Synthesis of cell-wall glycoproteins and their characterization in oat coleoptiles.

D-[U-14C]Glucosamine was rapidly taken up by oat coleoptile segments and metabolized to radioactive UDP-N-acetylglucosamine, which acted as specific glycosyl donor for the synthesis of glycolipids and cytosolic, membrane-bound and cell-wall glycoproteins. Cell-wall glycoproteins were solubilized from the walls by either cell-wall-degrading enzymes or chemical extractants. The solubilized cell-wall glycoproteins in the presence of peptide N-glycosidase F released oligosaccharide chains higher than seven glycosidic residues. The combined action of peptide N-glycosidase F and N-acetyl-beta-D-glucosaminidase on cell-wall glycoproteins indicated the presence of N-acetylglucosamine residues beta-1,2-linked to mannose. Less than 9% of the radioactive oligosaccharide chains was released from the solubilized cell-wall glycoproteins when treated with 0.5 M NaOH at 20 degrees, whereas more than 45% of the radioactivity was released in the presence of 1 M NaOH at 50 degrees. The high hydrolytic sensitivity of cell-wall glycoproteins to peptide N-glycosidase F, N-acetyl-beta-D-glucosaminidase and NaOH at 50 degrees indicated that most N-acetylglucosamine residues were incorporated into N-linked cell-wall glycoproteins. Further evidence of this was obtained by the use of inhibitors of biosynthesis and processing of N-linked glycoproteins.

Glucomannan synthesis in pea epicotyls: the mannose and glucose transferases.

Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a beta-1,4-[14C]mannan from GDP-D-[U-14C]-mannose, a mixed beta-1,3- and beta-1,4-[14C]glucan from GDP-D-[U-14C]-glucose and a beta-1,4-[14C]-glucomannan from both GDP-D-[U-14C]mannose and GDP-D-[U-14C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The beta-glucan synthase had different properties from other preparations which bring about the synthesis of beta-1,3-glucans (callose) and mixed beta-1,3- and beta-1,4- glucans and which use UDP-D-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-D-xylose in addition to GDP-D-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-D-glucose acted competitively in the presence of GDP-D-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-D-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-D-glucose and GDP-D-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-D-mannose and GDP-D-glucose to bring about the synthesis of the heteropolysaccharide.

A membrane-bound enzyme complex synthesising glucan and glucomannan in pine tissues.

Particulate membrane preparations isolated from cambial cells and differentiating and differentiated xylem cells of pine (Pinus sylvestris L.) trees synthesised [(14)C]glucans using either guanosine 5'-diphosphate (GDP)-D-[U-(14)C]glucose or uridine 5'-diphosphate (UDP)-D-[U-(14)C]glucose as glycosyl donors. Although these glucans had ß-(1→3) and ß-(1→4) linkages in an approximate ratio 1:1, the distribution of the linkages in the glucan synthesised from GDP-D-glucose was different from that synthesised from UDP-D-glucose. The synthesis of the mixed ß-(1→3) and ß-(1→4) glucan from GDP-D-[U-(14)C]glucose was changed to that of ß-(1→4) glucomannan in the presence of increasing concentrations of GDP-D-mannose. The glucan formed from UDP-D-[U-(14)C]glucose was not affected by any concentration of GDP-D-mannose. The membrane preparations epimerized GDP-D-glucose to GDP-D-mannose; however, the low amount of GDP-D-mannose formed was not incorporated into the polymer becaus the affinity of the synthase for GDP-D-glucose was much greater than that for GDP-D-mannose. The glucan formed from GDP-D-glucose and the glucomannan formed from GDP-D-glucose together with GDP-D-mannose were characterized. The apparent K m and V max of the glucan synthase for GDP-D-glucose were 6.38 µM and 5.08 µM·min(-1), respectively. No lipid intermediates were detected during the synthesis of either glucan or glucomannan. The results indicated that an enzyme complex for the formation of the glucomannan was bound to the membrane.

Efectos del aceite de hígado de bacalao como fuente de ácidos grasos n-3 sobre las lipoproteínas y la viscosidad sanguínea en pacientes con hiperlipoproteinemias primarias / Effects of cod liver oil as a source of fatty acids n-3 on lipoproteins and blood viscosity in patients with primary hyperlipoproteinemia

Las dietas ricas en AG-n-3 se consideran antiaterogénicas. El aceite de hígado de bacalao (AHB) contine 19 g/dl de AG-n-3 y 850 mg de colesterol, por lo cual estudiamos su efecto sobre las lipoproteínas plasmáticas y la viscosidad sanguínea en pacientes con hiperlipoproteinemias primas (clasificados según la OMS) y/o B-VLDL elevada, que se definió por la relación col-VLDL/TG > 0,35, beta ancha en el lipidograma electroforético y test de precipitación con heparina-MgCl2-NaCl. El grupo A, compuesto por pacientes de tipos IIa (n = 4), IIb (4), IV (2) y B-VLDL elevada, 1) recibió 50 ml/día de AHB. El grupo B, compuesto por pacientes por tipo IIa (4), IIb (9), IV (4), B-VLDL elevada (5) y V (2) recibió 25 ml/día. Un mes antes del tratameitno y durante éste dieta de los pacientes era baja en hidratos de carbono refinados, con menos de 300 mg de colesterol al día y supresión de etanol. en las muestras basales y a los 21 días de tratamiento se midieron TG, Col-en VLDL, LDL, HDL2 y HDL3, apo A1 y apo B, viscosidades sanguíneas, sérica y plasmática. En el grupo A disminuyeron TG (p < 0,05), pero col-VLDL/TG aumentó (p < 0,01) y apreció B-VLDL en 6 de 11 pacientes. En el grupo B, bajaron TG y col-VLDL (p < 0,02 y p < 0,05, respectivamente) pero los 5 pacientes con B-VLDL basal no mejoraron. Apo A1 aumentó (p < 0,05). El col-LDL no disminuyó en níngún grupo. El AHB no tiene efecto en pacientes con B-VLDL basal o hiperlipémicos de tipos IIa o IIb, en cambio los pacientes de tipo IV mejoraran. El AHB disminuye la viscosidad sanguínea (p < 0,05) pero no afecta la plasmática (AU)

Glucomannan-synthase activity in differentiating cells of Pinus sylvestris L.

Particulate membrane preparations have been isolated from cambial cells, and from differentiating and differentiated xylem cells of the main stem of pine trees. These preparations synthesise a ß1→4 glucomannan from guanosine 5'-diphosphate-mannose. The polysaccharide and the synthase have been characterized and the Km and Vmax for the synthase determined as 85 µM and 52.9 µM·min(-1), respectively. The enzymic activity was inhibited by the addition of guanosine 5'-diphosphate-D-glucose so that the presence of an epimerase on the particulate fraction in conjunction with the synthase probably allowed the heteropolymer to be formed with the optimal ratio of the concentrations of the nucleoside-diphosphate sugar donors. No evidence for a polyprenyl-phosphate derivative as an intermediate during the polymer synthesis was obtained. Part of the control mechanism for the deposition of the large amounts of the glucomannan during the secondary thickening of the tracheids of the vascular system is by an increase in the amount of synthase activity at the endomembrane system of the cells. This probably occurs by an increase in the amount of enzyme which is modulated by gene regulation during differentiation.

Xylan synthetase activity in differentiated xylem cells of sycamore trees (Acer pseudoplatanus).

Particulate enzymic preparations obtained from homogenates of differentiated xylem cells isolated from sycamore trees, catalyzed the formation of a radioactive xylan in the presence of UDP-D-[U-(14)C]xylose as substrate. The synthesized xylan was not dialyzable through Visking cellophane tubing. Successive extraction with cold water, hot water and 5% NaOH dissolved respectively 15, 5 and 80% of the radioactive polymer. Complete acid hydrolysis of the water-insoluble polysaccharide synthesized from UDP-D-[U-(14)C]xylose released all the radioactivity as xylose. ß-1,4-Xylodextrins, degree of polymerization 2, 3, 4, 5 and 6, were obtained by partial acid hydrolysis (fuming HCl or 0.1 M HCl) of radioactive xylan. The polymer was hydrolysed to xylose, xylobiose and xylotriose by Driselase which contains 1,4-ß xylanase activities. Methylation and then hydrolysis of the xylan released two methylated sugars which were identified as di-O-methyl[(14)C]xylose and tri-O-methyl-[(14)C]xylose, suggesting a 1→4-linked polymer. The linkage was confirmed by periodate oxidation studies. The apparent Km value of the synthetase for UDP-D-xylose was 0.4 mM. Xylan synthetase activity was not potentiated in the presence of a detergent. The enzymic activity was stimulated by Mg(2+) and Mn(2+) ions, although EDTA in the range of concentrations between 0.01 and 1 mM did not affect the reaction rate. It appears that the xylan synthetase system associated with membranes obtained from differentiated xylem cells of sycamore trees may serve for catalyzing the in vivo synthesis of the xylan main chain during the biogenesis of the plant cell wall.

Increase of xylan synthetase activity during xylem differentiation of the vascular cambium of sycamore and poplar trees.

The activity of a ß-(1-4)-xylan synthetase, a membrane-bound enzymic system, was measured in particulate enzymic preparations (1,000 g and 1,000-100,000 g pellets) obtained from homogenates of cambial cells, differentiating xylem cells and differentiated xylem cells isolated from actively growing trees of sycamore (Acer pseudoplatamus) and poplar (Populus robusta). The specific activity (nmol of xylan formed min(-1) mg(-1) of protein) as well as the activity calculated on a per cell basis (nmol of xylan formed min(-1) cell(-1)) of this enzymic system, markedly increased as cells differentiate from the vascular cambium to xylem. This increase is closely correlated with the enhanced deposition of xylan occurring during the formation of secondary thickening. The possible control of xylan synthesis during the biogenesis of plant cell wall is discussed.

Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in sycamore and poplar.

During the transition from primary wall formation to secondary thickening there is a marked shift in the synthesis of pectin, hemicellulose and cellulose. The activities of the enzymes [UDP-D-galactose 4-epimerase (EC 5.1.3.2)8 UDP-l-arabinose 4-epimerase (EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D--glucuronate decarboxylase (EC 4.1.1.35)] were measured in cambial cells, differentiating xylem cells and differentiated xylem cells isolated from sycamore and poplar trees, and phloem cells from poplar. At the final stage of the differentiation of cambium to xylem there was a decrease in activity of the enzymes directly involved in producing the soluble precursors of pectin (DUP-D-galactose 4-epimerase and UDP-L-arabinose 4-epimerase and an increase in those producing the precursors of hemicellulose (UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase). These results strongly suggest ahat the changes were correlated with the differences observed in the chemical composition of the wall during development. The changes found in the catalytic activity of the enzymes of nucleoside diphosphate sugar interconversion exert a coarse control over the synthesis of pectin and hemicelluloses. The tissues at all stages of development contained the necessary enzyme activities to produce all the precursors of pectin and hemicellulose, even at the final stage of differentiation when no pectin was formed.

Changes in enzymic activities of nucleoside diphosphate sugar interconversions during differentiation of cambium to xylem in pine and fir.

A protein fraction [precipitate obtained between 40 and 65% (NH4)2SO4 satn.] prepared from cambial cells, differentiating xylem cells and differentiated xylem cells of pine and fir trees contained all the enzymes required for the nucleoside diphosphate sugar interconversions. By using UDP-D-[U-14C]glucose or UDP-D-[U-14C]galactose, UDP-D-[U-14C-]glucuronic acid and UDP-D-[U-14C]xylose as substrates, the activities of UDP-D-galactose 4-epimerase (DC 5.1.3.2), UDP-D-xylose 4-epimerase(EC 5.1.3.5), UDP-D-glucose dehydrogenase (EC 1.1.1.22) and UDP-D-glucuronate 4-epimerase (EC5.1.3.6), UDP-d-glucuronate decarboxylase (EC 4.1.1.35) were measured at different stages of cell-wall development. The specific activities and the activities per cell of these enzymes varied during differentiation of cambium to xylem according to the type polysaccharide synthesized. Variations were also found between the two species investigated. These data, compared with those obtained in out previous work on angiosperms [see the preceding paper, Dalessandro & Northcote (1977) Biochem. J. 162, 267-279], suggest that some control of polysaccharide synthesis operates at the level of the formation of the precursors of pectin and hemicellulose syntheses.

Possible control sites of polysaccharide synthesis during cell growth and wall expansion of pea seedlings (Pisum sativum L.).

The activities of the enzymes of uridine diphosphate sugar interconversions (UDP-D-glucose 4-epimerase, UDP-D-glucuronate 4-epimerase, UDP-D-xylose 4-epimerase, UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase) were measured by using enzymic preparations (protein precipitated between 40-65% (NH4)2SO4 saturation) isolated from segments at different stages of elongation of the third internode of pea seedlings. All enzymic activities increased from dividing and non-elongated cells to fully elongated cells. At all stages of growth, the specific activity or the activity per cell of UDP-D-glucose dehydrogenase was much lower than that of UDP-D-glucuronate decarboxylase and this may represent a controlling step in the formation of UDP-D-xylose. During elongation, changes were also found in the activities of the epimerases. These could be correlated with the corresponding variations which occur in the chemical structure and physical properties of pectins during cell wall extension. However, the high levels of the epimerases present in cells which have completed elongation growth suggest that pectin synthesis is mainly controlled at the sites of the synthetase reactions.