Incorporation Of Carbohydrates Research Articles

Distribution of carbohydrate residues in normal skin.

Sections of biopsies of normal skin obtained from 11 individuals were incubated with 8 lectins using an avidin-biotin complex (ABC). All sections when incubated with the appropriate lectin showed the presence of the following carbohydrate residues: L-fucose, beta-(1-4)-D-GlcNAc)2 (N-acetylglucosamine), acetylneuraminic acid, Gal-beta-(1-3)-GalNAc (N-acetyl-galactosamine), beta-D-galactose, alpha-D-glucose, and alpha-D-mannose. In addition, sections of individuals with blood group A showed alpha-D-GalNAc and sections of individuals with blood group B showed alpha-D-galactose. In the stratum (str.) basale, carbohydrates were present in small quantities, but as the cells matured and moved upward, the incorporation of carbohydrates into the cell membranes increased considerably. In the str. granulosum, lectin reactivity was absent in many sections, probably due to masking by phospholipids. The dark cells in the eccrine glands showed reactivity with all lectins except in the one nonsecretor with blood group A1, whose dark cells showed no L-fucose and alpha-D-GalNAc. The endothelial cells of the blood vessels showed lectin reactivity except when incubated with concanavalin A. The sebaceous glands showed both cytoplasmic and membrane staining when incubated with various lectins.

Functional Properties of Lupin Seed (Lupinus mutabilis) Proteins and Protein Concentrates

ABSTRACTFunctional and electrophoretic properties of the seed flour and a protein concentrate prepared by alkaline extraction from lupin seeds (Lupinus mutabilis, cultivar H‐6) were investigated. SDS‐PAGE indicated presence of 13 and 12 subunits in the seed flour proteins and the protein concentrate, respectively. Lupin protein concentrate had good water and oil absorption and gelation properties. Solubility of lupin proteins was minimum at a pH of 4.0 but increased rapidly beyond pH 5.0. Foaming capacity of the protein concentrate could be improved by increasing concentration as well as by adding NaCl and was influenced by pH and incorporation of certain carbohydrates. Emulsion properties of lupin proteins were concentration and pH dependent. Moist heat improved the in vitro digestibility of the seed proteins. The seed flour as well as the protein concentrate did not have detectable trypsin, chymotrypsin, and α‐amylase inhibitory activities.

Metabolic Carbohydrate Labelling of Glycoproteins from Mitogen‐Stimulated Mouse Lymphocytes

CBA/J mouse lymphocytes from thymus, spleen and lymph nodes were separated into fractions enriched in either T (thymus‐derived) or B (bone‐marrow‐derived) cells. The cells were stimulated with ‘T cell mitogens’ (concanavalin A or phytohemagglutinin), ‘B cell mitogens’ (lipopolysaccharide or lipoprotein), and pokeweed mitogen to incorporate tritiated mannose, galactose or fucose. Mitogen‐induced incorporation of carbohydrates into acid‐precipitable material proved to be specific for the cell population.Carbohydrate‐labelled cells were extracted with dodecyl sulfate buffer and the extracts subjected to polyacrylamide gel electrophoresis and subsequent autoradiography. Depending on the carbohydrate used for labelling several glycoproteins specific for activated T cells (GP 200, GP 170, GP 125) and activated B cells (GP 210, GP 85 and GP 75) were noted. Also T or B cell subpopulations could be distinguished by their glycoprotein patterns. From these patterns the following conclusions were derived.a) Concanavalin‐A‐reactive T cells from thymus, spleen and lymph nodes comprise different subpopulations.b) Concanavalin A and phytohemagglutinin stimulate the same T cell subpopulations in spleen and lymph nodes.c) Lipopolysaccharide and lipoprotein stimulate different B cell subpopulations of the spleen.d) Lipopolysaccharide stimulated or lipoprotein‐stimulated B cells from lymph nodes differ from those in the spleen in that the latter show a markedly higher rate of labelling for GP 75, suggesting a higher rate of immunoglobulin M (IgM) synthesis.

Carbohydrate incorporation in plasma membranes of mouse thymocytes stimulated by concanavalin A.

Thymocytes from CBA/J mice were stimulated with concanavalin A. Incorporation of various carbohydrates, leucine and thymidine into trichloroacetic-acid-precipitable material was compared in stimulated and control cells. Incorporation of all carbohydrates, in particular of galactose, was enhanced in concanavalin-A-treated cells. Leucine and thymidine incorporation was increased 2 and 50-fold respectively in stimulated cultures. The kinetics of fucose, galactose, glucosamine, leucine and thymidine incorporation were studied using 10-h pulses at various times during the cultivation. The incorporation of fucose, galactose and thymidine showed two maxima 5 and 30 h after the beginning of the cultivation. In contrast, there were no pronounced maxima seen with glucosamine and leucine. Plasma membranes from stimulated cells were prepared by a modified procedure of McCollester (Cancer Res. 30, 2832–2840, 1970). The purity of the membrane fraction was examined by electron microscopy, by chemical analysis and by assay of marker enzymes. When prepared from cells radioactively labelled with fucose or galactose the plasma membrane fraction was enriched in carbohydrate label. Gel filtration experiments with papain treated, radioactively labelled membranes, solubilised in sodium dodecylsulphate buffer, showed that fucose was only attached to glycoprotein, while other carbohydrate label was distributed between glycoprotein and a fraction containing glycolipids. Analysis of hydrolysed, carbohydrate-labelled membranes by paper chromatography and paper electrophoresis revealed that the bulk of the fucose and galactose label had not been converted into other substances, although some (10%) galactose is converted into glucose. Mannose label had been partially (14%) converted into fucose. Glucosamine label was found to be distributed in sialic acid (25%), glucosamine (40%), galactosamine (30%) and neutral compounds (5%). The data are discussed in relation to the sequence of biochemical events occurring at the cell surface during stimulation of thymocytes with concanavalin A.

Thyroglobulin biosynthesis in "cold" and "hot" nodules in the human thyroid gland.

Studies on "cold" and "hot" nodules from human thyroid gland have been carried out to investigate thyroglobulin biosynthesis and to correlate carbohydrates incorporation and thyroid hormone formation in thyroglobulin. The aim of the investigation was to ascertain the role of carbohydrates of the protein molecule in its migration to the iodinating site of the cell. Thyroid slices from two cases with "cold" and two with "hot" nodules were incubated for 30 to 120 min with [3H]leucine, [3H]carbohydrates (ManNAc, Gal, Man, GlcNAc, GalNAc) and 125I. Soluble and particulate iodoproteins, solubilized by digitonin, were identified by density gradient centrifugation and immunoprecipitation; following hydrolysis carbohydrates and iodothyronines were identified by paper chromatography and chemically determined. While the rate of leucine incorporation into TG increased with time in both "cold" and "hot" nodules, the "cold" nodule, with respect to the "hot" tissue, showed: much lower efficiency incorporating carbohydrates and iodine into TG; lower thyroid/medium ratio of radioiodine; less total soluble protein and soluble TG; more particulate protein and solubilized TG; 1/3 of the carbohydrate and iodine chemical content; only minute amounts of labeled iodothyronines in TG compared to normal levels in "hot" tissue. The formation of thyroid hormone, in enzymatically iodinated TG, and the iodinating activity of the 105,000 X g pellet, incubated with low iodine TG, were similar in both "cold" and "hot" tissues. These results demonstrate that the "cold" nodule is able to synthesize thyroglobulin but the protein is defective in its carbohydrate content. The deficient iodine transport into "cold" tissue and impaired hormonal synthesis are confirmed. The incomplete incorporation of carbohydrates into thyroglobulin in the "cold" nodule could be important for the maturation and migration of the molecule to the iodinating site of the cell

Incorporation of carbohydrates into abnormal thyroglobulin in an experimental rat thyroid tumor.

Previous findings indicated that in line 1–1C2 of experimental rat thyroid tumor there is decreased secretion of 19S thyroglobulin associated with a lack of incorporation of sialic acid into thyroglobulin and sialyltransferase activity in the tumor. In the present work we studied another tumor line, 1–8, in which particle bound iodopro-tein is also increased. This tumor, however, secretes an abnormal form of thyroglobulin. Soluble and digitonin-solubilized iodoproteins were examined in tumor labeled in vitro with N-acetylmannosamine, galactose, mannose, N-acetyl-glucosamine, leucine and iodine. About half of each label except galactose and N-acetyl-mannosamine was in the pellet after digitonin extraction and the remainder was evenly distrib-uted between soluble and solubilized fractions. After galactose labeling, the soluble fraction con-tained relatively more labeled protein; after N-acetylmannosamine, the pellet content was higher but most of the remainder was in the soluble fraction. The soluble and so...

Immunochemical characterization of thyroglobulin-related components synthesized in cell-free systems

Various sheep thyroid subcellular fractions enriched either in rough microsomes or in Golgi structures (agranular microsomes) and isolated total polyribosomes have been incubated in the presence of various labeled precursors: 14C-labeled amino acids and nucleotides of 14C-labeled monosaccharides. Digitonin extracts of the microsomal fractions contained an acid-insoluble 14C-labeled material, a part of which can always be specifically bound by anti-19-S sheep thyroglobulin antibodies: 20 to 50% in the rough microsomal extracts labeled with 14C-labeled amino acids, [ 14C]mannose or N-[ 14 C] acetylglucosamine ; 45 to 70% in the agranular microsome extracts labeled with [ 14C]galactose, N-[ 14 C] acetylglucosamine or [ 14C]sialic acid. Polyribosomes incubated with 14C-labeled amino acids secreted into the medium an acid-insoluble labeled material containing up to 30% of thyroglobulin immunoreactive material. Sucrose gradient centrifugation showed that 14C-labeled proteins extracted from rough microsomes were essentially located in a 6–8-S peak accompanied, in the case of labeling with 14C-labeled amino acids, by a 2–4-S peak. Proteins synthesized by polyribosomes were in a 3–5-S peak. In agranular microsomal extracts a 17- to 19-S species was predominant. The proportion of 14C-labeled thyroglobulin immunologically detectable increased with increasing sedimentation coefficient (5–10% in the 2–4-S zone, up to 90% in the 17–19-S zone), whatever labeled precursor was used. In the case of rough microsomal extracts, an evolution has been observed in vitro: storage of the dialyzed extracts at −20°C or incubation in conditions favoring disulfide interchange generally increased the percent of immunologically-precipitable material, whereas 10- to 12-S and 17- to 19-S species often appeared. A parallelism between the acquisition of immunoreactive determinants, the evolution of the three-dimensional structure and the incorporation of carbohydrates, during the first steps of synthesis and secretion of the thyroglobulin is suggested and discussed.

Defective Thyroglobulin Synthesis in an Experimental Rat Thyroid Tumor: LACK OF MEMBRANE-BOUND SIALYLTRANSFERASE ACTIVITY

Abstract The incorporation of carbohydrates has been studied in vitro in the experimental rat thyroid tumor 1-1C2. The uptake of N-[3H]acetylmannosamine, a precursor of sialic acid, is less than 5% of that in normal thyroid gland; no label is found in 19 S thyroglobulin or its precursors either in the soluble or in the solubilized proteins. N-[3H]Acetylglucosamine is incorporated at a slower rate than in normal thyroid gland and is present in the membrane-bound thyroglobulin; there is no conversion into sialic acid in the soluble thyroglobulin of tumor as occurs in normal thyroid. [14C]Mannose and [14C]galactose are incorporated at an almost normal rate and [14C]fucose, at a subnormal rate; they are present mainly in the particle-bound proteins as ∼18 S thyroglobulin and its precursors. Measurement of sialyltransferase activity showed that, with desialylated thyroglobulin as acceptor, the 22,000–105,000 x g pellet of tumor contains ∼3% of normal sialyltransferase activity, and the soluble fraction contains ∼25% of normal activity. Desialylated fetuin and orosomucoid are generally better acceptors than desialylated thyroglobulin for tumor sialyltransferase. The findings indicate that defective thyroglobulin release and absence of incorporation of sialic acid into thyroglobulin coexist in this tumor. The very low activity of thyroglobulin-specific sialyltransferase activity in the tumor particulate fraction accounts for the failure of sialic acid incorporation into thyroglobulin and may explain the defect in thyroglobulin release.

Cell wall formation in yeast

Incorporation of tritiated glucose into cell walls of growingSaccharomyces cerevisiac andSchizosaccharomyces pombe was studied using electron microscopic autoradiography. The pattern and the extent of labelling ofS. cerevisiae cell walls depended on the cell stage in the cell cycle. Quantitative evaluation of autoradiographs showed that the highest rate of wall synthesis took place during bud growth. The incorporation of new material into the wall of growing bud showed an increasing rate with the magnitude of the bud. The incorporation into the mother cell wall was almost negligible during bud growth. The rate of wall synthesis in double cells decreased during cell division. This period and that before new bud initiation was found to be the time of substantially reduced rate of wall replication inS. cerevisiae. A significant random incorporation was observed into the walls of post-division adult cells, both parental and daughter. The cell walls ofS. pombe were labelled almost exclusively at growing tips. The incorporation of tritiated carbohydrates into non-extensile regions ofS. pombe cell walls was found to be only about 5% of the total wall labelling.

Incorporation of carbohydrates into endogenous acceptors of liver microsomal fractions

Radioactive sugar-nucleotides reacted with the microsomal fractions of rat and rabbit liver to form glycoproteins with endogenous acceptors. Triton X-100 caused 2- to 3-fold enhancement of incorporation, but the transfer enzyme system lost activity very rapidly under such conditions. Higher concentrations of sugar-nucleotides somewhat protected the enzymes. The transfer of hexosamine- N-acetyl from UDP-hexosamine-NAc was noncompetitively inhibited by UDP and UMP. Cyclic UMP, uridine, ADP, and AMP had no significant effect. The incorporation of mannose from GDP-mannose was inhibited by GDP, but UMP and UDP had only very slight inhibitory action. Snake venom phosphodiesterase also caused marked inhibition under conditions where it hydrolized only a fraction of UDP-hexosamine-NAc. Two products were observed in the reactions with UDP-galactose, UDP-hexosamine-NAc, and GDP-mannose; one was very labile to acid hydrolysis and could be extracted with lipid solvents, while the other was protein-bound sugar. The incorporation of hexosamine-NAc into the lipid was initially rapid then reached a plateau after about 10 min of reaction. The incorporation into protein was slower initially but proceeded longer at a steady rate. When microsomal fractions labeled previously with hexosamine-NAc- 14C were treated with UMP or UDP, the radioactivity of the sugar-lipid decreased rather rapidly. Phosphodiesterase had a similar action. In a pulse-chase experiment the radioactivity of the sugar-lipid decreased, as expected from a precursor of the protein-bound sugar, and the activity of the protein increased concomitantly. The incorporation of UDP- 3H and UMP- 3H into UDP-glucosamine-NAc was also measured. Under the conditions of the tests only a minute amount of 3H could be detected in UDP-glucosamine-NAc.

Acetoxycycloheximide effect on synthesis and metabolism of glucosamine-containing macromolecules in brain and in nerve endings.

AbstractCarbohydrate‐containing macromolecules of brain were substantially labeled after intracerebral injection of C14‐glucosamine. The “half‐life” of the labeled macromolecules, as measured in these experiments, was about one week. Acetoxycycloheximide inhibited incorporation of C14‐glucosamine into these substances. This appears to be secondary to inhibition of synthesis of polypeptide chains which act as “acceptors” for carbohydrate residues. Acetoxycycloheximide inhibited incorporation of C14‐glucosamine (and its derivatives) into the soluble carbohydrate‐containing macromolecules of nerve endings far less than that of the other subcellular fractions of brain. This suggests that the polypeptide “acceptors” of carbohydrate residues at nerve endings are not synthesized locally but are transported to the nerve endings. Final incorporation of carbohydrates into carbohydrate‐containing macromolecules occurs at nerve endings. The “turnover” of these substances in nerve endings was found to be relatively rapid. Evidence suggesting reversible incorporation and removal of some carbohydrate residues of carbohydrate‐containing macromolecules of brain was found and the possible regulatory role of this process is considered.

Environmental factors relating to tropolone formation by Penicillium stipitatum

The formation of stipitatic acid and other aromatic and troponoid metabolites during growth of Penicillium stipitatum under various conditions has been examined. The chemical and physical properties of several of these presumptive additional tropolone derivatives are described. The efficiency of incorporation of carbohydrates, of aromatic compounds, and of tricarboxylic acid cycle intermediates into stipitatic acid and into a class of substances entitled “acetate replacement” compounds, by the replacement technique, has been ascertained. The results of some preliminary isotopic tracer experiments have also been presented. The relationship of these findings to aromatic ring biosynthesis has been discussed.