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Biomedical subjects

D H Northcote

Publications and source records attributed to D H Northcote.

At least 19 recordsLinked to original sources
PubMed1993

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.

Acetyl Coenzyme A↗
PubMed1990

Spatial and temporal patterns of transcription of a wound-induced gene in potato.

Transcriptional fusions between the gene encoding win2 from potato and the reporter gene encoding beta-glucuronidase (GUS) have been used to study the spatial and temporal patterns of wound induced gene activity in transgenic potato and tobacco plants. Gene fusions containing a full length win2 promoter were found to be correctly regulated in response to mechanical wounding in transgenic potato, but not in the heterologous host, tobacco. Sequences greater than 560 bp upstream of the transcription start site of win2 were shown to be important for wound inducibility. The dramatic induction of GUS activity detected using fluorometric assays of extracts of wounded and aged leaves of several independent win2--GUS transformants was consistent with the kinetics of win2 mRNA accumulation. Histochemical analysis of wounded leaves showed that transcription first occurred in cells immediately adjacent to the wound, and was then progressively induced in cells associated with the vascular system at a distance from the wound site. In tubers, a localized response to wounding was observed, and this only spread to other parts of the tuber if it had started to sprout. It was concluded that active vascular transport was necessary for the spread of wound response. Win2--GUS fusions were also expressed as part of normal plant development, as GUS activity was detected in the developing buds and in a layer of cells associated with the lenticels of unwounded tubers.

Cloning, Molecular↗
PubMed1989

The mucilage secreted by roots and its possible role in cell-cell recognition for the adhesion of fungal pathogens to root surfaces of Zea mays L.

The outer cells of the roots of plants secrete a mucilage which lubricates the root and keeps it moist. The mucilage is secreted from the Golgi apparatus in vesicles which fuse at the plasma membrane. In maize roots a complex of at least three polysaccharides and glycoproteins are formed, some of which have a large proportion of fucose in their composition. The synthesis of these compounds can be readily monitored because fucose can be easily identified, and especially because exogenous fucose is not catabolized but is incorporated intact into the polymers. The synthesis of the polymers seems to be initiated in the endoplasmic reticulum in conjunction with polyprenoid oligosaccharides that contain fucose. Lipid-oligosaccharides of nine sugar residues can be obtained from the membrane preparations of the root cells. These compounds are polyprenyl diphosphate derivatives. A GDP-fucose:polyprenyl phosphate transfucosylase occurs in the endoplasmic reticulum, whereas fucosyl transferase that transfers fucose to a polymer occurs mainly in the Golgi apparatus. The indirect evidence suggests that oligosaccharides of polyprenyl diphosphate compounds are transferred to proteins, elaborated in the Golgi apparatus, and large molecular weight polysaccharides are finally exported as the mucus. Part of the mucus is acidic and in some respects resembles pectin. The presence of fucose in such large quantities in maize root mucilage suggested that this might have some significance for the recognition of these plants by parasitic root fungi. The adsorption of mucilage by pathogenic fungi was investigated with two types of fungi, a highly specialized ectotrophic root-infecting fungus, e.g. Phialophora radicicola and a vascular wilt fungus capable of attacking a great variety of tissues, e.g. Fusarium moniliforme. The adsorption of radioactively labelled and fluorescently labelled polymers by the pathogenic fungi was investigated. The character and proportion of fungal surfaces present in vitro were standardised by the production and semi-synchronous germination of populations of conidia. Changes in appearance of fungal walls, present before and after germination, were examined ultrastructurally. There was polyanionic material on hyphal but less on conidial surfaces of the ectotrophic root-infecting fungi. In contrast this material was present to similar extents on both hyphal and conidial surfaces of F. moniliforme.(ABSTRACT TRUNCATED AT 400 WORDS)

Adhesives↗
PubMed1986

Cell-cell recognition of host surfaces by pathogens. The adsorption of maize (Zea mays) root mucilage by surfaces of pathogenic fungi.

The adsorption of radioactive mucilage by pathogenic fungi was shown to be dependent upon time, the composition of mucilage, the type of fungal surface (conidia, hyphae, hyphal apices), fungal species, pH and bivalent cations. All fungal adhesins were inactivated by either proteinase or polysaccharase treatments. Adsorption was not inhibited by the numberous mono-, di- and oligo-saccharides that were tested individually, but it was inhibited absolutely by several polysaccharides. This suggested that adsorption of mucilage by pathogens involved conformational and ionic interactions between plant and fungal polymers but not fungal lectins bound to sugar residues of mucilage. Several fractionation schemes showed that pathogens bound only the most acidic of the variety of polymers that comprise mucilage. There was not any absolute distinction between ability to bind radioactive mucilage and type of pathogen or non-pathogen. However, there were notable differences in characteristics of adsorption between two types of pathogen. Differences were revealed by comparison of the adsorption capacities of conidia and germinant conidia and chromatography of radioactive mucilage on germinant conidia. An ectotrophic root-infecting fungus (a highly specialized pathogen) bound a greater proportion of mucilage than did a vascular-wilt fungus (of catholic host and tissue range) with more than one class of site for adsorption. In contrast with the vascular-wilt fungus, sites for adsorption on the specialized pathogen were present solely on surfaces formed by germination.

Adsorption↗
PubMed1985

In vitro glucan synthesis by membranes of celery petioles: the role of the membrane in determining the type of linkage formed.

Glucan synthesis was achieved with an in vitro membrane fraction from the petioles of celery (Apium graveolens). The optimum conditions for maximum synthesis were established. The Km and Vmax for the enzymic system were 1.0 mM and 0.19 microM min-1 mg protein-1, respectively. Mechanical damage to the membrane fraction altered the proportion of beta-(1----3) to beta-(1----4) glucosyl linkages that were synthesized. We suggest that cellulose synthesis (beta-(1----4)-linked glucan chains) is controlled by the availability of UDP-glucose at the plasma membrane surface in conjunction with an organized relationship between the synthase system and a specifically oriented glucosyl radical acting as an acceptor held on the membrane surface. An intact membrane is therefore necessary to direct synthesis for the beta-(1----4) bond by an enzyme that is capable of transglucosylation to the secondary alcoholic groups on C-2, C-3 or C-4 of the acceptor radical. The specificity of the system is controlled by the whole enzyme complex held on the membrane.

Carbohydrate Conformation↗
PubMed1984

Stability of the complex formed between French bean (Phaseolus vulgaris) phenylalanine ammonia-lyase and its transition-state analog.

Phenylalanine ammonia-lyase forms trans-cinnamate from L-phenylalanine, and thus stands at a gateway to secondary metabolism in higher plants. L-alpha-Amino-oxy-beta-phenylpropanoic acid (L-AOPP), a very effective competitive inhibitor of this enzyme, is most probably a transition-state analog for the elimination reaction. A preparation of phenylalanine ammonia-lyase (PAL), obtained from diluted suspension cultures of French bean cells, was used to investigate the binding of this compound in vitro. After extensive dialysis, the inhibitor remained tightly bound to the enzyme unless both an increased temperature and L-phenylalanine were provided, when the spectrophotometer trace of enzyme activity gradually approached linearity. Under such optimal catalytic conditions (37 degrees C; 25 mM L-phenylalanine; pH 8.8), dissociation of the enzyme-ligand complex took place with a half-time of approx 10 min. (This is much longer than reported for the enzyme from maize.) The consequences of these findings are discussed for investigations where L-AOPP is applied in vivo. These experiments have shown that the irreversible binding of the transition-state analog under appropriate conditions (0-4 degrees C, no L-phenylalanine) gave continued protection against attack on the enzyme by an excess of borohydride. By titrating the enzyme with increasing concentrations of analog and measuring the degree of protection afforded, the active-site concentration has been estimated. The turnover number (kcat = 0.8 s-1) given by this novel approach is of the same order of magnitude as previously reported from extensive purification of enzyme from other species.

Ammonia-Lyases↗
PubMed1983

Subunit structure and interactions of the phloem proteins of Cucurbita maxima (pumpkin).

The two major proteins from the phloem exudate of Cucurbita maxima (pumpkin), PP1 and PP2, were stable in the absence of reducing agents after modification of their accessible cysteine residues with iodoacetamide. This permitted their purification without precautions to prevent oxidation. PP2, a lectin specific for oligomers of N-acetyl-D-glucosamine, was shown by sedimentation-equilibrium ultracentrifugation to be a dimer of Mr of 48000. Neither dithiothreitol nor tri-(N-acetyl-D-glucosamine) altered this value. The constituent polypeptides were linked by two buried disulphide bridges. PP2 behaved aberrantly on gel-filtration on both Sephadex and Bio-Gel unless tri-(N-acetyl-D-glucosamine) was added to the elution buffer; the Mr was then measured as 46000. Other proteins which bind oligomers of N-acetyl-D-glucosamine are also retarded on gel-filtration. Soluble phloem filaments were prepared by collection of exudate into deaerated buffer containing iodoacetamide but no reducing agent. Oxidative gellation of the filaments was prevented by rapid modification of their many accessible cysteine residues, and is assumed to have maintained the degree of polymerisation found in vivo. Those disulphide bridges which were present allowed the incorporation of approximately 60% of the PP1 and 80% of the PP2 into polymeric material. It is concluded that PP1 and PP2 are both structural proteins present in the filaments observable in vivo. PP2 had an elongated binding-site for oligomers of N-acetyl-D-glucosamine. It is suggested that this lectin immobilises bacteria and fungi to the cross-linked filaments which seal wounded phloem sieve-tubes, and thus maintains sterility.

Chemical Phenomena↗
PubMed1983

Changes in the activity of acetyl-CoA carboxylase during rape-seed formation.

During the formation of rape-seeds, lipid accumulated in the cotyledons from 16 days after pollination, rising to a plateau after 28 days. The accumulation of lipid was preceded by a marked rise in acetyl-CoA carboxylase activity, which declined rapidly, correlating with the decline in rate of lipid formation. Incubation of rape-seed extracts with avidin-agarose resulted in a decrease in acetyl-CoA carboxylase activity in the extract. Polyacrylamide-gel electrophoresis of polypeptides bound to avidin-agarose showed the presence of a polypeptide of Mr 225 000. The intensity of this band increased during the period of increase of acetyl-CoA carboxylase activity in the seeds.

Acetyl-CoA Carboxylase↗
PubMed1983

Arabinan synthase and xylan synthase activities of Phaseolus vulgaris. Subcellular localization and possible mechanism of action.

Membrane fractions from bean hypocotyl or suspension cultures incorporated arabinose from UDP-beta-L-arabinose into arabinan and xylose from UDP-alpha-D-xylose in vitro; the level of each activity was dependent on the state of differentiation of the cells. These activities may be due to single transglycosylases, since no lipid or proteinaceous intermediate acceptors were found in either case. Subcellular fractionation studies showed that enzyme activity in vitro was localized in both Golgi-derived membranes and endoplasmic reticulum in similar amounts. However, incorporation into the polymers in vivo in suspension culture cells incubated with [1-3H]arabinose was considerably greater in the Golgi-derived membranes. Thus, although these enzymes may be translated and inserted at the level of the endoplasmic reticulum, their activities are under other levels of control, so that most of the activity in vivo is confined to the Golgi apparatus. Initiation of glycosylation in the endoplasmic activity may, however, occur.

Arabinose↗
PubMed1983

Induction by growth factors of polysaccharide synthases in bean cell suspension cultures.

Suspension cells of bean subcultured into medium that maintains the culture and stimulates cell division but not differentiation brings about an increase in arabinan synthase activity. Subculture into a medium that induces both cell division and xylogenesis brings about in addition an increase in xylan synthase. Both synthases are membrane-bound and are concerned with the formation of neutral pectin or hemicellulose of the cell wall respectively. During the rising phase of the induction of these activities in the appropriate culture medium, the increases in activities were inhibited by either actinomycin D (an inhibitor of transcription) or D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide (an inhibitor of translation). Thus the control for the induction of the enzyme activities involves transcription and possibly translation. Subculture of the cells brought about an increase, probably non-specific, in total membrane-bound translation, as indicated by increased amounts of bound polysomes and incorporation of [35S]methionine into membrane proteins. If the control of the appearance of specific mRNA molecules is partially effected by growth factors then these are probably operative during the period of the cell cycle that is stimulated by subculture and it is probably at this time that the growth factors act to bring about the changes necessary for differentiation.

Cell Division↗
PubMed1983

Sugar-nucleotide precursors of arabinopyranosyl, arabinofuranosyl, and xylopyranosyl residues in spinach polysaccharides.

Cultured spinach (Spinacia oleracea L. cv Monstrous Viroflay) cells incorporated exogenous l-[(3)H]arabinose sequentially into beta-l-arabinopyranose-1-phosphate, uridine diphospho-beta-l-arabinopyranose, uridine diphospho-alpha-d-xylopyranose and (in some experiments) alpha-d-xylopyranose-1-phosphate. The amount of (3)H in each of these compounds reached a plateau after a few minutes, and could be rapidly chased with nonradioactive l-arabinose, demonstrating rapid turnover. After a few minutes' lag, incorporation of (3)H into the arabinofuranosyl, arabinopyranosyl, and xylopyranosyl residues of polysaccharides was linear with respect to time. The kinetics of labeling were compatible with UDP-beta-l-arabinopyranose and UDP-alpha-d-xylopyranose being the immediate precursors of arabians (both the pyranose and the furanose residues) and xylans, respectively. No other radioactive nucleotides were formed; in particular, UDP-arabinofuranose was absent. There was no evidence for conversion of arabinopyranose to arabinofuranose within the polysaccharides, suggesting that this conversion occurs during polymer synthesis. The glycolipids detected showed too slow a turnover to be intermediates of pentosan synthesis.

Journal Article↗
PubMed1982

The use of acetyl-CoA carboxylase activity and changes in wall composition as measures of embryogenesis in tissue cultures of oil palm (Elaeis guineensis).

With some lines of oil-palm tissue cultures embryogenesis occurs spontaneously within the callus grown on a medium containing 2.5 mg of 3-naphthylacetic acid/litre. One of the initial biochemical events that occurs just before the embryoid can be seen is the accumulation of fat droplets within the cells. This accumulation of lipid is correlated with an increase in acetyl-CoA carboxylase activity. The carboxylase is thus probably a rate-limiting step in fatty acid synthesis in these cells and can be used as a quantitative marker of somatic embryogenesis within the tissue. During the development of the embryoid tissue there is an increase in cell division and the differentiation of vascular cells with secondary thickened walls. These stages of the differentiation may be monitored by measuring the ratio of pectin synthesis (polygalacturonic acid formation) to hemicellulose synthesis (xylan formation).

Acetyl-CoA Carboxylase↗
PubMed1981

The location of arabinosyl:hydroxyproline transferase in the membrane system of potato tissue culture cells.

Incubation of a particulate preparation from potato tissue culture cells with UDP-beta-L-[1-3H] arabinose yielded a glycoprotein fraction containing labelled material with the characteristics of hydroxyproline arabinosides. The sugar-protein linkage was resistant to hot alkaline hydrolysis, and the hydrolytic products showed similar electrophoretic and chromatographic behavior to authentic hydroxyproline-arabinosides prepared from potato tissue culture cell walls. Incorporation of arabinose into glycoprotein was stimulated by the addition of de-arabinosylated potato lectin. The product of the incubation co-migrated with native potato lectin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The subcellular distribution of the arabinosyl-transferase was investigated by fractionating potato tissue culture membranes on a discontinuous sucrose gradient in the presence or absence of Mg2+. Under both fractionation conditions the highest specific activity of the enzyme was found in the Golgi-enriched fraction. The results are discussed in relation to the synthesis of the hydroxy-proline-rich glycoprotein component of plant cell walls.

Arabinose↗
PubMed1981

The extraction from maize (Zea mays) root cells of membrane-bound protein with Ca2+-dependent ATPase activity and its possible role in membrane fusion in vitro.

Membrane fusion in vitro between Golgi apparatus- and plasma-membrane-rich fractions isolated from maize (Zea mays) roots was found to be dependent on Ca2+ and the membrane proteins. Trypsin treatment of mixed membrane fractions before the addition of Ca2+ inhibited their ability to fuse. It resulted also in a selective and progressive elimination of a characteristic intense polypeptide band (B1) on gel electrophoresis. This polypeptide was not removed by chymotrypsin or thermolysin. B1 is an integral membrane protein with an exposed portion to the outside. Sodium deoxycholate was used to solubilize the proteins of mixed membrane fractions. Extracted proteins analysed by non-SDS (sodium dodecyl sulphate) polyacrylamide-gel electrophoresis revealed the presence of four isolated bands. When re-electrophoresed in the presence of SDS, one of these bands exhibited the same mobility as polypeptide B1. Enzymic staining of non-SDS-polyacrylamide gels showed that this protein has Ca2+- and Mg2+-dependent ATPase activity. Its possible role in membrane fusion is discussed.

Amino Acids↗