesterol synthesis on the activity of cerebroside sulfotransferase in cultured glioblastoma cells treated with estradiol

Cultured glioblastoma cells were inoculated with estradiol in concentrations of 0.5–10 μl/ml medium in order to check the effect of this hormone on the activity of cerebroside sulfotransferase, an enzyme whose activity is strongly related to myelination. Thereby we could show that the cerebroside-sulfotransferase activity increases to a value of 200% of normal. Concomitant to this effect, the cholesterol content of the membrane bearing cerebroside sulfotransferase activity decreases to 60% of normal. The effect is fully reversible: after 48 h, cholesterol synthesis as well as cerebroside sulfotransferase activity reach normal values again. We suggest that cerebroside sulfotransferase activity is modulated by the changing cholesterol/phospholipid ratio in the cells during the inoculation period.

Brain reaggregate cultures: Biochemical evidence for myelin membrane synthesis

Myelin membrane synthesis was studied using mechanically dissociated fetal rodent CNS which formed spherical reaggregates while being maintained in rotating culture flasks. These reaggregate cultures exhibited myelinogenesis in vitro after precisely the same period of time needed for myelin synthesis to commence in vivo. The myelin membrane related enzymes, 2', 3' cyclic nucleotide phosphohydrolase (CNP) and cerebroside sulfotransferase (CST), appear similar in their specific activities and follow the same developmental patterns that these enzymes exhibit in vivo. In addition, phosphorylation of myelin basic protein occurs by the third week in vitro which agrees with previously published in vivo studies. These experiments indicate that this nerve-cell culture system may be a appropriate model for studying the biological regulation of myelinogenesis as well as a variety of other nervous-system functions.

Age-dependent modulation of 3'-phosphoadenosine-5'-phosphosulfate -galactosylceramide sulfotransferase by lipids extracted from the microsomal membranes and artificial lipid mixtures

The 3'-phosphoadenosine-5'-phosphosulfate-galactosylceramide-sulfotransferase (cerebroside sulfotransferase) is a microsomal enzyme, which shows a definite developmental activity pattern. This report gives evidence that the enzyme activity of partially delipidated microsomes is modulated by the cholesterol: phospholipid ratio of the extracted microsomal lipids in an age-dependent manner. These findings suggest that in vivo the enzyme activity is modulated by the lipid surrounding.

Myelinogenesis in optic nerve. A morphological, autoradiographic, and biochemical analysis.

Morphological, autoradiographic, and biochemical methods were used to study the time of appearance, distribution, and nature of sulfated constituents in the developing rat optic nerve. Electron microscope studies showed that myelination begins (6 days postnatal) shortly after the appearance of oligodendroglia (5 days postnatal). Over the ensuing 3 wk, myelination increased rapidly. During the 1st postnatal wk, mucopolysaccharides and glycoproteins were labeled with 35S and autoradiographs showed grains over arachnoidal cells, astroglia, and the glia limitans. These results indicated that astroglia synthesize sulfated mucopolysaccharides of the glia limitans. After the onset of myelination, however, the major portion of [35S]sulfate was incorporated into sulfatide. Autoradiographs showed a shift of radioactive grains from astroglia and arachnoidal cells to myelin, indicating that actively myelinating oligodendroglia incorporate [35S]sulfate into myelin sulfatide; there was a concomitant increase in the activity of cerebroside sulfotransferase. In addition, the increasing amounts of proteolipid protein and myelin basic protein corresponded with the morphological appearance of myelin. These results point to a strict correlation between the structural and biochemical changes occurring during myelination. This system provides a useful model for studies designed to evaluate the effects of various perturbations on the process of myelination.