Induction by hormones of phenylalanine ammonia-lyase in bean-cell suspension cultures. Inhibition of superinduction by actinomycin D.
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Ammonia-Lyases↗
Biomedical subjects
D H Northcote
Publications and source records attributed to D H Northcote.
Partial characterization of the polyisoprenoid carrier of N-acetylglucosamine in Glycine max (soya bean).
Dolichyl phosphate (C55) and dolichyl phosphate prepared from liver were incubated with an enzyme prepared from soya-bean protoplasts. They both stimulated the transfer of radioactivity from UDP-D-glucose to lipid, but the stimulation was greater with liver dolichyl phosphate. Liver dolichyl phosphate with the soya-bean enzyme stimulated the transfer of radioactivity from UDP-N-acetyl-D-[U-14C]glucosamine to acidic lipid. UDP-D-[U-14C]glucose and UDP-N-acetyl-D-[U-14C]glucosamine were used with the soya-bean enzyme to prepare the glycosylated-acid-lipid acceptor of each sugar. Mild acid hydrolysis revealed that the radioactivity in the lipid glucosylated from UDP-glucose was present exclusively as glucose. That in the lipid glycosylated from UDP-N-acetylglucosamine was present mostly as N-acetylglucosamine. The soya-bean acidic-lipid acceptors of glucose and N-acetylglucosamine were stable to both catalytic hydrogenation and treatment with hot aqueous phenol; they behaved in a similar way as authentic alpha-saturated glucosylated polyisoprenyl phosphates. The soya-bean acidic-lipid acceptors of glucose and N-acetylglucosamine were eluted as deoxycholate complexes from a Sephadex column. In comparison with glucosylated polyisoprenyl phosphates of known size, the patterns of elution indicated that both soya-bean lipids contained a polyisoprenoid chain of 18 isoprene units.
Acetylglucosamine↗
Glucosylation of phosphorylpolyisoprenol and sterol at the plasma membrane of soya-bean (Glycine max) protoplasts.
Protoplasts were prepared from cells of soya-bean (Glycine max) suspension cultures and the plasma membrane was labelled with diazotized [G-3H]sulphanilic acid. Homogenates were fractionated by differential and isopycnic centrifugation, and membrane fractions in a density gradient were characterized by enzymic markers and the radioactive label. When fractions containing a large amount of protein were incubated with UDP-[U-14C]glucose, radioactive material soluble in chloroform/methanol was formed and this separated into acidic and neutral fractions on ion-exchange chromatograms of DEAE-cellulose. The acidic fraction was shown to consist of dolichol phosphate glucose, and the neutral fraction sterol glucosides and acylsterol glucosides. Optimum conditions for glucosylation of dolichol phosphate were established as 5 mM-MgCl2, pH 6.0, and the enzyme had a Michaelis constant of 1.5 x 10(-5) m-UDP-glucose. Optimum conditions for glucosylation of sterol were 5 mM-MgCl2, pH 8.0 GDP-[U-14C]glucose was a poor substrate for the synthesis of both acidic and neutral lipids. Although the synthesis of dolichol phosphate glucose and sterol glucosides occurred throughout the sucrose gradient, the specific activities of both glucosyltransferases were greatest in a fraction coincident with the radioactively labelled plasma membrane. Results are discussed in relation to the likely role fo these transglucosylase activities.
Carbohydrates↗
A reliable method for obtaining matched replicas of freeze-fractured cell suspensions.
A method that overcomes the majority of practical problems involved in performing matched freeze-fracture replication of cell suspensions is described. Specimens are rapidly frozen between copper support plates. The prior attachment of gold grids to the plates using Formvar ensures the exact alignment of corresponding grid squares and the production of a central fissure running through the specimen upon fracturing. Grids, with replicas permanently attached, are removed from the support plates by dissolving the Formvar. Sample digestion and rinsing of replicas is by a gentle procedure employing glass capillaries as a means of handling the grids. Matched replication of plant and animal cells has been achieved by this methods. Apart from the excellent preservation of the membrane ultrastructure of unfixed cells and the capacity for matching both membrane halves this technique also enhances the fracture characteristics of certain specimens, notably isolated protoplasts, providing larger fracture faces than are obtained by the knife-splintering method.
Cell Membrane↗
Plasma membrane ultrastructure during plant protoplast plasmolysis, isolation and wall regeneration: a freeze-fracture study.
The freeze-fracture morphology of the plasma membrane of cells and isolated protoplasts of plant callus suspensions has been investigated. Plasmolysis of suspension cells leads to the formation of 2 types of hexagonal arrays of intramembrane particles situated on the inner fracture face (PF). These arrays are interpreted as proteins that have 'crystallized' in the plane of the membrane as the area of surrounding lipid bilayer is reduced during protoplast retraction from the cell wall. Time-course studies have revealed no positive relationship between the distribution of hexagonal arrays and the occurrence of microfibrils regenerated around isolated protoplasts during periods of culture. No evidence for the specialized transport functions attributed to hexagonal arrays of plant cells by previous workers has been found.
Cell Membrane↗
Isolation and characterization of membranes from the cells of maize root tips.
A discontinuous sucrose density gradient was used to separate membrane fractions from a homogenate of maize root tips. Endoplasmic reticulum-, Golgi apparatus-, plasma membrane- and mitochondria-rich fractions were identified by their enzymic characteristics and by their appearance under the electron microscope. Maize roots were incubated in vivo with D-[U-14C]glucose, [Me-14C]choline chloride and diazotized [U-3H]sulphanilic acid. The pattern of incorporation of radioactivity into the various membrane fractions was investigated. Analyses of the polypeptide chains of the membrane fractions by SDS-polyacrylamide gel electrophoresis showed that the mitochondria-rich fraction had a different pattern of polypeptides from that of the other membrane fractions. The results are discussed in relation to the hypothesis of endomembrane flow and differentiation.
Amino Acids↗
Measurement and characteristics of fusion of isolated membrane fractions from maize root tips.
Maize root tips were incubated in vivo with radioactive glucose, choline or diazotized sulphanilic acid. Membrane fractions were prepared from radioactive and non-radioactive roots. The transfer of radioactivity between mixed membrane fractions has enabled a quantitative system to be developed to study in vitro membrane fusion between Golgi apparatus and plasma membrane-rich fractions. Membrane fusion was found to be dependent on time, temperature, Mn2+ and Ca2+. Mn2+ was as effective as Ca2+, other divalent cations had a moderate or no effect. The effect of various substances, including inhibitors of microtubular and microfilament assembly and blocking reagents for sulphydryl group on membrane fusion has been investigated. The process appears to be dependent on the membrane proteins or glycoproteins; trypsinization of mixed membranes prior to the addition of Ca2+ inhibited significantly the fusion process. SDS-polyacrylamide gel electrophoresis of trypsin-treated membranes revealed the selective loss of one particular polypeptide which could play a role in membrane fusion.
Acid Phosphatase↗
Polyprenyl phosphate sugars synthesized during slime-polysaccharide production by membranes of the root-cap cells of maize (Zea mays).
Two types of experiments were carried out; either maize roots were incubated in L-[1-3H]fucose or membranes were prepared from root tips and these were incubated with GDP-L-[U-14C]fucose or UDP-D-[U-4C]glucose. The radioactively labelled lipids that were synthesized in vivo and in vitro were extracted and separated into polar and neutral components. The polar lipids had the characteristics of polyprenyl phosphate and diphosphate fucose or glucose derivatives, and the neutral lipids of sterol glycosides (fucose or glucose). A partial separation of the glycolipid synthetase reactions was achieved. Membranes were fractionated into material that sedimented at 20,000g and 100,000g. Most of the polar glycolipid synthetase activity (for the incorporation of both fucose and glucose) was located in the 100,000 g pellet, and this activity was probably located in the endoplasmic reticulum. The neutral lipid, which contained fucose, was synthesized mainly by membranes of the 20,000g pellet, and the activity was probably associated with the dictyosomes, whereas the neutral glucolipids were synthesized by all the membrane fractions. It is suggested that the polar (polyprenyl) lipids labelled with fucose could act as possible intermediates during the synthesis of the glycoproteins and slime in the root tip.
Cell Membrane↗
The involvement of the Golgi apparatus in the biosynthesis and secretion of glycoproteins and polysaccharides.
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Animals↗
The loss of morphogenetic potential and induction of phenylalanine ammonia-lyase in suspension cultures of Phaseolus vulgaris.
The loss of morphogenetic potential in bean suspension cultures has been investigated by measuring the amounts of phenylalanine ammonia-lyase activity induced in the cells when they are transferred from a medium in which they are grown and maintained to an induction medium. The tissue has been grown in 2 types of medium: (1) supplemented with 2,4-dichlorophenoxyacetic acid as the only growth hormone, and (2) supplemented with 2,4-dichlorophenoxyacetic acid and coconut milk. When cells were grown in medium with only 2,4-dichlorophenoxyacetic acid for a period of 5--10 subcultures and samples were transferred to the induction medium at intervals during the subcultures, the amounts of phenylalanine ammonia-lyase activity and the number of xylem elements induced progressively declined. Cells grown in the presence of coconut milk did not lose the ability to induce phenylalanine ammonia-lyase or xylem elements. Cells grown in the presence of coconut milk were cloned and clones capable of producing different amounts of phenylalanine ammonia-lyase when transferred to induction medium were obtained. However, clones producing low amounts of activity did not grow faster in the medium lacking coconut milk and no evidence was obtained to show that selective growth of non-inducible cells was responsible for the loss of morphogenetic potential. In addition to the induction brought about by the presence of naphthylacetic acid and kinetin in the induction medium the cells could also be stimulated to produce phenylalanine ammonia-lyase activity by dilution at subculture. This increase in activity occurred within 10 h of the dilution, whereas that produced by the hormones in the induction medium occurred after 120 h. The induction produced by dilution also occurred in these cells which had lost their ability to respond to the hormonal induction. Thus the mechanism that produced the increase in phenylalanine ammonia-lyase activity was intact but had lost its ability to respond to the hormones of the induction medium. The loss of inducibility was therefore probably not due to a genetic change in the cells brought about by continuous growth in a medium lacking coconut milk, but to reversible changes in the hormonal requirements of the cells necessary for induction.
2,4-Dichlorophenoxyacetic Acid↗
Location of fucosyl transferases in the membrane system of maize root cells.
There are two fucosyl transferase activities present within the endomembranes of the cells of maize root-tips. One transfers fucose to polyprenyl phosphate and occurs in the endoplasmic reticulum, the second transfers fucose probably to polysaccharide or glycoprotein. In order to show an association of this second fucosyl transferase activity with the endoplasmic reticulum as well as the Golgi apparatus, a method of fractionating the membranes in a discontinuous sucrose gradient was used. Membranes were prepared in the presence of Mg2+, which maintained the attachment of ribosomes to the endoplasmic reticulum, and also in the presence of EDTA, which removed most of the ribosome complex. This caused a shift in density of these membranes. Two types of experiments were carried out; either maize roots were incubated in L-[1-3H]fucose and then membranes prepared and the amount of polymer synthesized in vivo determined or isolated membranes were incubated with GDP-L-[U-14C]fucose in vitro and the amount of polymer synthesized was found. The results showed that the Golgi apparatus had the highest amount of this fucosyl transferase activity, but there was a significant amount of activity associated with the endoplasmic reticulum and the latter was shifted in the sucrose gradient depending on the conditions used.
Endoplasmic Reticulum↗
A protein-glucan intermediate during paramylon synthesis.
A sodium deoxycholate extract containing glucosyltransferase activity was obtained from a particulate preparation from Euglena gracilis. It transferred glucose from UDP-[14C]glucose into material that was precipitated by trichloroacetic acid. This material released beta-(1 leads to 3)-glucan oligosaccharides into solution on incubation with weak acid, weak alkali and beta-(1 leads to 3)-glucosidase. The products of the incubation of the deoxycholate extract with UDP-[14C]glucose were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioactive bands were obtained that had the properties of beta-(1 leads to 3)-glucan covalently linked to protein by a bond labile to weak acid. High-molecular-weight material containing a beta-(1 leads to 3)-glucan was also shown to be present by gel filtration. The bond linking glucan to aglycone is possibly a pyrophosphate linkage. It is proposed that in Euglena gracilis beta-(1 leads to 3)-glucan (paramylon) is synthesized on a protein primer.
Animals↗
The structure and function of glycoproteins synthesized during slime-polysaccharide production by membranes of the root-cap cells of maize (Zea mays).
The synthesis of the maize root slime polysaccharides was investigated by using [1-3H]-fucose as a marker for slime production. Three fractions were separated by centrifugation in a CsCl density gradient. Two of these were glycoproteins and occurred within the membranes of the cells of the root tip; the third was the slime polysaccharides. Radioactive pulse-chase experiments showed that the glycoproteins were precursors of the slime polysaccharides, and the carbohydrate portion of the glycoproteins had a similar composition to that of the free slime. The linkage between the protein and the carbohydrate of one of the glycoproteins was shown to be a xylose-threonine bond. It is postulated that the slime polysaccharides are synthesized and transported on proteins within the membrane system of the root tip.
Cell Membrane↗
Influence of cations at the plasma membrane in controlling polysaccharide secretion from sycamore suspension cells.
1. Three soluble polysaccharides and a soluble protein containing hydroxyproline were secreted by sycamore suspension cultures. l-[1-(3)H]Fucose was incorporated solely into the fucose of fucoxyloglucan and l-[1-(14)C]arabinose mainly into the arabinose of arabino-galactan. [U-(14)C]Glucose was a general precursor for soluble protein and polysaccharides. 2. The steady-state rate of secretion of all the polymers was increased within seconds of adding various electrolytes and polyelectrolytes to the growth medium. The increased secretion was induced by cations at the outer surface of the plasma membrane. It was brought about by a stimulation of the normal mechanisms of cell-wall polysaccharide secretion. It was partly inhibited by anaerobiosis or sodium arsenate and was unaffected by temperature changes in the range 0-35 degrees C. 3. The precursor pool from which secretion was induced contained completely synthesized polysaccharides and was probably located in the Golgi-derived vesicles. The results indicated that the endoplasmic reticulum did not secrete polysaccharide directly to the cell exterior. 4. The various cations probably induced secretion by causing a depolarization of the negative electric potential of the cell surface, and this resulted in the fusion of vesicles with the plasma membrane. 5. Analogy with exocytosis and pinocytosis in various animal tissues suggested that the decreased surface potential brought about membrane fusion by causing an increase in plasma-membrane permeability to Ca(2+). 6. The results showed that the fusion of vesicles with the plasma membrane was rate-limiting and a potential control point. Auxin-stimulated cell-wall deposition could be a result of a stimulated influx of Ca(2+) causing vesicle fusion with the plasma membrane.
Calcium Chloride↗
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.
Arabinose↗
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.
Carbohydrate Epimerases↗
An ultrastructural study of the relationship between the plasma membrane and the cell wall of the coenocytic alga hydrodictyon africanum.
A wall/plasma membrane unit was prepared from Hydrodictyon africanum by microdissection. A replica of the inner surface of the membrane was made and by freeze-fracture of the whole cell, 2 corresponding internal fracture faces were obtained. The large coenocytes of the alga were plasmolysed and the wall separated by cutting it away. Its inner surface was directly viewed in the electron microscope after shadowing with Pt/C. Particles were found on the outer half of the internal fracture face of the membrane which were oriented in the same 2 directions as the microfibrils laid down at the inner surface of the wall. No structures were found at the inner surface of the membrane. Some evidence was obtained for a structural connexion between the innermost layers of the wall and the plasma membrane.
Cell Membrane↗
The isolation of plasma membrane from protoplasts of soybean suspension cultures.
A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1-14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.
Cell Fractionation↗