Ecophysiological comportment of the tropical CAM-tree Clusia in the field: II. Modes of photosynthesis in trees and seedlings.

Measurements were performed on leaves of Clusia rosea Jacq. trees in the moist central mountains (330 to 365 m above sea level) and at the dry south coast of St John Island (US Virgin Islands, Lesser Antilles). Seedlings of C. rosea were also studied in the central hills. During the study period (March 1989) all trees showed crassulacean acid metabolism (CAM), in which net CO2 uptake extended for a remarkably long time in the morning (phase II of CAM: until about 11 to 12 h) and contributed about 1/3 of total net CO2 -uptake. During the night (phase I of CAM) malic acid and citric acid were accumulated concurrently at a molar ratio of malic: citric acid of about 1.6. Internal recycling of respiratory CO2 was 20% of total CO2 fixed during the night. Water-use-efficiency (mol CO2 taken up: mol H2 O transpired) was 0.014 to 0.022. The pH of leaf-cell sap at the end of the dark period was 2.85. This would still allow an H+ -ATPase at the tonoplast to transport 2H+ into the vacuole per ATP hydrolysed when operating near thermodynamic equilibrium. Free sugars, glucose and fructose, and starch were used as precursors for the CO2 -acceptor phosphoenolpyruvate during the dark period; contributions of the two hexoses were about equal and together four-times that of starch. Xylem tensions showed increases of up to 8 bar during day-time. Leaf-sap osmotic pressures did not change significantly; the trend was a small decline during day-time. Among the seedlings, three different modes of photosynthesis were encountered, namely C3 -photosynthesis in terrestrial and in epiphytic seedlings, continuous stomatal opening and CO2 -uptake day and night in epiphytic seedlings, and CAM in seedlings growing in the tanks of Aechmea lingulata (L.) Baker.

Ecophysiological comportment of the tropical CAM-tree Clusia in the field: I. Growth of Clusia rosea Jacq. on St John, US Virgin Islands, Lesser Antilles.

Clusia rosea Jacq. is abundant in the moist parts of the Caribbean island of St John (US Virgin Islands, Lesser Antilles) but relatively rare along the dry south coast. Three types of seedlings were encountered, terrestrial seedlings, seedlings growing as humus-epiphytes on other trees, and seedlings growing inside the tanks of the bromeliad Aechmea lingulata (L.) Baker. Free-living trees grow from terrestrial seedlings or from epiphytic seedlings strangling and shading their host trees. Leaf-Na+ levels were always low (1-4 mequiv I-1 tissue water); trees close to the shore were not affected by salinity. In leaves of mature C. rosea trees, levels of Ca2+ , Mg2+ and K+ were about 60-90, 40-50, 45-55 mequiv I-1 tissue water, respectively. Epiphytic seedlings tended to contain lower levels of these inorganic cations than seedlings growing terrestrially or in the tanks of Ae. lingulata. Epiphytic seedlings contained significantly less nitrogen than terrestrial seedlings. In the leaves of mature trees N-levels were independent of altitude and location on the island, but shaded leaves had significantly higher N-levels than exposed leaves. Light compensation point of photosynthesis in epiphytic seedlings performing C3 -photosynthesis was 17-5 (µmol photons m-2 s-1 ), photosynthesis was saturated at about 300µmol photons m-2 s-1 showing a maximum rate of CO2 -uptake of 2-3 µmol m-2 s-1 .

Cyclitols as cryoprotectants for spinach and chickpea thylakoids.

Thylakoid membranes isolated from either spinach or chickpea leaves were used as a model system for evaluating the capacity of cyclitols to act as cryoprotectants. The effect of freezing for 3 h at -18 degrees C on cyclic photophosphorylation and electron transport was measured. The cyclitols, ononitol, O-methyl-muco-inositol, pinitol, quebrachitol and quercitol at 50-150 mol m(-3) decreased membrane damage by freezing and thawing to a similar degree as the well known cryoprotectants sucrose and trehalose. On addition of the cryotoxic solute NaCl (100 mol m(-3)) to the test system these methylated cyclohexanhexols again provided a protection comparable to that of the two disaccharides. Quercitol (cyclohexanpentol) was not effective when added in lower concentrations (50-100 mol m(-3)) and in case of this cyclitol a ratio of membrane toxic to membrane compatible solute of 0.66 was apparently needed to prevent a loss of cyclic photophosphorylation. Little difference was observed in the results from spinach or chickpea thylakoids although these plants naturally accumulate different cyto-solutes (spinach: glycinebetaine; chickpea: pinitol).

Occurrence of alpha 1-2-fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver. Aberrant type of fucosylation in a malignant tissue.

A comparative study was undertaken to characterize the linkages of L-fucose in N-glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum. After in-vivo radiolabelling of rats with L-[6-3H]fucose, the asparagine-linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride. Two specific alpha-L-fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of L-fucose in the oligosaccharide chains. Fucose alpha 1-2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total L-[3H]fucose in N-glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose alpha 1-3/4 linked to N-acetyl-D-glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained alpha-fucosyl residues linked at the C6 position of the proximal N-acetyl-D-glucosamine.

Purification and characterization of trimming glucosidase I from pig liver.

Trimming glucosidase I has been purified about 400-fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enzyme has an apparent molecular mass of 85 kDa, and is an N-glycoprotein as shown by its binding to concanavalin A-Sepharose and its susceptibility to endo-beta-N-acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non-specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N-linked high-mannose sugar chain. Purified pig liver glucosidase I hydrolyzes specifically the terminal alpha 1-2-linked glucose residue from natural Glc3-Man9-GlcNAc2, but is inactive towards Glc2-Man9-GlcNAc2 or nitrophenyl-/methyl-umbelliferyl-alpha-glucosides. The enzyme displays a pH optimum close to 6.4, does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (Ki approximately 2.1 microM), N,N-dimethyl-1-deoxynojirimycin (Ki approximately 0.5 microM) and N-(5-carboxypentyl)-1-deoxynojirimycin (Ki approximately 0.45 microM), thus closely resembling calf liver and yeast glucosidase I. Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to cross-react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross-reactivity.

Purification and properties of a secreted and developmentally regulated alpha-L-fucosidase from Dictyostelium discoideum.

During its development the eukaryotic microorganisms Dictyostelium discoideum secretes an alpha-L-fucosidase (EC 3.2.1.51). In cells of the growth phase almost no alpha-L-fucosidase activity is detectable. The activity increases steadily up to the aggregation stage and accumulates also in the extracellular medium. The developmental regulation is mediated by pulsatile cAMP signals. The alpha-L-fucosidase was purified from extracellular medium. The isolation procedure started with concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulfate precipitation, followed by Sephacryl S-300 gel filtration and further purification by fast protein liquid chromatography on Mono Q, phenyl-Superose, and finally Superose 12. The purified preparation was found to be essentially free of activities of six other glycosidases also secreted by D. discoideum. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed one major band with an apparent molecular mass of 62 kilodalton. Gel filtration of the enzyme on a Superose 12 column was consistent with an active monomer. A monoclonal antibody was produced, which recognizes a carbohydrate epitope shared by all lysosomal enzymes in D. discoideum. The pH optimum of the alpha-L-fucosidase is at 3.7. The apparent Michaelis constant for p-nitrophenyl alpha-L-fucoside as substrate is 1.2 mM. The enzyme catalyzes preferentially the hydrolysis of alpha 1----6GlcNAc but also of alpha 1----2Gal and alpha 1----3Glc fucosyl linkages.

Incorporation of the hexose analogue 2-deoxy-D-galactose into membrane glycoproteins in HepG2 cells.

The incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of glycoproteins and the consequences of 2-deoxy-D-galactose treatment on the fucosylation of glycoproteins were investigated in the human hepatoma cell line HepG2. Using different methods, it was shown that treatment of HepG2 cells with 2-deoxy-D-galactose leads to an incorporation of 2-deoxy-D-galactose and a decrease of L-fucose incorporation into the oligosaccharides of glycoproteins. The extent of labeling by L-[3H]fucose was determined by removing L-[3H]fucose from labeled cells with the aid of a purified alpha 1,2-fucosidase from Aspergillus niger. Using this method, it was shown that 2-deoxy-D-galactose markedly inhibits alpha 1,2-fucosylation. Measurement of the amount of 2-deoxy-D-galactose incorporated, however, showed that replacement of D-galactose by 2-deoxy-D-galactose does not entirely account for the decrease in alpha 1,2-fucosylation. In addition, a hitherto unreported compensatory increase of alpha 1,3/alpha 1,4-fucosylation was found to occur when alpha-1,2-fucosylation was inhibited by treatment with 2-deoxy-D-galactose.