Frog brain uridine diphosphate galactose–N-acetylgalactosaminyl-N-acetylneuraminylgalactosylglucosylceramide galactosyltransferase

1. The enzyme that catalyses the transfer of galactose from UDP-galactose to N-acetylgalactosaminyl-(1-->4)-N-acetylneuraminyl-(2-->3)-galactosyl-(1-->4)-glucosylceramide (G(M2)) was found mainly in the heavy- and light-microsomal fractions of the adult frog brain. 2. The subcellular distribution of the enzyme, UDP-galactose-G(M2) galactosyltransferase, parallels that of gangliosides in adult frog brain. 3. The enzymic activity was first detected at late gastrulation (Shumway stage 11(1/2)) and increased until the completion of the operculum (Shumway stage 25) and then decreased in the tadpoles. 4. In adult frog brain, the enzyme exhibited a pH optimum of 7.2-7.3 in both cacodylate and tris buffers. The enzyme required 10mm-Mn(2+) for maximal activity and the K(m) for Mn(2+) was determined as 2.2mm. The half-maximal velocity was obtained at a G(M2) concentration of 0.18mm. Inhibition of the enzymic reaction was found when the G(M2) concentration was greater than 1.38mm. 5. The enzymic activity was also inhibited by the products in the pathway of ganglioside synthesis, i.e. either by a mixture of gangliosides or by individual ganglioside components. The most active inhibitor was disialoganglioside. The degree of inhibition is a function of the individual ganglioside concentration. 6. A product-inhibition mechanism for the regulation of ganglioside biosynthesis is discussed.     

Drastically Abnormal Gluco- and Galactosylceramide Composition Does Not Affect Ganglioside Metabolism in the Brain of Mice Deficient in Galactosylceramide Synthase

Mice that are genetically deficient in UDP-galactose: ceramide galactosyltransferase are unable to synthesize galactosylceramide. Consequently, sulfatide, which can be synthesized only by sulfation of galactosylceramide, is also totally absent in affected mouse brain. -Hydroxy fatty acid-containing glucosylceramide partially replaces the missing galactosylceramide. A substantial proportion of sphingomyelin, which normally contains only non-hydroxy fatty acids, also contains -hydroxy fatty acids. These findings indicate that -hydroxy fatty acid-containing ceramide normally present only in galactosylceramide and sulfatide is diverted to other compounds because they cannot be synthesized into galactosylceramide due to the lack of the galactosyltransferase. We have examined brain gangliosides in order to determine if -hydroxy fatty acid-containing glucosylceramide present in an abnormally high concentration is also incorporated into gangliosides. The brain ganglioside composition, however, is entirely normal in both the total amount and molecular distribution in these mice. One feasible explanation is that UDP-galactose: glucosylceramide galactosyltransferase does not recognize -hydroxy fatty acid-containing glucosylceramide as acceptor. This analytical finding is consistent with the relative sparing of gray matter in the affected mice and provides an insight into sphingolipid metabolism in the mouse brain.     

Rat liver Golgi galactosyltransferases. Distinct enzymes for glycolipid and glycoprotein acceptor substrates.

Two enzymes that catalyse the transfer of galactose from UDP-galactose to GM2 ganglioside were partially purified from rat liver Golgi membranes. These preparations, designated enzyme I (basic) and enzyme II (acidic), utilized as acceptors GM2 ganglioside and asialo GM2 ganglioside as well as ovalbumin, desialodegalactofetuin, desialodegalacto-orosomucoid, desialo bovine submaxillary mucin and GM2 oligosaccharide. Enzyme II catalysed disaccharide synthesis in the presence of the monosaccharide acceptors N-acetylglucosamine and N-acetylgalactosamine. The affinity adsorbent alpha-lactalbumin-agarose, which did not retard GM2 ganglioside galactosyltransferase, was used to remove most or all of galactosyltransferase activity towards glycoprotein and monosaccharide acceptors from the extracted Golgi preparation. After treatment of the extracted Golgi preparation with alpha-lactalbumin-agarose, enzyme I and enzyme II GM2 ganglioside galactosyltransferase activities, prepared by using DEAE-Sepharose chromatography, were distinguishable from transferase activity towards GM2 oligosaccharide and glycoproteins by the criterion of thermolability. This residual galactosyltransferase activity towards glycoprotein substrates was also shown to be distinct from GM2 ganglioside galactosyltransferase in both enzyme preparations I and II by the absence of competition between the two acceptor substrates. The two types of transferase activities could be further distinguished by their response to the presence of the protein effector alpha-lactalbumin. GM2 ganglioside galactosyltransferase was stimulated in the presence of alpha-lactalbumin, whereas the transferase activity towards desialodegalactofetuin was inhibited in the presence of this protein. The results of purification studies, comparison of thermolability properties and competition analysis suggested the presence of a minimum of five galactosyltransferase species in the Golgi extract. Five peaks of galactosyltransferase activity were resolved by isoelectric focusing. Two of these peaks (pI 8.6 and 6.3) catalysed transfer of galactose to GM2 ganglioside, and three peaks (pI 8.1, 6.8 and 6.3) catalysed transfer to glycoprotein acceptors.     

ENZYMIC SYNTHESIS OF PSYCHOSINE SULPHATE

—An enzyme which catalyses the synthesis of psychosine sulphate by the transfer of [³⁵S]sulphate from 3′-phosphoadenosine 5′-phosphosulphate to galactosyl-sphingosine has been demonstrated in the brain of the young mouse. The enzyme activity appears to be bound to a microsomal fraction which is spun down with the synaptosomes. The product of the incubation mixture has been characterized as psychosine sulphate by a variety of TLC separations and other chemical procedures. Several parameters (detergent, cations, substrate and 3′-phosphoadenosine 5′-phosphosulphate concentrations, pH and incubation time), affecting the 3′-phosphoadenosine 5′-phosphosulphate-psychosine sulphotrans-ferase activity, have also been investigated. In the normal mouse brain there is a maximum enzyme activity at 17–19 days after birth, which is the period of most rapid myelin formation. In the brains of Jimpy mice, mutants with myelin deficiency, the activity is reduced and reaches a maximum around the 13th day. The lower activity correlates with the small amounts of sulphatides in Jimpy mouse brains. The results are discussed and related to present knowledge of galactolipid biosynthesis.     

A simple and specific assay of glycosyltransferase and glycosidase activities by an enzyme-linked immunosorbent assay method, and its application to assay of galactosyltransferase activity in sera from patients with cancer.

A simple, sensitive, and specific assay method for glycosyltransferase and glycosidase activities has been established by means of an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody, H-11 directed to lactoneotetraosylceramide (nLc4Cer). Enzyme activity was determined by assaying the amount of reaction product, nLc4Cer with the ELISA method. For the assay of galactosyltransferase activity, lactotriaosylceramide (Lc3Cer) immobilized on a 96-well microtiter plate was incubated with bovine milk galactosyltransferase in cacodylate buffer (pH 6.8) containing Triton CF-54, Mn2+, and UDP-galactose. Optimum incubation conditions for the enzyme were determined. Glycosidase activity was also assayed by the ELISA method by using Clostridium perfringens sialidase and neolacto-series gangliosides as substrates, and the substrate specificities towards the gangliosides were examined. By this method, 3-100 pmol of reaction product could be determined. The assay method has several advantages as follows: 1, the method is simple; 2, separation of the reaction product is not required; 3, quantification and identification of the reaction product were done simultaneously; 4, naturally occurring substrates are available (especially for glycosidase); 5, many samples can be assayed in one microplate; 6, sensitivity is very high. The present method was applied for the detection of galactosyltransferase in human sera. Significant elevations of the galactosyltransferase levels were observed in the sera from cancer patients. The formation of nLc4Cer was confirmed by employing the TLC-immunostaining method for bands of Lc3Cer after incubation of the bands with serum and cofactors on an HPTLC plate.     

UDP-galactose:glycoprotein galactosyltransferase activity in tissues of developing rat

UDP-galactose:glycoprotein galactosyltransferase activity has been measured in several tissues of the rat, ranging in age from 16 days embryo to 35 days postnatal. The enzyme activity was found to be high in fetal liver, lungs, and brain tissues but the concentration decreased with gestational age with no further changes after birth. The enzyme activity in the serum of newborns was higher than in pregnant and nonpregnant adult rats. There was no qualitative difference (optimum pH, cation requirements, affinity for the substrate UDP-galactose, or requirement for Triton X-100) between the enzyme from embryonic liver and that from adult rats. During the embryonic stage nearly half of the enzyme activity was localized in a plasma membrane-rich fraction and only a minor part in the microsomal fraction, while in the adult most of the activity was present in the microsomal fraction. Under certain conditions of assay the incorporation of galactose into glycoprotein in liver homogenates was greatly stimulated by CDP-choline or ATP. However, CDP-choline showed a considerably greater effect than ATP at 5 days after birth but this effect could be eliminated by solubilizing the homogenates in deoxycholate.     

Biosynthesis of galactosylsphingosine (Psychosine) in the twitcher mouse

In attempts to elucidate the origin of accumulated galactosylsphingosine in the twitcher mouse, a murine model of human globoid cell leukodystrophy (Krabbe's disease), UDP-galactose: sphingosine galactosyltransferase activity was assayed in tissues from normal and twitcher mice. Among several tissues from normal, 20 day postnatal mice, the highest galactosyltransferase activity was found in the brainstem and spinal cord, followed by cerebrum, kidney and liver, in that order. Chronologically, the enzyme activity in the central nervous tissue increased with age, reached a maximum at 25 postnatal days, and declined thereafter. In the kidney and liver, however, the activity remained much the same during development. In the twitcher mouse, developmental change in the enzyme activity was similar to that seen in control mouse, but the decrease in activity in the central nervous tissue after the 25 postnatal days was more rapid. The galactosyltransferase activity and the accumulation of galactosylsphingosine in the tissue of the twitcher mouse were closely related; where and when the enzyme activity was higher, the greater was the accumulation of galactosylsphingosine in the tissue of the twitcher mouse. These results strongly suggest that the accumulated galactosylsphingosine in the twitcher mouse is synthesized mainly by UDP-galactose: sphingosine galactosyltransferase.     

Purification of uridine diphosphate-galactose:glucosyl ceramide, beta 1-4 galactosyltransferase from human kidney.

A galactosyltransferase that transfers galactose from UDP-galactose to glucosylceramide was purified 440-fold to apparent homogeneity from normal human kidney "buffy coat" preparation employing detergent extraction, ultrafiltration, and Sepharose Q column chromatography. On reducing and nonreducing gels, the enzyme resolved into two bands with apparent molecular weights on the order of 60,000 and 58,000, respectively. The activity of the enzyme was also associated with these two bands following separation on polyacrylamide gels. Analytical isoelectric focusing revealed that the pI of this enzyme is approximately 4.55. Product characterization and substrate specificity studies employing chromatography, enzymatic digestion with various glycosidases, and use of a variety of glycosphingolipid substrates revealed that the major product synthesized by this enzyme was Cer1-1 beta Glc4-1Gal, and Cer1-1 beta Glc was the preferred substrate. Digestion of the 60- and 58-kDa proteins with Staphylococcus aureus (V-8) protease revealed at least six peptides having identical electrophoretic migration. This finding suggests that the two proteins may be related to each other. Western immunoblot assays revealed that the antibody against UDP-galactose:GlcCer, beta 1-4 galactosyltransferase (GalT-2) but not galactosyltransferase UDP-Gal:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyltransferase (EC 2.4.1.38) (B-GT) immunoprecipitated (recognized) the kidney GalT-2. In contrast, antibody against B-GT did not immunoprecipitate GalT-2. Thus our data indicate that GalT-2 and B-GT are two distinct enzymes. The availability of the enzyme GalT-2 and corresponding antibody will allow functional studies in the near future.     

UDP-glucose: ceramide glucosyltransferase of rat brain: an association with smooth microsomes and requirement of an intact membrane for enzyme activity

Subcellular distribution of rat brain UDP-glucose:ceramide glucosyltransferase, the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The activity of the enzyme was highest in the fraction rich in microsomes. Subfractionation of crude microsomal fractions resulted in a further enrichment of the enzyme activity in the fraction which contained smooth microsomes, thus suggesting that the enzyme is associated with microsomal membranes. The enzyme does not appear to be associated with synaptosomes or myelin. Treatment of the microsomal fraction with phospholipase A and C or detergents resulted in the loss of enzyme activity. Preincubation of the microsomal fraction at 37 °C also resulted in a loss of enzyme activity. These results suggest the requirement of specific membrane structure for the activity of the enzyme UDP-glucose:ceramide glucosyltransferase of rat brain. The amount of the enzyme activity lost during preincubation was dependent on the composition of the incubation medium and the age of the rats from which microsomal fractions were obtained.     

Initiation of chondroitin sulphate synthesis by beta-D-galactosides. Substrates for galactosyltransferase II.

beta-Galactosides were found to initiate chondroitin sulphate chain synthesis in chick-embryo cartilage in vitro and thereby relieve inhibition by cycloheximide of [3H]-acetate incorporation into chondroitin sulphate. beta-Galactosides with an apolar aglycan group such as phenyl O-beta-galactoside were active, whereas those with a charged or polar aglycan group such as pyridine 3-O-beta-galactoside or those with sulphur instead of oxygen in the glycosidic linkage (phenyl beta-thiogalactoside) were not. beta-Galactosides also serve as substrates for microsomal galactosyltransferase activity from chick-embryo cartilage. Phenyl O-beta-galactoside and pyridine 3-O-beta-galactoside were effective substrates for this enzyme, but phenyl S-beta-thiogalactoside and pyridine 2-S-beta-thiogalactoside were only slightly active. This galactosyltransferase was shown to be a separate enzyme from galactosyltransferase I, which catalyses transfer of galactose from UDP-galactose to beta-xylosides. It is proposed that the enzyme catalysing this reaction is galactosyltransferase II, responsible for transfer of the second galactose residue of the chondroitin sulphate linkage oligosaccharide. No transfer of glucuronic acid from UDP-glucuronic acid to beta-galactosides, catalysed by the microsomal preparation could be detected.     

Reversal of non-hydroxy:alpha-hydroxy galactosylceramide ratio and unstable myelin in transgenic mice overexpressing UDP-galactose:ceramide galactosyltransferase

The sphingolipids galactosylceramide and sulfatide are important for the formation and maintenance of myelin. Transgenic mice overexpressing the galactosylceramide synthesizing enzyme UDP-galactose:ceramide galactosyltransferase in oligodendrocytes display an up to four-fold increase in UDP-galactose:ceramide galactosyltransferase activity, which correlates with an increase in its products monogalactosyl diglyceride and non-hydroxy fatty acid-containing galactosylceramide. Surprisingly, however, we observed a concomitant decrease in alpha-hydroxylated galactosylceramide such that total galactosylceramide in transgenic mice was almost unaltered. These data suggest that UDP-galactose:ceramide galactosyltransferase activity does not limit total galactosylceramide level. Furthermore, the predominance of alpha-hydroxylated galactosylceramide appeared to be determined by the extent to which non-hydroxylated ceramide was galactosylated rather than by the higher affinity of UDP-galactose:ceramide galactosyltransferase for alpha-hydroxy fatty acid ceramide. The protein composition of myelin was unchanged with the exception of significant up-regulation of the myelin and lymphocyte protein. Transgenic mice were able to form myelin, which, however, was apparently unstable and uncompacted. These mice developed a progressive hindlimb paralysis and demyelination in the CNS, demonstrating that tight control of UDP-galactose:ceramide galactosyltransferase expression is essential for myelin maintenance.     

The localization of galactosyltransferases in polyacrylamide gels by a coupled enzyme assay

A coupled enzyme assay for GlcNAc¹: UDP-galactose galactosyltransferase has been developed that allows this enzyme to be assayed spectrophotometrically and in nondenaturing polyacrylamide gels. Utilizing three, intermediate enzymes, galactosyltransferase activity has been coupled to the production of NADH with a stoichiometry of 2 mol of NADH produced for each mol of galactose transferred to GlcNAc. The enzyme reactions coupled to the production of UDP by galactosyltransferase can be summarized as follows:

The activities of partly purified bovine milk galactosyltransferase and galactosyltransferase in dialyzed fetal calf serum have been determined spectrophotometrically by measuring NADH production at 340 nm. The reaction is dependent on N-acetylglucosamine, UDP-galactose, and Mn²⁺. For both enzyme sources, activities calculated from NADH production are similar to those determined from assays that use radioactive sugar nucleotide substrates. Both galactosyltransferase activities have been localized on 7.5% nondenaturing polyacrylamide gels after electrophoresis by incubating the gel with an agarose indicator gel containing the coupled enzyme system. Enzyme activity is marked by NADH fluorescence, which is dependent on the presence of N-acetylglucosamine in the indicator gel. The intensity of fluorescence increases with increasing galactosyltransferase activity applied to the gel.     

Topology of glucosylceramide synthesis in Golgi membranes from porcine submaxillary glands

The topology of ceramide glucosyltransferase and de novo synthesized glucosylceramide was studied in sealed and 'right-side-out' vesicles of porcine submaxillary glands derived from Golgi apparatus. Pronase treatment which did not cause any breakdown of the luminal glycoprotein galactosyltransferase activity, inhibited the ceramide glucosyltransferase to more than 50% at a ratio proteinase to Golgi protein 1:100. Trypsin at the same concentration, while producing no inactivation of luminal galactosyltransferase, caused a complete loss of ceramide glucosyltransferase activity. The membrane-impermeable compound, DIDS, which did not cause any inhibition of the galactosyltransferase, inhibited the ceramide glucosyltransferase (70% reduction at 80 microM DIDS). Thus, the enzyme ceramide glucosyltransferase is accessible from the cytoplasmic side of the Golgi vesicles. The orientation of the newly synthesized glucosylceramide is studied by the ability of the enzyme glucosylceramidase to hydrolyse this compound both on intact and on disrupted vesicles. The same percentage (respectively, 36 and 30%) of hydrolysis was obtained during an incubation of 3 h, showing that glucosylceramide is not at all protected from external hydrolysis. Pronase-treated vesicles revealed an increase in glucosylceramidase hydrolysis (up to 45%), which indicates that glucosylceramide that glucosylceramide may be cryptic. All these results indicate that the ceramide glucosyltransferase, as well as related glucosylceramide, are cytoplasmically oriented in Golgi vesicles from porcine submaxillary glands.     

Purification and properties of two enzymes catalyzing galactose transfer to GM2 ganglioside from rat liver Golgi

An enzyme activity which catalyzed the transfer of galactose from UDP-galactose to GM2 ganglioside was demonstrated in rat liver homogenate and enriched 38-fold in specific activity by preparation of Golgi membranes. This activity could be solubilized from Golgi membranes by sonication and extraction with 1% Triton X-100. The solubilized activity catalyzed the formation of GM1 ganglioside and was completely dependent upon the addition of acceptor. The rate of galactose incorporation was constant for up to 5 h at 30 degrees C. This enzyme activity was further purified by gel filtration on Sepharose CL-6B and ion exchange chromatography on DEAE-Sepharose. The elution position on gel filtration corresponded to a molecular weight for the enzyme of 38,000 which was in good agreement with that obtained by sedimentation velocity studies. Ion exchange chromatography resolved GM2 ganglioside galactosyltransferase into two species. The more basic enzyme (I) comprising 28% of the recovered activity was not retarded by the column, whereas enzyme II was eluted from the resin following the application of a salt gradient. Net purification was 120- to 140-fold for each enzyme with a total recovery of 42%. Unlike the activity in the Golgi extract, the purified enzymes I and II were labile to freezing and could be stored at -20 degrees C only in the presence of 50% glycerol. Both enzymes I and II had similar molecular weights and Michaelis constants and both had a strict requirement for Mn2+. Properties which distinguish the two enzymes included pH optima (enzyme I 7.0, enzyme II 6.0) and surfactant requirements. Neither enzyme was active following removal of Triton X-100 from the preparation. Among a series of glycolipids tested for ability to serve as substrates for galactose transfer only GM2 and asialo-GM2 ganglioside served as acceptors.     

Mucin biosynthesis. Characterization of UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase from human tracheal epithelium

We have characterized the UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase in human tracheal epithelium using asialo ovine submaxillary mucin as the acceptor. Maximal enzyme activity was obtained at pH 6.0-7.5 and at 20-25 mM MnCl2 and at 2% Triton X-100. Cd2+ could substitute for Mn2+ as the divalent ion cofactor. Spermine, spermidine, putrecine, cadaverine, and poly-L-lysine stimulated the enzyme activity at low (2.5 mM) MnCl2 concentration. The apparent Michaelis constants for N-acetylgalactosamine, asialo ovine submaxillary mucin, and UDP-galactose were 15.5, 1.14, and 1.36 mM, respectively. The enzyme activity was not affected by alpha-lactalbumin. The alpha-N-acetygalactosaminide beta 3 galactosyltransferase was shown to be different from the N-acetylglucosamine galactosyltransferase by acceptor competition studies. The product of galactosyltransferase was identified as Gal beta 1 leads to 3GalNAc alpha Ser (Thr) by (a) isolation of [14C]Gal-GalNAc-H2 after alkaline borohydride treatment of the 14C-labeled product, (b) establishment of the beta-configuration of the newly synthesized glycosidic bond by its complete cleavage by bovine testicular beta-galactosidase, and (c) assignment of the 1 leads to 3 linkage by identification of threosaminitol obtained from the oxidation of the disaccharide with periodic acid followed by reduction with sodium borohydride, hydrolysis in 4 N HCl, and analysis on an amino acid analyzer. The 1 leads to 3 linkage was confirmed by its resistance to jack bean beta-galactosidase and by the presence of a m/e 307 ion fragment and the absence of a m/e 276 ion by gas-liquid chromatography-mass spectrometry analysis. When acid and beta-galactosidase-treated human tracheobronchial mucin was used as the acceptor, 3.3% of the product was found as [14C]Gal-GalNAc-H2. The remainder of the [14C]Gal was found in longer oligosaccharides formed by a different beta-galactosyltransferase. This galactosyltransferase is slightly inhibited by alpha-lactalbumin and stimulated by spermine.     

Studies on the Golgi apparatus. Cumulative inhibition of protein and glycoprotein secretion by d-galactosamine

1. The administration of d-galactosamine leads to inhibition of protein and glycoprotein secretion by rat liver. To test the secretory function, the secretion times for galactose-and fucose-containing glycoproteins were determined; they were lengthened from 6 to 9min and from 8 to 13min respectively. 2. The Golgi apparatus was enriched 100-120-fold relative to the homogenate. A new linked-assay system for the marker enzyme, UDP-galactose-N-acetyl-d-glucosamine galactosyltransferase, is presented. The activity of the enzyme was measured spectrophotometrically by following the formation of UDP coupled to nicotinamide nucleotide reduction. The Michaelis constants were calculated to be 0.11mm for UDP-galactose with N-acetyl-d-glucosamine as exogenous acceptor and 19mm for N-acetyl-d-glucosamine itself. 3. The physiological substrate of the galactosyltransferase, UDP-galactose, can be replaced by UDP-galactosamine, which accumulates after d-galactosamine administration. Under conditions in vitro the rate of d-galactosamine transfer to an endogenous acceptor protein of the Golgi fraction reaches 9% of that with d-galactose; this finding is noteworthy, because normally a non-acetylated amino sugar does not occur in glycoproteins. 4. The albumin content of the Golgi-rich fraction was diminished to 55% of the reference value 6h after the injection of 375mg of d-galactosamine hydrochloride/kg body wt. The transfer of d-[1-(14)C]galactose to an endogenous acceptor protein fell to 60% compared with Golgi-rich fractions from untreated animals. Analysis of the Golgi-rich fraction by polyacrylamide-gel electrophoresis showed a decrease or loss of several protein bands. 5. Protein synthesis can be restored by up to 80% if the UTP pool, decreased after d-galactosamine administration, is filled up by several injections of uridine. 6. From the results presented it can be concluded that the disturbed secretion of proteins and glycoproteins was due to a cumulative effect of galactosamine by: (a) inhibition of protein synthesis leading to a diminution of the endogenous acceptor pool of the galactosyltransferase; (b) inhibition of the galactosyltransferase activity by galactosamine metabolites and (c) replacement of UDP-galactose by UDP-galactosamine.     

Characterization of a soluble glycolipid galactosyltransferase which occurs in bovine milk.

Bovine milk was found to contain, in soluble form, an enzyme which transfers galactose from UDPgalactose to glucosylceramide. This enzyme was partially purified by the same procedure used to isolate the galactosyltransferase of lactose synthetase. The partially purified enzyme required detergents for activity, had a pH optimum of 7.2--7.3 and required Mn2+. The apparent Km calculated for glucosylceramide was 1.33 . 10(-4) M. With glucosylceramide as acceptor the product of the reaction was identified as lactosylceramide by autoradiography on thin-layer chromatograms. Lactosylceramide was also an effective acceptor for the transferase reaction but neutral glycosphingolipids or gangliosides with terminal galactose of N-acetylgalactosamine residues were ineffective or poorly effective as acceptors. Addition of alpha-lactalbumin inhibited the transferase reaction.     

Neuraminidase Activities in Oligodendroglial Cells of the Rat Brain

Neuraminidase activities in oligodendroglial cells were characterized using rats of different ages. Rat oligodendroglial cells had intrinsic neuraminidase activities directed toward GM3 and N-acetylneuramin(2-3)lactitol (NL). Developmental profiles of the neuraminidase activities toward the two substrates in oligodendroglial cells were different from each other. The neuraminidase activity toward GM3 increased rapidly with the onset of active myelination and, after 26 days of development, reached the adult level which was about 18 times higher than that in myelin. At the adult age, oligodendroglial cells had the highest neuraminidase activity toward GM3 among the individual brain cell types examined. The activity of NL-neuraminidase showed a less remarkable developmental profile, with a peak value at 26 days. The UDP-galactose:ceramide galactosyltransferase activity in oligodendroglial cells increased during the period of active myelination and, afterward, returned to the basal level. The enrichment and unique developmental profile in oligodendroglial cells of the neuraminidase activity toward GM3 suggest that this enzyme may play an important role in the formation and maintenance of the myelin sheath.     

DEVELOPMENTAL PROFILES OF GANGLIOSIDES IN HUMAN AND RAT BRAIN

Abstract— The developmental profiles of individual gangliosides of human brain were compared with those of rat brain. Interest was focused mainly on the pre- and early postnatal development. Human frontal lobe cortex covering the period from 10 foetal weeks to adult age and the cerebrum of rat from birth to 21 days were analysed. Lipid-NANA and lipid-P were followed; in the rat, also protein and brain weight. A limited number of samples of human cerebral white matter and cerebellar cortex were also studied. The following major results were obtained:

• 1 The ganglioside concentration increased approximately three-fold within a short period: in rat cerebrum, from birth to the 17th day; in human cerebral cortex, from the 15th foetal week to the age of about 6 months. The largest increase in the rat brain occurred by the 11th to the 13th day; in human brain by term. The relative increase of gangliosides during this period was more rapid than that of phospholipids.
• 2 A hitherto unknown distinct early period of ganglioside and phospholipid formation in rat occurred by the second to fourth day.
• 3 The changes in brain ganglioside pattern, characteristic of the developmental stages of the rat, were found to be equally pronounced in the human brain.
• 4 Regional developmental differences in the ganglioside pattern were demonstrated in human brain. A characteristic white matter pattern, rich in monosialogangliosides, had developed by the age of 1 year. The increase in ganglioside concentration and the formation of the definitive ganglioside pattern of cerebellar cortex occurred later than in cerebral cortex. This cerebellar pattern was characterized by a very large trisialoganglioside fraction.
• 5 The two periods of rapid ganglioside metabolism in rat brain preceded the two periods of rapid protein biosynthesis.

     

Photoaffinity labeling of lactose synthase with a UDP-galactose analogue

A photoaffinity analogue of UDP-galactose, 4-azido-2-nitrophenyluridylyl pyrophosphate (ANUP), has been synthesized for the investigation of the binding topography of alpha-lactalbumin on galactosyltransferase. Results obtained from steady state kinetics show that ANUP is an effective competitive inhibitor against UDP-galactose in the reactions of lactose and N-acetyllactosamine syntheses. The specific binding of ANUP to the UDP-galactose-binding site is further demonstrated by its ability to facilitate the formation of the lactose synthase complex on solid supports, either alone or in the presence of glucose or N-acetyl-glucosamine. ANUP inactivates galactosyltransferase on irradiation. One mole of ANUP was incorporated per mol of enzyme inactivated. This process is Mn2+-dependent and can be prevented by UDP-galactose. Glucose and N-acetylglucosamine render only partial protection. Photoaffinity labeling of lactose synthase either free in solution or immobilized on Sepharose does not result in any reduction of the alpha-lactalbumin modifier activity. In addition, no incorporation of radioactivity into alpha-lactalbumin was observed when radioactive ANUP was used, whereas galactosyltransferase was labeled. These data indicate that alpha-lactalbumin does not bind to galactosyltransferase in the region of the ANUP site, suggesting that the location of protein-protein interaction between the two subunits of lactose synthase may be removed from the UDP-galactose-binding domain.