Radioactive Sugars Research Articles

Identification and characterisation of two surface glycoproteins on cultured cerebellar cells

Two high molecular weight surface glycoproteins of cerebellar cells which are selectively labeled by lactoperoxidase-catalyzed iodination of monolayer cultures from the developing mouse cerebellum, have been identified and partially characterised. Both molecules, called peak 2 and peak 3 proteins, were the major glycoprotein species detected in cerebellar cell cultures after labelling with various radioactive sugars. The freshly iodinated molecules were firmly bound to the cells, but they were released into the medium upon prolonged incubation of the cultures. The soluble peak 2 and peak 3 proteins recovered from the medium comigrated on SDS-polyacrylamide gels with their cell-bound counterparts. Thus, their release results from mechanisms other than extensive degradation. The soluble proteins eluted from gel columns corresponding to molecular weights of over 500,000 and around 300,000, for peaks 2 and 3, respectively. They bound to and were specifically eluted from concanvalin A-Sepharose columns. Peak 3 protein could be easily identified as the most prominent iodinatable polypeptide in cerebellar cell cultures. Its surface expression depended on the presence of neuronal cells. After degenerations of neuron-like cells, a component of greater molecular weight than peak 3 or 2 was predominantly labeled by surface iodination. Peak 2 protein was quantitatively precipitated from labeled culture medium by two heterologous antisera. Anti-peak 2 activity was removed from the antiserum by absorption with adult mouse brain, but not by liver, spleen, thymus, kidney, heart and] lung. Thus, peak 2 protein may be considered as a brain-specific glycoprotein.

Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the 'high-mannose' type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the 'high-mannose' type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.

THE INCORPORATION OF GLUCOSE INTO α‐1, 4‐GLUCAN PROTEINS OF BOVINE RETINA MEMBRANES 1

Abstract—Incubation of bovine retina membranes with UDP‐[14C]glucose resulted in the incorporation of [14C]glucose into endogenous α‐1, 4‐glucan proteins. The transferring system was concentrated in membranes that floated at 0.94 and 1.10m‐sucrose when centrifuged in a discontinuous sucrose density gradient and was almost absent in the rod outer segment (ROS) and the 100, 000 g supernatant fractions. The glucan proteins labelled by incubation with the radioactive sugar nucleotide at micromolar concentrations were distinguished in two fractions by their solubilities in trichloroacetic acid (TCA): glucan protein‐I (GP‐I), insoluble in TCA, and glucan protein‐II (GP‐II), soluble in TCA and precipitable by ethanol from the TCA soluble fraction. GP‐I and GP‐II were precipitated by trichloroacetic acid‐phosphotungstic acid (TCA‐PTA). A third fraction, glucan protein‐III (GP‐III) was found when incubations were carried out with UDP‐[14C]glucose at millimolar instead of micromolar concentrations. GP‐III was soluble in TCA and in TCA‐PTA and precipitable by ethanol from the TCA soluble fraction. GP‐II was excluded from a Sephadex G‐200 column and showed a greater size than GP‐I in a Sepharose 2B column. The radioactive residues obtained from the glucan proteins after digestion with pronase were totally included in a Sephadex G‐25 column and were of a greater size than the labelled residues released with salivary α‐amylase. Only radioactive maltose was found after a‐amylase treatment. When membranes containing labelled GP‐I and GP‐II were incubated with unlabelled UDP‐glucose at millimolar concentrations, GP‐I was converted into GP‐II and GP‐III was formed.

The effect of tunicamycin on the glycosylation of lactating-rabbit mammary glycoproteins.

1. Tunicamycin inhibited the incorporation of d-[2-(3)H]mannose into dolichol-linked oligosaccharide and glycoprotein of lactating-rabbit mammary explants by approximately the same extent (approx. 30% of control value), suggesting that lipid-linked intermediates are involved in the mannosylation of mammary glycoproteins. 2. The incorporation of radioactivity from N-acetyl-d-[1-(14)C]glucosamine into dolichol-linked oligosaccharide was inhibited by tunicamycin to 32% of the control value, whereas the incorporation of the radiolabel into glycoprotein was only inhibited to 72% of the control value. 3. Considerable redistribution of label from N-acetylglucosamine to N-acetylgalactosamine was found to occur in the explants. In the presence of tunicamycin approx. 76% of the radioactivity incorporated into glycoprotein from N-acetyl-d-[1-(14)C]glucosamine was present as N-acetylgalactosamine, compared with approx. 61% in the absence of the inhibitor. Thus tunicamycin selectively inhibits the incorporation of N-acetylglucosamine into glycoprotein. 4. Radioactivity from N-acetyl-d-[1-(14)C]glucosamine was incorporated into a glycoprotein that was identified as casein by the use of a casein-specific antiserum, and also into a group of glycopolypeptides with apparent mol.wts. ranging between 40000 and 80000. N-Acetylgalactosamine was the only radioactive sugar released on strong-acid hydrolysis of the immunoprecipitated casein, whereas N-acetylglucosamine was the major radioactive residue present in the non-casein glycoproteins. Glucosamine and galactosamine were the only radiolabelled sugars detected by paper chromatography of the strong-acid hydrolysate of the protein fraction. 5. Tunicamycin inhibited the incorporation of radioactivity from N-acetyl-d-[1-(14)C]glucosamine into the glycopolypeptides with mol.wts. between 40000 and 80000 as described by polyacrylamide-gel electrophoresis, but did not affect the incorporation of label into casein. It appears that tunicamycin inhibits the incorporation of mannose and N-acetylglucosamine into a number of mammary glycoproteins by inhibiting the formation of lipid-linked intermediates, but does not inhibit the incorporation of N-acetylgalactosamine into casein.

Host cell- and virus strain-dependent differences in oligosaccharides of hemagglutinin glycoproteins of influenza A viruses

The effect of the host cell and virus strain on the glycosylation of influenza A virus hemagglutinin glycoproteins was investigated by analysis of Pronase-digested glycopeptides labeled with radioactive sugar precursors. When influenza A/WSN virus was grown in MDBK cells, HA 1 contained both complex galactose-containing (type I) and mannose-rich (type II) glycopeptides, but HA 2 lacked type II glycopeptides. In contrast, type I and type II glycopeptides were found in HA 1 as well as HA 2 isolated from virus grown in primary chick embryo fibroblast cells, indicating that whether type I or type II oligosaccharide chains are attached to HA polypeptides is determined in part by the host cell type. However, analyses of glycopeptides from various influenza A strains representative of all human antigenic subtypes grown in a single host cell type, MDCK cells, revealed that the oligosaccharide types associated with HA polypeptides also depend on the virus strain. Both type I and type II glycopeptides were found in all strains tested, and there was no significant difference in the sizes of these glycopeptides among the strains. However, strain-dependent differences were observed in the relative amounts of type I and type II glycopeptides in virions as well as isolated glycoproteins HA 1 or HA 2. Such differences were evident among influenza A strains of the same as well as distinct HA subtypes, which suggests that changes in primary structure of the HA polypeptides induced by either antigenic drift or major antigenic shifts have caused the variation in oligosaccharide chains associated with these polypeptides.

Ultrastructural cytochemistry of atrial muscle cells. VII. Radioautographic study of synthesis and migration of glycoproteins

Rats were injected intravenously with l-fucose 3H to study the synthesis and migration of glycoproteins in atrial cardiocytes and to evaluate the fate of this radioactive sugar in both serum and right atrium at various time intervals. Radioactivity was decreased in serum by 50% within 20 min of the injection and by a 100-fold within 1 h. There was a low uptake of radioactivity by the right atrium and it showed a 5-fold reduction between 20 min and 1 h post-injection. Ultrastructural radioautography revealed a high relative specific radioactivity in the Golgi complex at 5 min and a rapid decrease thereafter. The radioactivity increased steadily in specific granules and cell surface to reach maxima at 4 h. Low levels of radioactivity were present at all time intervals in mitochondria, cytosol, myofilaments and Z-bands. These results indicate that atrial specific granules contain glycoproteins synthetized in the Golgi complex of atrial cardiocytes.

Biosynthesis of the oligosaccharides of influenza viral glycoproteins

Glycosylation of influenza viral glycoproteins was investigated by pulse-labeling of infected BHK21-F cells with radioactive sugar precursors and by cell fractionation and analysis of Pronase-digested viral glycopeptides by gel filtration. The results with short pulses of [ 3H]mannose suggested that the initial event in glycosylation is the en bloc transfer of oligomamiosyl cores to viral glycoproteins associated with rough membranes. The molecular weight of the glycopeptides which represent the cores was estimated to be approximately 1600–2200. Some mannose residues appear to be subsequently removed from oligosaccharide cores. [ 3H]mannose-labeled glycopeptides obtained either from cells pulsed for brief periods or from rough membranes, which contain predominantly oligosaccharide cores, were sensitive to digestion by endo-β- N-acetylglucosaminidase H (endo-H). On the other hand, glycopeptides larger than oligosaccharide cores, which appeared during chases or after migration of viral glycoproteins from rough to smooth membranes, were resistant to endo-H treatment. The branched sugars (glucosamine, galactose, and fucose), which are contained only in the complex (type I) oligosaccharide chains of virions, appear to be added in a stepwise manner to the trimmed oligosaccharide cores primarily on smooth membranes. Mannoserich glycopeptides of virions (type II) are similar in size to oligosaccharide cores detected in infected cells and are totally sensitive to endo-H, suggesting that type II glycopeptides may represent oligomannosyl cores which escape trimming as well as addition of branched sugars. Comparison of glycopeptides of infected and uninfected BHK21-F cells suggests that influenza viral glycoproteins contain oligosaccharide chains similar in size to those of host cells except for the absence of sialic acid in viral glycoproteins. Further, we observed that intracytoplasmic membranes from infected cells contain much less sialic acid than those from uninfected cells, indicating that viral neuraminidase present in the interior of infected cells possesses enzymatic activity.

The sugar chain structures of ABO blood group active glycoproteins obtained from human erythrocyte membrane.

The glycoproteins of human erythrocyte membrane have two groups of sugar chains with blood type ABH determinants, which are quite distinct in their molecular sizes. A neutral sugar chain and an acidic sugar chain, which belong to the small size group, were isolated from the glycoproteins obtained from the erythrocyte of blood type O individuals, and their structures were elucidated as Fucalpha1 leads to 2Galbeta1 leads to 3N-acetylgalactosaminitol and Fucalpha1 leads to 2Galbeta1 leads to 3(AcNeualpha2 leads to 6)N-acetylgalactosaminitol, respectively. The molecular weight of the large sugar chains with ABH determinants were estimated to be more than 4000. Both large and small neutral sugar chains of membrane glycoproteins obtained from blood type O erythrocyte could serve as acceptors of alpha-N-acetylgalactosaminyltransferases purified from milk of blood type A1 and A2 individuals, producing the same radioactive sugar chain distribution patterns. However, the acidic sugar chain with the H determinant could not serve as an acceptor of these enzymes.

Mannosyl transferases of the retina: Mannolipid and complex glycan biosynthesis I. Kinetic properties; Product identification

The retina was shown to contain enzymes that catalyse the transfer of mannose from GDP-[U 14C]mannose to endogenous acceptors resulting in the synthesis of a mannose-containing lipid. This product had the properties of the polyprenyl-phosphate monosaccharides. In addition, the transfer to a mannose-containing complex glycan was also observed. The optimal conditions for both of these enzymatic reactions were similar. The optimal pH for these reactions in 0·2 m-TES buffer was 7·0. Enzymatic activity did not require the addition of a metal ion, although both reactions were stimulated optimally by 3·3 m m-MnCl 2. Both reactions were inhibited by EDTA. A high degree of specificity was observed for GDP-mannose as the substrate. The apparent K m for GDP-mannose in the formation of the mannolipid was 1·4 μ m. Mannose was the only radioactive sugar detected in both products. The complex glycan, present as an acid insoluble, chloroform/methanol (2:1)-extracted residue, was solubilized by pronase, from which a low molecular weight (about 7000), mannose containing product was obtained. The mannolipid had chromatographic and acid-base stability properties which were similar to those of dolichyl-phosphate mannose.

Carbohydrates of influenza virus. I. Glycopeptides derived from viral glycoproteins after labeling with radioactive sugars.

The carbohydrate moiety of the influenza glycoproteins NA, HA(1), and HA(2) were analyzed by labeling with radioactive sugars. Analysis of glycopeptides obtained after digestion with Pronase indicated that there are at least two different types of carbohydrate side chains. The side chain of type I is composed of glucosamine, mannose, galactose, and fucose. It is found on NA, HA(1), and HA(2). The side chain of type II contains a high amount of mannose and is found only on NA and HA(2). The molecular weights of the corresponding glycopeptides obtained from virus grown in chicken embryo cells are 2,600 for type I and 2,000 for type II. The glycoproteins of virus grown in MDBK cells have a higher molecular weight than those of virus grown in chicken embryo cells, and there is a corresponding difference in the molecular weights of the glycopeptides. Under conditions of partial inhibition of glycosylation, virus particles were isolated that contained hemagglutinin with reduced carbohydrate content. Glycopeptide analysis indicated that this reduction is due to the lack of whole carbohydrate side chains and not to the incorporation of incomplete ones. This observation suggests that glycosylation of the viral glycoproteins involves en bloc transfer of the core sugars to the polypeptide chains.

Purification and properties of a beta-mannosidase from Aspergillus niger.

A beta-mannosidase (beta-D-mannoside mannohydrolase, EC 3.2.1.25) was purified to apparent homogeneity from the culture filtrate of the fungus, Aspergillus niger. The enzyme had an estimated molecular weight of about 120,000 and was a glycoprotein. Radioactive enzyme was prepared by growing the fungus in [14C]fructose, and this enzyme was used for the preparation of 14C-glycopeptides. The glycopeptides were purified on Sephadex G-25 and G-50 and were then hydrolyzed for sugar analysis. Two radioactive sugars were found in the glycopeptides and these were identified as mannose and glucosamine in a ratio of 2.5 or 3:1. Based on susceptibility of the enzyme to alkaline treatment and the formation of [3H]glucosaminitol in the presence of NaB3H4, the oligosaccharide is apparently attached to the protein in a GlcNAc-asparagine linkage. The beta-mannosidase had good activity on p-nitrophenyl-beta-D-mannoside but was inactive on p-nitrophenyl-alpha-D-mannoside as well as on other p-nitrophenyl glycosides. It also showed good activity on the beta(1 leads to 4)-linked trisaccharide of mannose and somewhat lower activity of the corresponding disaccharide. With each of these substrates the Km was about 1 mM, whereas with the p-nitrophenyl-beta-D-mannoside the Km was about 2 mM. The beta-mannosidase also released [14C]mannose from the Man-GlcNAc-GlcNAc trisaccharide isolated from the lipid-linked oligosaccharides of aorta and released mannose from the disaccharides, Man-(beta1 leads to 4)GlcNAc and Man-(beta1 leads to 4)ManNAc. The pH optimum for the enzyme was about 3.5 to 4.0 in glycine or acetate buffer.

Uptake and metabolism of carbohydrates by epidermal tissue

Isolated epidermis of Commelina communis L. and Tulipa gesneriana L. assimilated (14)CO2 into malic acid and its metabolites but not into sugars or their phosphates; epidermis could not reduce CO2 by photosynthesis and therefore must be heterotrophic (Raschke and Dittrich, 1977). If, however, isolated epidermis of Commelina communis was placed on prelabelled mesophyll (obtained by an exposure to (14)CO2 for 10 min), radioactive sugars appeared in the epidermis, most likely by transfer from the mesophyll. Of the radioactivity in the epidermis, 60% was in sucrose, glucose, fructose, 3-phosphoglyceric acid and sugar phosphates. During a 10-min exposure to (14)CO2, epidermis in situ incorporated 16 times more radioactivity than isolated epidermal strips. Isolated epidermis of Commelina communis and Tulipa gesneriana took up (14)C-labelled glucose-1-phosphate (without dephosphorylation), glucose, sucrose and maltose. These substances were transformed into other sugars and, simultaneously, into malic acid. Carbons-1 through-3 of malic acid in guard cells can thus be derived from sugars. Radioactivity appeared also in the hydrolysate of the ethanol-insoluble residue and in compounds of the tricarboxylic-acid cycle, including their transamination products. The hydrolysate contained glucose as the only radioactive compound. Radioactivity in the hydrolysate was therefore considered an indication of starch. Starch formation in the epidermis began within 5 min of exposure to glucose-1-phosphate. Autoradiograms of epidermal sections were blackened above the guard cells. Formation of starch from radioactive sugars therefore occurred predominantly in these cells. Epidermis of tulip consistently incorporated more (14)C into malic and aspartic acids than that of Commelina communis (e.g. after a 4-h exposure to [(14)C]glucose in the dark, epidermis, with open stomata, of tulip contained 31% of its radioactivity in malate and aspartate, that of Commelina communis only 2%). The results of our experiments allow a merger of the old observations on the involvement of starch metabolism in stomatal movement with the more recent recognition of ion transfer and acid metabolism as causes of stomatal opening and closing.

Glycosylation of vesicular stomatitis virus glycoprotein in virus-infected HeLa cells

Glycosylation of the envelope glycoprotein of vesicular stomatitis virus was examined using virus-infected HeLa cells that were pulse-labeled with radioactive sugar precursors. The intracellular sites of glycosylation and the stepwise elongation of the carbohydrate side chains of the G protein were monitored by membrane fractionation and gel filtration of Pronase-digested glycopeptides. The results with short pulses of sugar label (5 to 10 mtein linkage (glucosamine and mannose) are added to G which was associated with the rough endoplasmic reticulum-enriched membrane fraction, whereas the more distal sugars (galactose, sialic acid, fucose, and possibly more glucosamine) are added in the light-density internal membrane fraction. Accumulation of mature G was observed in the plasma membrane-enriched fraction. The gel filtration studies indicated that the initial glycosylation event may be the en bloc addition of a mannose and glucosamine oligomer, followed by the stepwise addition of the more distal sugars.

The repair of the surface structure of animal cells.

Experiments were performed to determine if animal cells in culture possess specific mechanisms to repair surface molecules damaged by enzymes. The surface membranes of a primary cell culture, chick fibroblasts, a permanent hamster cell line, BHK21/C13, and its virally transformed counterpart, C13/B4 were damaged by exposure to trypsin or to neuraminidase. Following digestion with trypsin, the incorporation of radioactive amino acids or sugars into purified surface membrane of cells was monitored. No differences were noted in rates of incorporation when control and trypsin-damaged cells were compared. Neuraminidase damage to the surface of BHK21/C13 and C13/B4 cells was evidenced by altered gel filtration profiles of surface glycopeptides, i.e., delayed elution because of reduction in size. By labelling cells with 14C-L-fucose prior to neuraminidase treatment and following the incorporation of 3H-L-fucose into cell surface glycopeptides after neuraminidase digestion, we were able to monitor the synthesis and turnover of fucose-containing glycopeptides in the same cells. Gel filtration profiles indicated that little or no desialylated glycoproteins were resialylated (repaired) by specific replacement of sialic acid. Comparing neuraminidase-digested and control cells we observed no difference in rates of 3H-L-fucose incorporation or of 14C-L-fucose loss from these cells; nor did we find differences in the rate of incorporation of isotopic glucosamine into sialic acid. Neuraminidase treatment failed to alter the rate of cell growth or the pattern of isotopic incorporation into various cell surface components. These results support the suggestion that return of sialic acid (repair) was effected by turnover which serves as a non-specific repair mechanism to replace damaged cell surface molecules (Warren and Glick '68; Warren, '69).

Rhodopsin as a glycoprotein: a possible role for the oligosaccharide in phagocytosis

Isolated bovine photoreceptor outer segment preparations are capable of transferring both galactose and fucose from the appropriate sugar nucleotides to rhodopsin. Both manganese chloride and Triton, a non-ionic detergent, are required for full activity. A further stimulation of transfer is produced by ATP. No effect is produced by cyclic-AMP, cyclic-GMP, taurine or calcium chloride on either galactose or fucose transfer. The incorporated radio-active sugars co-migrate with rhodopsin on agarose chromatography. Intact retinas incubated in vitro also incorporate galactose into opsin and rhodopsin. Visual pigment labeled with galactose by either method can be purified on agarose columns and hydrolyzed, releasing 94% of the label which co-chromatographs with authentic galactose in two solvent systems. Neither fucose nor galactose is found in rhodopsin but may be added to rhodopsin as a prelude to disc shedding, thereby providing a new recognition site which allows the pigment epithelium to initiate phagocytosis.

Further analysis of the moving strand model of translocation, using a numerical calculation

Numerical computer results for the moving strand model of phloem translocation are in excellent agreement with the analytical results of Canny and Phillips. Numerical results are presented for the case of constant loading of radioactive sugar, for which Canny and Phillips failed to specify the appropriate boundary condition. Numerical results are obtained for a pulse time variation of loading and for a pulse time variation of sugar in the loading region reservoir, cases which were not solved by Canny and Phillips, and the results are compared with experimental data. We show that the moving strand model does not predict the kind of moving pulse of tracer observed in morning glory (Fisher 1975) after pulse labeling a source leaf with 14CO2 except when transport parameters are specified that clearly conflict with those measured and proposed by Canny. We see little theoretical justification for retaining the moving strand model.

Effect of impaired glycosylation on the biosynthesis of Semliki forest virus glycoproteins.

The glycoproteins of Semliki Forest virus, grown in chicken embryo cells, were labeled with radioactive sugars. The data indicate a high mannose content of the nonstructural precursor glycoprotein NSP 63. This protein can also be readily labeled with 2-deoxy-D-glucose. The envelope glycoproteins E1 and E2 are relatively rich in galactose, glucosamine, and fucose. Glycosylation can be impaired by 2-deoxy-D-glucose or D-glucosamine or by omission of sugars in the culture medium. Under these conditions characteristic changes in the electrophoretic profile of the viral polypeptides are observed: in the regions of glycoproteins NSP 97, NSP 63, and E1 and E2 new protein peaks can be detected. These polypeptides seem to be aberrant forms of the glycoproteins. When compared with the normal molecules they have lower molecular weights and contain less carbohydrates, especially mannose. Pulse-chase experiments indicate that the altered glycoproteins are degraded very slowly if at all. If, however, impairment is caused by omission of sugars in the culture medium, the radioactivity is chased after addition of glucose from the region between NSP 63 and E1 + E2 into the E1 + E2 peak. This suggests a completion of the carbohydrate chains under these conditions.

Étude de l'activité de transfert de glucose, à partir d'UDP‐glucose, dans les membranes microsomiques des hépatocytes de rat

Microsomal preparations from rat liver mediate transfer of glucosyl units from UDP-glucose to three different kinds of acceptors: an endogenous glycoprotein, exogenous glycogen and collagen. Both glucosyl transferases work at acidic pH, 6.5 for transfer on endogeneous acceptor and glycogen and at pH 5.5 for transfer on collagen. None of these enzymes require divalent cations for activity. While transfers on endogenous acceptor and glycogen are inhibited by the presence of a non-ionic detergent, Triton X-100, the transfer on collagen is activated by the same detergent. Glycogen-synthase activity requires glucose 6-phosphate at an optimal concentration of 1 mM. The Km values for UDP-glucose are respectively: 0.5 mM, 0.33 mM, and 1 mM for transfer on endogenous acceptor, glycogen and collagen. Characterisation of the product indicates that a protein-bound alpha1-4 glucan is formed when no primer is added. Enzymatic and acidic hydrolyses of radioactive glycogen and collagen show only glucose as a radioactive sugar identified by thin layer chromatography on cellulose. Pre-treatment of microsomal membranes by alpha-amylase demonstrates that glucosyltransferases are not adsorbed on endogenous glycogen and seem to be really membranous enzymes.

Studien an Cuscuta reflexa Roxb.: III. Zur Frage einer cytokininabhängigen Nährstoffaufnahme )

Summary The content of cytokinin-active substances was shown to be lower in haustoria bearing sections of the parasite Cuscuta reflexa than in the tissue of the attacked axes of the host Vicia faba. The transport of radioactive sugars from the host axis to its roots is drastically decreased by the parasite, and removal of the Toot is without influence on the sugar uptake of Cuscuta. From these observations the mechanism of uptake into the root and Cuscuta is suggested to be different. Pretreating a host leaf with kinetin did not decrease the transport of [U-14C]-sucrose to the parasite. When kinetin was applied to Cuscuta axis it maintained the green colour at the site of application, inhibited stem lengthening, and increased stem thickening. Papillae similar to the early stages of haustoriae were formed. The reason for the strong parasitic deprivation of nutrients is regarded as the action of enzymes mediating a direct connexion of the parasite to the phloem of the host, and consequently, a rapid uptake of the nutrients. A hormone-induced “attraction power” seems not to be the mechanism involved in the host-parasite relations.

Inhibition of glycosylation of the influenza virus hemagglutinin.

d-Glucosamine and 2-deoxy-d-glucose interfere with the biosynthesis of the hemagglutinin glycoproteins. With increasing inhibitor concentrations a progressive decrease in size of the precursor HA and the cleavage products, HA(1) and HA(2) can be observed. The shift in molecular weight is paralleled by a decrease of the carbohydrate content. This was shown by labeling studies with radioactive sugars which revealed that the inhibitors block the incorporation into glycoproteins, whereas they have no or only slight effects on the uptake and activation of sugars. Under conditions of maximal inhibition, the hemagglutinin proteins lack all or most of their carbohydrates. These findings indicate that the inhibitory effect of d-glucosamine and 2-deoxy-d-glucose is due to an impairment of glycosylation. When glycosylation is inhibited, the precursor polypeptide is synthesized at normal rates. Its cleavage products, however, are very heterogeneous. This suggests that carbohydrate protects the hemagglutinin from proteolytic degradation.