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.     

Cell surface glycosyl transferase activities in liver cells of developing chicken embryos

Summary The cell surface of embryonic chick liver cells contains transferases for mannose, fucose, galactose, N-acetyl-glucosamine and N-acetyl-neuraminic acid. Liver cells obtained by trypsin-dissociation of the tissue use the corresponding exogenous sugar nucleotides as substrates. The activities of the enzymes tested do not depend neither on the dissociation procedure nor onde novo protcin synthesis. They vary considerably during development of the embryos, reaching maximal values at the 8th ± 1 day and at the 12th ± 1 day. Glycoproteins are the final stable endogenous acceptors for all sugars. Mannose transfer proceeds via a two or multistep reaction sequence. In a first step labile lipophilic intermediates are formed. Mannose can be liberated by treating the intermediates with 0.1n HCI at 100°C. In a second reaction step mannose becomes attached to glycoproteins. From embryonic chick liver cells a glycopeptide fraction has been obtained by pronase digestion followed by several purification steps. The purified glycopeptides inhibit all transferase systems and act as exogenous acceptors for mannose transfered from exogenous GDP-mannose.     

Comparison of hepatic, renal and intestinal bilirubin UDP-glucuronyl transferase activities in rat microsomes

1. Bilirubin UDP-glucuronyltransferase activity and its dependence on substrate concentrations in rat liver, renal cortex and intestinal mucosa microsomes were studied. 2. Bilirubin monoglucuronide synthesis from unconjugated bilirubin was a higher capacity, lower affinity step in comparison with bilirubin diglucuronide formation in the three tissues tested. 3. Bilirubin glucuronide formation in liver microsomes showed a higher capacity but a lower affinity than extrahepatic ones. Renal cortex and intestinal mucosa exhibited similar kinetics parameters. 4. In vitro bilirubin glucuronidation in renal cortex and intestinal mucosa was quantitatively important as compared with the hepatic one.     

Surface biochemical changes accompanying primary infection with Rous sarcoma virus. I. Electrokinetic properties of cells and cell surface glycoprotein:glycosyl transferase activities

A special class of polysomes synthesizing tubulin was determined using embryos of the sea urchin, Hemicentrotus pulcherrimus. Three criteria were established for identification of polysomes carrying nascent tubulin, i.e., nascent tubulin on polysomes should have (i) colchicine binding activity, (ii) precipitability with vinblastine and (iii) coincidence in mobility by electrophoresis with tubulin. Two classes of polysomes had polypeptides which accorded with the three criteria. One was tetramers and the other was composed of 15–20 ribosomes. From data reported previously on the molecular weight and amino acid composition of completed microtubule proteins, it was suggested that the class of polysomes composed of 15–20 ribosomes constituted the polysome-synthesizing tubulin of sea urchin embryos. The nature of the nascent polypeptides carried by the tetramer polysomes having colchicine binding activity and precipitability with vinblastine could not be clarified.     

Carbohydrate contents, and glycosidase and glycosyl transferase activities in tissues from streptozotocin diabetic mice

The contents of hexoses and hexosamines in brain, liver, and kidney of streptozotocin diabetic mice are significantly increased in comparison to the controls. These differences for hexoses contents in the heart are not significant. N-acetyl-beta-D-glucosaminidase and beta-D-glucosidase activities in brain, liver and kidney of diabetic mice are significantly higher when compared to the controls. However, beta-D-galactosidase activity is significantly lower in brain, liver, spleen and kidney of the diabetic mice, in comparison to the controls and similar in heart. alpha-D-Mannosidase activity of diabetic mice is significantly increased in spleen and heart and significantly decreased in liver and kidney. alpha-L-Fucosidase of diabetic mice shows higher activities, with significant differences, in liver and spleen; however, in heart and kidney the activities are significantly lower. Brain sialyltransferase and galactosyltransferase activities are significantly increased in diabetic mice; but for heart and kidney these differences are not significant. The activity for brain and kidney fucosyltransferase is not significant and that for the other assayed organs is significantly higher in comparison to the controls.     

Sequence and expression of Thai Rosewood beta-glucosidase/beta-fucosidase, a family 1 glycosyl hydrolase glycoprotein

Dalcochinin-8'-O-beta-glucoside beta-glucosidase (dalcochinase) from the Thai rosewood (Dalbergia cochinchinensis Pierre) has aglycone specificity for isoflavonoids and can hydrolyze both beta-glucosides and beta-fucosides. To determine its structure and evolutionary lineage, the sequence of the enzyme was determined by peptide sequencing followed by PCR cloning. The cDNA included a reading frame coding for 547 amino acids including a 23 amino acid propeptide and a 524 amino acid mature protein. The sequences determined at peptide level were found in the cDNA sequence, indicating the sequence obtained was indeed the dalcochinase enzyme. The mature enzyme is 60% identical to the cyanogenic beta-glucosidase from white clover glycosyl hydrolase family 1, for which an X-ray crystal structure has been solved. Based on this homology, residues which may contribute to the different substrate specificities of the two enzymes were identified. Eight putative glycosylation sites were identified, and one was confirmed to be glycosylated by Edman degradation and mass spectrometry. The protein was expressed as a prepro-alpha-mating factor fusion in Pichia pastoris, and the activity of the secreted enzyme was characterized. The recombinant enzyme and the enzyme purified from seeds showed the same K(m) for pNP-glucoside and pNP-fucoside, had the same ratio of V(max) for these substrates, and similarly hydrolyzed the natural substrate, dalcochinin-8'-beta-glucoside.     

In situ determination of the molecular weight of hepatic gamma-glutamyl transferase and gamma-glutamyl hydrolase activities

The molecular weight of gamma-glutamyl transferase from normal rat liver and hepatoma tissue was determined by radiation-inactivation and found to be approx 100000 in each case. The molecular weight previously reported for the subunit containing the gamma-glutamyl binding site (22000) is considerably less than that of the holoenzyme, suggesting that in situ the large subunit is implicated in both transferase and hydrolase activities.     

ADP-ribosyl transferase and NAD glycohydrolase activities in rat liver mitochondria

ADP-ribosyl transferase and NAD glycohydrolase activities have been estimated in mitochondria in mitoplasts as well as in other submitochondrial fractions. A high activity of these two enzymes was present in mitoplasts as compared to the outer membrane preparation or intermembrane compartment. Inhibitor studies provide strong evidence for the involvement of ADP-ribosyl transferase in the process of ADP-ribosylation of mitochondrial proteins. When NAD glycohydrolase was blocked by nicotinamide or 3-aminobenzamide, the incorporation of ADP-ribose into mitochondrial proteins still occurs. ADP-ribosyl transferase activity could also be detected when NAD glycohydrolase was separated by hydroxylapatite chromatography. The protein-linked ADP-ribose moiety appears to be an oligomer in mitochondria.     

Sialyl transferase activities of rat ascites hepatoma cells and rat liver

Sialyl transferase activities of the homogenate of rat ascites hepatome cells were compared with normal rat liver homogenate. The former had only 20% of the activity of the latter when an exogenous acceptor was added in the reaction mixture.Toward endogenous receptors, the activity of the hepatoma cell homogenate was 50% of that of the normal cell homogenate. A stimulation of the activity toward endogenous acceptors was observed when the homogenate of rat ascites hepatoma cells and that of rat liver were mixed. This stimulatory effect seems to be the consequence of utilization of acceptors from ascites hepatoma cells by the sialyl transferases of the rat liver.     

Schistosoma mansoni: glycosyl transferase activity and the carbohydrate composition of the tegument.

Homogenates of adult Schistosoma mansoni contain enzymes which transferred [¹⁴C]mannose, [¹⁴C]glucose, and [¹⁴C]galactose from GDP-[U-¹⁴C]mannose, UDP-[U-¹⁴C]glucose, and UDP-[U-¹⁴C]galactose respectively to a lipid acceptor; in comparison, free [¹⁴C]mannose, GDP-[U-¹⁴C]fucose, and UDP-[U-¹⁴C]acetyl-glucosamine were poorly transferred. The lipid acceptor is believed to be an intermediate in the glycosylation of the worm's glycoproteins and in the biosynthesis of oligosaccharides and glycolipids. The tegument of adult worms was isolated by the freeze-thaw procedure and sugars associated with macromolecules in this fraction were analyzed; the major monosaccharide components were glucose, galactose, and mannose. These results suggest that the mechanism of glycosylation of the adult schistosome's tegumental macromolecules may occur through the glycosyl transferase system. The schistosome mannosyl transferase (EC 2.4.1), which is membrane bound was solubilized with 0.1% Triton X-100 without loss of activity; after density gradient centrifugation there was a peak of enzymic activity in a region of density 1.08, which could not be associated with any particular organelle.     

Cellular distribution of lysosomal hydrolase activities in the regenerating rat liver

Cathepsins B and D, beta-galactosidase, and acid phosphatase activities were found to be decreased in the regenerating rat liver, the reduction being maximal around the peak of hepatocyte mitoses (30 h). To investigate whether these changes could be heterogeneously distributed among hepatic cells, total cell populations from control or two-thirds hepatectomized rat livers were dissociated by the collagenase perfusion technique and analysed by different procedures. Isopycnic centrifugation in a Metrizamide gradient satisfactorily resolved hepatocytes and non-parenchymal cells from control animals but was not adequate when applied to 30-h regenerating liver cells. Colchicine treatment of the hepatectomized animals, resulted in substantial accumulation of phase M-hepatocytes. Subpopulations considerably enriched in fast-sedimenting phase M-cells were obtained by sedimentation at 1 g of the total liver cell population, and subsequently analysed by isopycnic equilibration. Phase M-hepatocytes were shown to have markedly reduced levels of beta-galactosidase, acid phosphatase, and cathepsin B activities in comparison, not only with control hepatocytes, but also with those parenchymal cells which were not metaphase-arrested in the same regenerating livers. Therefore, in partially-hepatectomized rats, hepatocytes progressing up to metaphase in the first mitotic cycle exhibited a selective depletion of lysosomal enzyme activities. The mechanism(s) underlying this change remain(s) presently unknown.     

Cultured neonatal rat oligodendrocytes are enriched in acid hydrolase activities

We measured the activity of several acid hydrolases in oligodendrocyte and mixed glial (predominantly astrocytic) cell cultures prepared from neonatal rat cerebra. When compared with the mixed glial cultures, the cultured oligodendrocytes exhibited higher levels for all the hydrolases when activities were normalized to protein content. When enzymic activities were examined as a function of DNA content, oligodendrocytic -l-fucosidase, -d-glucuronidase, arylsulfatase, and N-acetyl--d-glucosaminidase were higher than in mixed glial cultures, whereas the activities of -d-glucosidase, -d-galactosidase and acid phosphatase were not elevated. These differences could not be accounted for by the fetal bovine serum present in the culture medium. The enrichment in acid hydrolase specific activities in the oligodendrocytes may be associated with a rapid turnover of at least some of the extensive myelin-like membranes formed by these cultured cells. Alternatively, the enrichment of acid hydrolase activity in the oligodendrocytes may be associated with intracellular vesicles of lysosomal origin which may play a role in myelin-like membrane assembly. Exactly which of the above two processes, or possible combinations thereof, is responsible for the present finding is not known.