Influence of thyroid on formation of myelin lipids

—The formation of lipids found primarily in the myelin sheath was investigated in rats made hypothyroid, at birth. A marked reduction in cerebroside, sulphatide, and cholesterol of brains was found in 18-day-old hypothyroid animals. These lipids were also reduced, although to a lesser degree, in 30-day-old ex-hypothyroid animals allowed to return to the euthyroid state at age 18 days. The onset of formation of sulphatide in vivo was delayed in the hypothyroid animals. Sulphatide formation reached a peak at a later time and was greatly reduced in comparison to control animals. The activity in vitro of galactolipid sulphotransferase, which forms sulphatide from cerebroside and PAPS, was reduced only when exogenous PAPS was not added to the assay medium. This finding suggests a defect in the formation of PAPS from ATP and sulphate in the hypothyroid brain. In addition, T₃ (tri-iodothyronine) had a stimulatory effect in vivo of formation brain sulphatide when administered to rats during the first 5 days of life.     

MYELIN SUBFRACTIONS IN DEVELOPING RAT BRAIN: CHARACTERIZATION AND SULPHATIDE METABOLISM

Centrifugation of isolated myelin on discontinuous sucrose gradients resulted in a separation into three bands and a pellet. The three bands were morphologically identical to myelin, whereas the pellet consisted primarily of vesicular membranes. These four fractions differed from one another in their lipid-to-protein ratios and in molar ratios of cholesterol:phospholipid:galactolipid. All of the fractions contained proteins typical of myelin, although the proportions of the proteins varied, with the pellet being the lowest in basic protein and proteolipid protein. High activity of 2′,3′-cyclic nucleotidase and low activity of cerebroside sulphotransferase further distinguished these fractions from the microsomal fraction. Distribution of radioactive sulphatide in the subfractions at 15 min after intracranial injection of radioactive sulphate indicated that newly-labelled sulphatide first appeared in the lipid-poor fractions, followed by the lipid-rich fractions; results of pulse-chase experiments also suggested this relationship. Several days or weeks after the injection of radioactive sulphate, most of the radioactive sulphatide was in the lipid-rich fractions.     

Regulation of arylsulphatase A and sulphogalactolipid turnover by cortisol in myelinogenic cultures of cells dissociated from embryonic mouse brain

Myelinogenic cultures of cells dissociated from embryonic mouse brain were used to study the regulation of myelination-associated molecules by cortisol. Cortisol in physiological concentrations (0.03 microM) caused an increased accumulation of myelination-associated sulphogalactolipids. It also stimulated the myelin- and oligodendroglia-specific cyclic nucleotide phosphohydrolase. The increase in sulphogalactolipid content was caused by a cortisol-concentration-dependent inhibition in arylsulphatase A activity and not by an increase in either cerebroside sulphotransferase activity or an increase in availability of adenosine 3'-phosphate 5'-phosphosulphate. Of several steroid hormones tested only the glucocorticoid types brought about these changes. The relationship between net sulphogalactolipid accumulation and arylsulphatase A inhibition induced by cortisol was confirmed by sulphogalactolipid turnover studies. Depending on whether a single-phase or a two-phase decay calculation is used, the turnover of sulphogalactolipid with cortisol present was decreased at 22 days in culture by either 62% or 65% respectively of that without cortisol. This decrease in turnover can be attributed completely to the decrease of arylsulphatase activity by cortisol to 63% of the value for normal cells grown under the same conditions.     

Sulpholipid metabolism in developing chicken retina.

Glycolipid analysis of chicken retina and brain indicated the presence of cerebroside, cerebroside 3-sulphate and sulphogalactosylglycerolipid In retina, the ratio of cerebroside to cerebroside 3-sulphate was approximately half compared to brain. During chicken retina ontogenesis the ratio of cerebroside 3-sulphate to sulphogalactosylglycerolipid increased rapidly and in the adult animal, the amount of cerebroside 3-sulphate was 14 times higher than that of sulphogalactosylglycerolipid. The activity of PAPS: cerebroside sulphotransferase and arylsulphatase A in developing chicken retina indicated that the general ontogenic profiles of retinal PAPS: cerebroside sulphotransferase and arylsulphatase A were similar to those obtained for the brain. Both the enzymes showed the highest activity just before hatching. The significance of occurrence of sulpholipids in retina is discussed.     

Kinetics of entry of galactolipids and phospholipids into myelin.

Abstract— Brain slices from 17 day rats were incubated with [³H]galactose and [³⁵S]sulphate to label cerebroside and sulphatide. Myelin was isolated by centrifugation on a sucrose density gradient. Following lipid extraction and alkaline methanolysis, cerebroside and sulphatide were isolated by tic, and radioactivity was measured. Appearance of [³H]cerebroside and [³H]sulphatide in myelin showed a lag of less than 15min, while appearance of [³⁵S]sulphatide in myelin showed a longer lag of about 30min. In chase experiments, the rate of appearance of [³H]cerebroside and [^3SS]sulphatide in the non-myelin fraction and of [³H]cerebroside in the myelin fraction slowed markedly after the chase. In contrast, [³⁵S]sulphatide continued to increase in myelin at a normal rate for 30min after the chase, then stopped, while ³H from galactose continued to accumulate in myelin sulphatides for 60 min. These data are interpreted to demonstrate an interval of 30 min between synthesis of cerebroside and its sulphation in the non-myelin fraction, and another delay of 30 min between sulphation and appearance in myelin. The distribution of newly synthesized cerebroside and sulphatide between myelin and non-myelin fractions also supported the concept that a complex metabolic pool of cerebroside in the non-myelin fraction is precursor to sulphatide of myelin. For comparison, entry of phosphatidyl choline and phosphatidyl ethanolamine into myelin was followed with [2-³H]glycerol as precursor. Like cerebroside, both phospholipids showed little delay in their initial appearance in myelin, and prompt cessation of their addition after a chase with unlabeled precursor. These results are consonant with either rapid entry of these three lipids into myelin after synthesis at an extra-myelin site, or synthesis of the lipids within myelin itself.     

The biosynthesis of sulphogalactosyldiacylglycerol of rat brain in vitro

Triton X-100 extracts of rat brain microsomal fraction catalyse the formation of sulphogalactosyldiacylglycerol from galactosyldiacylglycerol and adenosine 3'-phosphate 5'-sulphatophosphate. Of the various subcellular fractions of brain assayed, the microsomal fraction contained most (79%) of the adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase activity. The enzyme activity was stimulated by Triton X-100 and showed linearity with increasing time, concentrations of enzyme and added substrates. ATP and KF prolonged the linearity of the activity with time, but ATP had an overall inhibitory effect on the sulphotransferase. Both ATP and KF inhibit the degradation of adenosine 3'-phosphate 5'-sulphatophosphate, which probably causes the increased linearity of the sulphotransferase reaction with time. The enzyme preparation did not catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to either cholesterol or galabiosyldiacylglycerol (galactosylgalactosyldiacylglycerol). Significant differences between the formation of sulphogalactosyldiacylglycerol and cerebroside sulphate catalysed by the same enzyme preparation were noted. ATP and Mg(2+) strongly inhibit the formation of sulphogalactosyldiacylglycerol but equally strongly stimulate the synthesis of cerebroside sulphate. The apparent K(m) for galactosyldiacylglycerol is 200mum, and that for cerebroside is 45mum. Galactosyldiacylglycerol and cerebroside are mutually inhibitory toward the synthesis of sulphated derivatives of each. These data do not necessarily lead to the conclusion that two sulphotransferases are present, but they do indicate a possible means of controlling the synthesis of these two sulpholipids.     

Hymenolepis diminuta: lack of sulphate activation and sulphotransferase activity

There is no evidence that Hymenolepis diminuta can carry out sulphoconjugation reactions. Neither whole worms nor worm extracts were able to sulphate 4-methylumbelliferone. No sulphotransferase activity could be demonstrated in H. diminuta using a variety of substrates, nor was H. diminuta capable of synthesising the sulphate donor 3'-phosphoadenosine-5'-phosphosulphate from ATP and inorganic sulphate. Possible alternative sources of active sulphate in this parasite are discussed.     

Development and characteristics of an oestrogen sulphotransferase in placenta and uterus of the pregnant mouse. Comparison between mouse and rat.

The mouse placenta possesses a soluble oestrogen sulphotransferase activity which increases markedly from at least 12 days of gestation until term. At about 16 days of gestation, a similar activity is found in the uterus. This activity also increases until term and disappears rapidly post partum. The uterine enzyme activity appears to require the presence of the foetal unit for its onset, since unoccupied horns, whether their endometrial stromal cells are differentiated to decidual cells or not, are essentially devoid of it. Uterine cytosols from non-pregnant mice are also inactive in this respect. In late gestation, the uterine sulphotransferase is confined to the decidua basalis, the areas to which the placentas are attached. The sulphotransferase(s) of placenta and uterus has an absolute requirement for 3'-phosphoadenosine 5'-phosphosulphate, and possesses little activity in the absence of exogenous thiol groups. Stimulation is also seen in the presence of Mn2+, Mg2+ or Ca2+. Oestrone and oestradiol, and to a lesser degree oestriol, are substrates for the enzyme(s), whereas testosterone, cortisol and dehydroepiandrosterone are not. Oestrone and oestradiol at higher concentrations (1.0-1.5 microM) completely inhibit the enzyme(s). These enzymes could play a role in altering tissue concentrations of active oestrogens during gestation in the mouse. Oestrogen sulphotransferase activity is low or absent in reproductive tissues of the pregnant rat.     

Sulphatide synthesis in isolated oligodendroglial and neuronal cells

—Cerebroside sulphotransferase activity in oligodendroglia from calf brain is 8-fold greater per cell than in calf neurons isolated at the same time under similar conditions. However, neuronal cell fractions from calf or rat brain have significant sulphotransferase activity, and in neurons isolated from rat brain at various ages, the capacity to synthesize sulphatide increases during myelination. The neuronal and oligodendroglial enzymes have similar substrate specificities and pH optima. Less of the enzyme could be extracted with Triton X-100 from the isolated cells than from microsomes prepared from whole brain.     

3''-phosphoadenosine-5''-phosphosulphate synthesis and involvement in sulphotransferase reactions in the insect, Spodoptera littoralis.

1. Synthesis of 3'-phosphoadenosine-5'-phosphosulphate from ATP and 35SO4(-2) was demonstrated by homogenates of gut. Malpighian tubules and fat body of Spodoptera littoralis. 2. The enzyme system was most active in the gut tissue, and was primarily located in the cytosol fraction of the cell. Gut cytosol preparations were used as a source of the 3'-phosphoadenosine-5'-phosphosulphate generating system for more detailed studies. 3. Maximum synthesis required an incubation mixture containing Tris/HCl buffer (pH 7.5), ATP (20 mM), MgCl2 (13.0 mM) and K2SO4 (3 mM). 4. The specific activity of 3'-phosphoadenosine-5'-phosphosulphate synthesizing activity in gut cytosol increased during development of the sixth instar larva, reaching a peak at day 4. A sudden fall in specific activity was observed in the prepupal stage. 5. 3'-Phosphoadenosine-5'-phosphosulphate formation is the rate limiting process in the overall sulphation of p-nitrophenol in the gut cytosol preparations from S. littoralis. 6. It is concluded that the properties of the sulphate-activating system in this insect are similar to those reported for vertebrates.     

SUBCELLULAR AND SUBMICROSOMAL DISTRIBUTION OF GLYCOLIPID-SYNTHESIZING TRANSFERASES IN YOUNG RAT BRAIN

The subcellular and submicrosomal distributions of four glycolipid-synthesizing transferases were studied in young rat brains. (1) Two galactosyl transferases involved in the synthesis of cerebrosides, the cerebroside sulphotransferase which catalyses the synthesis of sulphatides, and the glucosyl transferase which plays an important role in the ganglioside biosynthesis were localized essentially in the microsomal fraction. Only low activities were detected in the crude mitochondrial and synaptosome-enriched fractions. (2) A comparison of the activities of these enzymes in the crude myelin and two myelin subfractions showed that the galactosyl transferases and the cerebroside sulphotransferase had similar activities in the crude myelin and myelin-like fractions. A considerable galactosyl transferase activity was found in purified myelin. In this respect these two enzymes were different from cerebroside sulphotransferase, whose activity was much lower in purified myelin. On the other hand, glucosyl transferase had a relatively low specific activity in all three myelin fractions. Analysis of different markers showed that the activities were considerably higher than those expected from the maximum microsomal contamination calculated. (3) Subfractionation of the microsomes demonstrated that the galactosyl transferases were more concentrated in the lower parts of the gradient, containing vesicles with attached ribosomes. Cerebroside sulphotransferase and glucosyl transferase were found predominantly in the upper and intermediate parts of the gradient, which were composed essentially of smooth-surfaced vesicles and membrane fragments. Chemical analysis of submicrosomal fractions confirmed the morphological observations.     

The effects of inhibitors of protein synthesis on incorporation of lipids into myelin

Abstract— Following intracranial injections of puromycin, the incorporation of [³H]leucine into brain protein was inhibited by 80 per cent. Conversely, incorporation of [³⁵S]sulphate into sulphatide or [2-³H]glycerol into phosphatidyl choline was not inhibited. Under these conditions, appearance of labelled protein in myelin was inhibited by 90 per cent, while the appearance of newly labelled sulphatide and phosphatidyl choline in myelin membrane was not greatly affected. Experiments with cycloheximide gave similar results with phosphatidyl choline, but incorporation of [³⁵S]sulphate into total sulphatide was decreased by about 30 per cent in animals given cycloheximide. Neither puromycin nor cycloheximide had any inhibitory effect on galactocerebroside sulphotransferase.     

Sodium ion diffusion through liposome membranes containing cerebroside

Na⁺ efflux from liposomes (small unilamellar vesicles, SUV) of various compositions was studied, using ²²Na⁺ and ³H-labelled stachyose in simultaneous dual isotope measurements, stachyose being used as a measure of liposome disintegration. Dialysis was utilised to separate liposomes from extra-liposomal activity.Liposomes were made from egg lecithin and sphingomyelin and from mixtures of egg lecithin, sphingomyelin, cerebroside, sulphatide and cholesterol. All mixtures produced more leaky and less stable SUVs than pure lecithin and pure sphingomyelin. The incorporation of cerebroside is significantly smaller than that of the phospholipids including sphingomyelin. It was found that membranes containing cerebroside had a significantly higher Na⁺ permeability than membranes without cerebroside.     

SULPHATE METABOLISM IN ACUTE EAE1 RATS USING ISOLATED BRAIN PERFUSION TECHNIQUE

—Metabolism of glycolipids and glycosaminoglycans were studied in rats in the acute stage of experimental allergic encephalomyelitis (EAE) using isolated brain perfusion technique. It was observed that there was a significant decrease in the concentration of cerebroside, sulphatide and GAG (hyaluronic acid and low sulphated GAG) when compared to normal and pairfed control rats. The radioactive sulphate incorporation into the cerebroside sulphate and sulphated GAG was significantly higher in the case of rats in the acute stage of EAE than the normal and pairfed control rats.     

THE SULPHATION OF VANILYLMANDELIC ACID AND HOMOVANILLIC ACID BY LIVER, SMALL INTESTINE AND BRAIN

Abstract— The sulpho-conjugates of vanilylmandelic acid (VMA) and homovanillic acid (HVA) were biosynthesized in vitro from ATP and sodium sulphate and from 3'-phosphoadenosine-5'-phosphosulphate (PAPS) obtained commercially or generated in vitro . Hydrolysis with sulphatase indicated the sulphate nature of these conjugates. In decreasing order of activity, the liver, small intestine and brain of various animals exhibited this sulpho-conjugatory ability. The K _m, values of VMA and HVA for the sulphotransferase of mouse liver were, respectively, 1740 and 93 μM. Competition studies suggested that the same enzyme may be involved in the sulphation of these two acids.     

A kinetic study of the effects of galactocerebroside 3-sulphate on human spleen glucocerebrosidase. Evidence for two activator-binding sites.

Extraction of control human spleen glucocerebrosidase with sodium cholate and butan-l-ol reversibly inactivates the enzyme in terms of its ability to hydrolyse the water-soluble substrate 4-methylumbelliferyl beta-D-glucopyranoside (MUGlc). The acidic brain lipid galactocerebroside 3-sulphate (sulphatide) reconstitutes beta-glucosidase activity in a strongly concentration-dependent manner. In this study we show that sulphatide exhibits three critical micellar concentrations (CMCs): CMC1, 3.72 microM; CMC2, 22.6 microM; CMC3, 60.7 microM. We designate the aggregates formed at these CMCs as primary, secondary and tertiary micelles respectively. From the results of kinetic studies performed at various sulphatide concentrations (0.012-248 microM), we found that sulphatide monomers (less than 3 microM) decreased the Km (for MUGlc) of control glucocerebrosidase from 11 to 4.6 mM, and lowered the Vmax. 2-fold. However, secondary and tertiary micelles were required for expression of high control glucocerebrosidase activities. Glucocerebrosidase prepared from the spleen of a patient with non-neuronopathic type 1 Gaucher's disease exhibited a very low Km (2.8 mM) even in the absence of exogenous lipid, and sulphatide monomers had no effect on the mutant enzyme's Km or Vmax. However, secondary or tertiary micelles markedly increased the Vmax. of the type 1 glucocerebrosidase to 60% of the corresponding control enzyme value. In contrast, for the glucocerebrosidase of the neuronopathic type 2 case, although sulphatide decreased the Km from 9.2 to 1.7 mM, the Vmax. never reached more than 5% that of the control enzyme, even at high concentrations of sulphatide. In addition, we found that secondary and tertiary sulphatide micelles enhanced the rate of inactivation of all three glucocerebrosidase preparations by chymotrypsin. Collectively, these results indicate the presence of two sulphatide-binding sites on glucocerebrosidase: one that enhances substrate binding, and another that enhances catalysis.     

The activator of cerebroside-sulphatase. A model of the activation.

The activator of cereboroside-sulphatase (cerebroside-3-sulphate-3-sulphohydrolase, EC 3.1.6.8) is necessary for the enzymic hydrolysis of sulphatides (cerebroside sulphates) at ionic concentrations in the physiological range. The pH optimum of the reaction is 4.5--4.8. Under similar incubation conditions, a complex is formed between activator and sulphatides which is partially inhibited, due to competitive binding in the presence of cerebrosides of phosphatidylserine. Inhibition depends upon the concentration of the lipids and is of the same order of magnitude as the inhibition (by these lipids) of enzymic sulphatide hydrolysis in the presence of activator. Complex formation between activator and sulphatides is reversible since the complex dissociates partially when certain concentrations of phosphatidylserine are added. Moreover, the rate of sulphatide hydrolysis increases with the concentration of the activator.sulphatide complex in the reaction mixture. This indicates that the activator.sulphatide complex is the substrate for the enzyme and a model for this activation is presented.     

Enzymic synthesis of steroid sulphates. XVII. On the structure of bovine estrogen sulphotransferase

Estrogen sulphotransferase plays a major role in controlling intracellular levels of 17 beta-estradiol in human mammary cancer cells and human endometrium. Bovine estrogen sulphotransferase c-DNA has recently been cloned; the encoded protein having a maximum Mr of 35,000 (Nash, A.R. et al. (1988) Aust. J. Biol. Sci. 41, 507-516). Enzyme of Mr 35,000 by SDS-PAGE has now been isolated and cyanogen bromide-cleaved peptides sequenced. The latter were identified in the c-DNA-predicted amino acid sequence which confirms that the active enzyme (Mr approximately 70,000) exists as a dimer of identical subunits. Sequence data on similar peptides isolated from an enzyme preparation containing a protein of Mr 74,000 as the major species on SDS-PAGE, which was previously thought to represent the enzyme, suggested that this protein was transferrin. This was confirmed by PAGE, SDS-PAGE, susceptibility to neuraminidase and reaction with bovine transferrin antibody. Isoelectric focusing experiments show that active enzyme exists in two or three polymorphic forms (pI values 5.3, 5.7 and possibly 5.9) having similar physicochemical properties of polymorphic forms of transferrin so that they overlap on ion-exchange chromatography and PAGE. The enzyme shows some homology to the amino acid sequence close to the Fe-binding site in lactoferrin and the question is raised as to the possible presence of a tightly bound metal in estrogen sulphotransferase involved in the binding of adenosine 3'-phosphate 5'-phosphosulphate.     

Human liver steroid sulphotransferase sulphates bile acids.

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.     

SPHINGOLIPIDS AND PHOSPHOLIPIDS IN MICROSOMES AND MYELIN FROM NORMAL AND PATHOLOGICAL BRAINS

—Myelin and microsomes were separated from human cerebral white matter and cortex respectively using the technique of 15% caesium chloride and their sphingolipid and phospholipid contents estimated. Normal brains as well as cerebral material from cases of metachromatic leucodystrophy, Krabbe's disease and Tay-Sachs’disease were studied. Gangliosides were not present in normal myelin but were found in microsomes and in myelin from the pathological material. The ratio of cerebroside to sulphatide in myelin was 4 to 1 in normal, 1 to 20 in metachromatic and 7 to 1 in Krabbe's disease. The results in the human material are briefly discussed.