Glycosyl transferases of microsomal fractions from brain: synthesis of glucosyl ceramide and galactosyl ceramide during development and the distribution of glucose and galactose transferase in white and grey matter

Abstract— The substrate specificity for glycosyl transferases of microsomal fractions from brain was investigated. Ceramides were found to be better acceptors than sphingosine for both glucose and galactose when a Celite dispersion of lipid substrate was used. For galactose transfer only hydroxy fatty acid ceramide served as an acceptor. For transfer of glucose both non-hydroxy and hydroxy fatty acid ceramide served as acceptors, but the hydroxy fatty acid ceramide was the more effective of the two. Glucose transferase activity was found to be highest between birth and 15 days of age and declined slowly with later development. Galactose transferase activity did not appear until the 10th day of postnatal age and reached a peak at about the 30th day. Galactose transferase activity was present principally in white matter microsomes, but glucose transferase activity was present in the microsomal fractions of both white and grey matter. The developmental alteration in the activities of galactosyl and glucosyl transferases and their distribution in white and grey matter correlated with development and distribution of cerebroside and ganglioside, respectively.     

Glycosyl transferases of baby-hamster-kidney (BHK) cells and ricin-resistant mutants. N-glycan biosynthesis

Five cell lines of ricin-resistant BHK cells have been assayed for gross carbohydrate analysis of cellular glycoproteins, for the activities of several glycosidases and of specific glycosyl transferases active in assembly of N-glycans of glycoproteins. The latter enzymes include sialyl transferase using asialofetuin as glycosyl acceptor, fucosyl transferases using asialofetuin and asialoagalactofetuin acceptors, galactosyl transferases using ovalbumin, ovomucoid and N-acetylglucosamine as acceptors and N-acetylglucosaminyl transferases using ovalbumin and glycopeptides as acceptors. Cell line RicR14, binding less ricin than normal BHK cells, contains reduced amounts of sialic acid, galactose and N-acetylglucosamine in cellular glycoproteins and lacks almost completely N-acetylglucosamine transferase I, an essential enzyme in assembly of ricin-binding carbohydrate sequences of N-glycans. These cells also contain reduced levels of N-acetylglucosamine transferase II active on a product of N-acetylglucosamine transferase I action. Sialyl transferase activity is severely depressed while fucose-(alpha 1 leads to 6)-N-acetylglucosamine fucosyl transferase activity is increased. Cell lines RicR15, 17, 19 and 21 showed partial deficiencies in galactosyl and N-acetylglucosaminyl transferases. A hypothesis is put forward to account for the different carbohydrate compositions and ricin binding properties of glycoproteins synthesised by these cells in terms of the determined enzyme defects, the normal level of sialyl transferases detected in RicR15 and RicR21 cells and the elevated levels of sialyl and fucosyl transferases detected in RicR17 and 19 cells. None of the above changes in glycosyl transfer reactions in the RicR cell lines are due to enhanced glycosidase or sugar nucleotidase activities in the mutant cells.     

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.     

Separation of membranes from semiprotoplasts of suspension-cultured sycamore maple (Acer pseudoplatanus) cells

Semiprotoplasts were produced from suspension-cultured Acer pseudoplatanus (sycamore maple) cells prior to cell disruption by passing them through a 60 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on linear sucrose density gradients. Samples were collected by gradient fractionation and subcellular fractions were assayed for membrane markers and glycosyl transferase activities. Results of standard marker assays (cytochrome c reductase for endoplas-mic reticulum. uridine and inosine diphosphatases for Golgi. and eosin-5'-maleimide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of ¹⁴C-labeled sugars from UDP-glucose and UDP-xylose into ethanol precipitated polysaccharides was used to detect glucan synthases I & II (glucosyl transferases) and xylosyl transferase activities in Golgi membrane fractions. All three glycosyl transferases overlapped in fractions corresponding to both Golgi and plasma membrane markers, although peak activities for all three occurred in different fractions. More than one peak of glucan synthase I activity was found. Glucan synthase II, associated with ß-l.3 glucan (cullose) synthesis in plasma membranes, was also detected and exhibited a 10-fold stimulation in the presence of Ca²⁺.     

Glycoprotein biosynthesis: Folic acid effects on glycoprotein:glycosyl transferase activities of rat kidney and liver

Folic acid at 14 μM to 1.4 mM increased the activity of the collagen:glc and fetuin:gal and decreased the activity of the fetuin:NANA glycoprotein:glycosyl transferases of rat liver and kidney in vitro; highest effects were found with 1.4 mM folic acid. 1.4 mM folic acid increased kidney fetuin:gal activity 5-fold and decreased fetuin:NANA activity 3-fold. At 1.4 mM, folinic acid and p-methylaminobenzoic acid were totally inactive toward the transferases, methasquin was moderately active, and homofolic, tetrahydrohomofolic and methotrexate were very active toward the transferases. In all instances, however, the fetuin:gal and collagen:glc transferases were activated while the fetuin: NANA transferase was inhibited. From the data presented, folic acid is viewed as a possible control molecule in the synthesis of glycoprotein.     

Subcellular Localization of Glycosyl Transferases Involved in Glycoprotein Biosynthesis in the Cotyledons of Pisum sativum L

Subcellular membrane fractions from 21-day-old pea (Pisum sativum) cotyledons that have associated UDP-N-acetylglucosamine N-acetylglucosaminyl transferase and GDP-mannose mannosyl transferase activities have been isolated and identified. The rough endoplasmic reticulum (RER) is the principal location of glycosyl transferases involved in the assembly of lipid-linked sugar intermediates and glycoproteins. Antimycin A-insensitive NADH-cytochrome c reductase activity was used to identify RER at a density of 1.165 g/cc in sucrose gradients. The high proportion of RER in this fraction was confirmed by electron microscopy.

Other mannosyl transferases are found at a density of 1.123 g/cc and 1.201 g/cc but these glycosyl transferases do not appear to be involved with the formation of lipid-linked sugar intermediates utilized in glycoprotein biosynthesis.

     

Sphingomyelin-metabolizing enzymes and protein kinase C activity in liver plasma membranes of rats fed with cholesterol-supplemented diet.

The effect of cholesterol-supplemented diet on the activities of rat liver plasma membrane sphingomyelin-metabolizing enzymes and protein kinase C was studied. Protein kinase C, phosphatidylcholine:ceramide-phosphocholine transferase, and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase activities were found to increase continuously and almost in parallel during the experimental period on cholesterol diet (days 10, 20, and 30). Linear regression analysis showed a positive correlation between these activities with correlation coefficients r = 0.959 for protein kinase C and phosphatidylcholine:ceramide-phosphocholine transferase, and r = 0.998 for protein kinase C and phosphatidylethanolamine:ceramide-phosphoethanolamine transferase. On the other hand, protein kinase C activation does not correspond to sphingomyelinase activity changes. These data suggest that protein kinase C activation observed in cholesterol-enriched plasma membranes is due to increased production of diacylglycerol and increased acylation of sphingosine to ceramide.     

Isolation and characterization of the multiple glutathione S-transferases from human liver. Evidence for unique heme-binding sites

Thirteen forms of glutathione S-transferase were isolated from human liver in high yields by glutathione-affinity chromatography and chromatofocusing. Apparent isoelectric points ranged from 4.9 to 8.9 and included neutral forms. All 13 forms appeared to be identical immunochemically in a quantitative enzyme-linked immunosorbent assay. These forms were immunochemically distinct from the major acidic glutathione S-transferase found in placenta and erythrocyte and were immunochemically distinct from two forms of higher molecular weight glutathione S-transferase found in some but not all liver samples. The 13 forms exhibited similar activities with 1-chloro-2,4-dinitro-benzene as substrate, specific activities of 33-94 mumol/min/mg. Likewise, these forms all exhibited glutathione peroxidase activity with cumene hydroperoxide, specific activities of 1.5-8.3 mumol/min/mg. All 13 forms bound bilirubin with subsequent conformational changes leading to states devoid of transferase activity, a process prevented by the presence of foreign proteins. As hematin-binding proteins, however, these multiple transferases exhibited a very broad range of binding extending from nonbinding to high-affinity binding (KD approximately 10(-8) M). Hematin binding was noncompetitive with transferase activity and did not involve the bilirubin-binding site, suggesting the existence of unique heme-binding sites on these proteins. The two forms of the immunochemically distinct glutathione S-transferases transferases found in some liver samples also exhibited both transferase and peroxidase activities. In addition, they also have separate sites for binding bilirubin and hematin.     

Hypoxanthine-guanine phosphoribosyltransferase deficiency and erythrocyte synthesis of pyridine coenzymes.

Purine and pyridine metabolism were studied in ten Lesch-Nyhan patients, with virtually no hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity in erythrocytes. Increased NAD erythrocyte concentrations were found in all patients. Raised activities of two enzymes catalysing NAD synthesis from nicotinic acid (nicotinic acid phosphoribosyltransferase: NAPRT, and NAD synthetase: NADs) was found in erythrocyte lysates from all patients. The two enzymes had normal apparent Km for their substrates and increased Vmax. The rate of synthesis of pyridine nucleotides from nicotinic acid by intact erythrocytes in vitro was also increased in most patients. These findings suggest that raised NAD concentrations in HPRT- erythrocytes are due to enhanced synthesis as a result of increased enzyme activities.     

Glycolytic enzyme activities in murine sarcoma virus-transformed cultures of Balb 3T3. The role of culture density.

The effects of transformation by murine sarcoma virus and of increasing cell density on the activities of several key glycolytic enzymes in Balb 3T3 cells were tested. Hexokinase levels increased with culture density in the uninfected and in the two virus-transformed (HB2 and KA31) cells. Phosphofructokinase did not increase with culture density in the uninfected cells but rose dramatically in dense cultures of virus-transformed cells. 6-Phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase levels were high in sparse cultures of uninfected cells and decreased steadily with increased culture density. Pyruvate kinase levels increased with density only in KA31 cultures. A density-dependent decrease in the level of hexokinase type II with a concomitant increase in type I isozyme was seen in uninfected 3T3 cultures. This change was negligible in HB2 cells.     

Modification of the ADP-ribosyltransferase and NAD glycohydrolase activities of a mammalian transferase (ADP-ribosyltransferase 5) by auto-ADP-ribosylation.

Mono-ADP-ribosylation, a post-translational modification in which the ADP-ribose moiety of NAD is transferred to an acceptor protein, is catalyzed by a family of amino acid-specific ADP-ribosyltransferases. ADP-ribosyltransferase 5 (ART5), a murine transferase originally isolated from Yac-1 lymphoma cells, differed in properties from previously identified eukaryotic transferases in that it exhibited significant NAD glycohydrolase (NADase) activity. To investigate the mechanism of regulation of transferase and NADase activities, ART5 was synthesized as a FLAG fusion protein in Escherichia coli. Agmatine was used as the ADP-ribose acceptor to quantify transferase activity. ART5 was found to be primarily an NADase at 10 microM NAD, whereas at higher NAD concentrations (1 mM), after some delay, transferase activity increased, whereas NADase activity fell. This change in catalytic activity was correlated with auto-ADP-ribosylation and occurred in a time- and NAD concentration-dependent manner. Based on the change in mobility of auto-ADP-ribosylated ART5 by SDS-polyacrylamide gel electrophoresis, the modification appeared to be stoichiometric and resulted in the addition of at least two ADP-ribose moieties. Auto-ADP-ribosylated ART5 isolated after incubation with NAD was primarily a transferase. These findings suggest that auto-ADP-ribosylation of ART5 was stoichiometric, resulted in at least two modifications and converted ART5 from an NADase to a transferase, and could be one mechanism for regulating enzyme activity.     

Effect of clofibrate treatment on carnitine acyltransferases in different subcellular fractions of rat liver.

The subcellular distribution of carnitine acetyl-, octanoyl-, and palmitoyl- transferase in the livers of normal and clofibrate-treated male rats was studied with isopycnic sucrose density gradient fractionation. In normal liver 48% of total carnitine acetyltransferase activity was peroxisomal, 36% of the activity located in mitochondria and 16% in a membranous fraction containing microsomes. Carnitine octanoyltransferase and carnitine palmitoyltransferase were confined almost totally (77--81%) to mitochondria in normal liver. Clofibrate treatment increased the total activity of carnitine acetyltransferase over 30 times, whereas the total activities of the other two transferases were increased only 5-fold. From the three different subcellular carnitine acetyltransferases the mitochondrial one was most responsive to clofibrate treatment, i.e. the rise in mitochondrial activity was over 70-fold as contrasted to the 6- and 14-fold rises in peroxisomal and microsomal activities, respectively. After treatment mitochondria contained 79% of total activity. It is concluded that the clofibrate-induced increase of carnitine acetyltransferase activity is not due to the peroxisomal proliferation that occurs during clofibrate treatment. The rise in peroxisomal activity contributed only 8% to the total increase. After clofibrate treatment the greatest part of carnitine octanoyl- and palmitoyltransferase activities were located in mitochondria but a considerable amount of both activities was found also in the soluble fraction of liver.     

Effects of brefeldin A on the localization of chondroitin sulfate-synthesizing enzymes. Activities in subfractions of the Golgi from chick embryo epiphyseal cartilage.

Membranes from brefeldin A-treated and untreated chick embryo epiphyseal cartilage were fractionated separately by equilibrium sucrose density gradient centrifugation. Fractions were assayed for Gal I transferase, Gal II transferase, Gal ovalbumin transferase, chondroitin polymerization on endogenous acceptors, GalNAc transfer to exogenous chondroitin hexasaccharide, and sulfate transfer to exogenous chondroitin. Gal I transferase and Gal II transferase activities were found in heavier cis- and medial-Golgi fractions, but with distributions different from each other. Brefeldin A had no effect on either their distribution or their total activity. Gal ovalbumin transferase activity in fractions from untreated cartilage was found as a dual peak in medial- and trans-Golgi areas. The latter peak was diminished in the fractions from the brefeldin A-treated cartilage, whereas the former peak was correspondingly increased. A similar dual medial- and trans-Golgi distribution for chondroitin polymerization on endogenous acceptors was seen with fractions from untreated cartilage. This was modified in fractions from brefeldin A-treated cartilage with a complete loss of synthesis in the trans-Golgi peak and a slight increase in synthesis in the medial-Golgi peak. However, the distribution of GalNAc transferase activity using exogenous chondroitin hexasaccharide indicated that considerable chondroitin-synthesizing activity still remained in these trans-Golgi fractions. This demonstrated that brefeldin A had caused a block in movement of endogenous proteochondroitin acceptors to the trans-Golgi site of synthesis. Sulfotransferase activity was also found in a dual distribution similar to that of the chondroitin polymerization and GalNAc transferase, with a small reduction in activity in the trans-Golgi fractions of brefeldin A-treated cartilage. Thus, treatment of cartilage with brefeldin A resulted in the loss of considerable trans-Golgi chondroitin sulfate-synthesizing enzyme activity and a block in the transport of one form of proteochondroitin precursor to the trans-Golgi membranes.     

Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture.

Primary cultures of embryonic chick skeletal myogenic cells were used as an experimental model to examine the possible role of mono(ADP-ribosyl)ation reactions in myogenic differentiation. Initial studies demonstrated arginine-specific mono(ADP-ribosyl)transferase activity in the myogenic cell cultures. We then examined the effect of a novel inhibitor of cellular arginine-specific mono(ADP-ribosyl)transferases, meta-iodobenzylguanidine (MIBG), on differentiation of cultured embryonic chick skeletal myoblasts. MIBG reversibly inhibited both proliferation and differentiation of embryonic chick myoblasts grown in culture. Micromolar (15-60 microM) concentrations of MIBG blocked myoblast fusion, the differentiation-specific increase in creatine phosphokinase activity, and both DNA and protein accumulation in myogenic cell cultures. Meta-iodobenzylamine, an analog of MIBG missing the guanidine group, had no effect. Low concentrations of methylglyoxal bis-guanylhydrazone, a substrate for cholera toxin with a higher Km than MIBG, also had no effect, but higher concentrations reversibly inhibited fusion. These findings suggest a possible role for mono(ADP-ribosyl)ation reactions in myogenesis. In addition, the total arginine-specific mono(ADP-ribosyl)transferase activity increased with differentiation in the myogenic cell cultures, and this increase was also blocked by MIBG treatment. Because high levels of activity were found in the membrane fraction derived from later, myotube cultures, the membrane fraction from 96-h cultures was incubated with [32P]NAD+ and subjected to electrophoresis and autoradiography. Three proteins, migrating at 21, 20, and 17 kDa, that were ADP-ribosylated in the absence, but not the presence, of MIBG were identified. These proteins may be endogenous substrates for this enzyme.     

Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases.

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.     

Ceramide galactosyltransferase and cerebroside sulphotransferase in chicken brain cellular fractions and glial and neuronal cells in culture.

Enzymes involved in the synthesis of cerebrosides and sulphatides were assayed in cultured cells of neuronal and glial origin and their activity compared to that found in analogous fractions prepared from chicken brain. High activity was observed for both enzymes in chicken neuronal and glial fractions. However ceramide galactosyltransferase could not be detected in normal glial cells or neuroblastoma cells. A very low activity was found in the glioblastoma cells. Although sulphotransferase was absent from normal glial cells, a notable activity was found in glioblastoma or neuroblastoma cells.     

Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. III. Changes of enzyme activities on cell membranes during culture

When liver cells were dispersed with collagenase, their 5'-nucleotidase activity decreased to half the initial level, but it increased to the original level again on culture of the cells for a few days. The activity of another membrane enzyme, alkaline phosphatase, did not decrease on dispersion of the cells, but it increased about 10-fold on culture of the cells. These inductions did not require any hormone, but the effects were greater at a high cell density. These enzymes are located in both the plasma membranes and the cytoplasm, but the enzymes in these two locations can be distinguished by differences in their pH optima, substrate specificities, and susceptibilities to inhibitors. The increases were found to be due to increases in the activity of only the enzymes in the plasma membranes. The increases in enzyme activities were inhibited by actinomycin D, cycloheximide, and puromycin. The activities of leucine aminopeptidase and aminopeptidase B, other membrane enzymes, remained constant during dispersion and culture of the cells. These results show that enzymes in the cell membranes are affected in different ways by cell dispersion with collagenase and subsequent culture of the cells.     

The glutathione transferase activity and tissue distribution of a cloned Mr28K protective antigen of Schistosoma mansoni.

A protective Mr28K antigen of Schistosoma mansoni, expressed from its cDNA, has been purified in a single step and shown to possess glutathione (GSH) transferase activity as predicted from sequence homologies with two mammalian GSH transferase multigene families. It is notable for its high 1-chloro-2,4-dinitrobenzene GSH transferase and linoleic acid hydroperoxide GSH peroxidase activities. The major GSH transferase of S. mansoni has been purified and its subunit is identical to this Mr28K antigen by criteria of Mr, immunochemistry, substrate specificity and peptide sequence analysis. In the parasite, the antigen is present in the tegument, protonephridial cells and subtegumental parenchymal cells. No significant immunological cross-reactivity between the S.mansoni and mammalian (human and rat) GSH transferases was observed.     

Identity of malonyl and palmitoyl transferase of fatty acid synthetase from yeast. Functional interrelationships between the acyl transferases.

Functional interrelationships between the acyl transferases of yeast fatty acid synthetase were investigated. In binding assays with synthetase modified by 5,5'-dithiobis(2-nitrobenzoic acid), 4--5 malonyl transferase entities per multienzyme complex molecule could be titrated. In the presence of palmitoyl-CoA these malonyl transferases were found inaccessible to malonyl-CoA, whereas the acetyl transferases were reactive towards acetyl-CoA. Between four and five palmitoyl transferase entities per synthetase equivalent were found reactive towards palmitoyl-CoA, the palmitoyl binding being inhibited by malonyl-CoA. Following palmitoyl binding the acetyl transferases were found towards acetyl-CoA. Substrate model assays were consistent with these data. It is concluded that malonyl and palmitoyl transferases are closely coupled enzyme components of the multienzyme complex which are fairly independent of the acetyl transferase entities. The molecular basis for the observed coupling will be given in the following paper.