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.

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.

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.

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.

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).

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.

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.

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.

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 × 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.

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.

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.

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.

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°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.

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.

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.

A plasma-membrane fraction was isolated from the alga Hydrodictyon africanum by micro-dissection and its phospholipid components were analysed. Phosphatidylcholine was the major phospholipid of the preparation. Both phosphatidylserine and diphosphatidylglycerol were enriched in the fraction compared with the whole cell, but the relative amount of phosphatidylglycerol present was less than that in the whole cell. Phosphatidylinositol was absent from the plasma-membrane preparation.

Cell-wall enzymes were assayed by the difference between enzyme activities in the whole cell and the protoplast. Both peroxidase (85.2%) and acid phosphatase (21.9%) were located in the wall. However, malate dehydrogenase was found only in the protoplast. A study of the time-course of the release of peroxidase and malate dehydrogenase into the incubation medium from cells either treated with cellulase or untreated, also indicated that peroxidase and not malate dehydrogenase was located in the wall. Only two anodic isoenzymes of peroxidase were present in the cell wall. These were more negatively charged than those of horseradish peroxidase.

1. Endosperm extracts were prepared at various times during germination from intact castor-bean seeds and from seeds from which the embryos had been removed. The sterilized seeds were incubated either on solid water agar or on agar containing 0.3 mM-gibberellic acid. 2. Isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase had very low activities in the mature seeds, but increased 44-fold and 27-fold respectively during germination. In contrast, the extracts of mature seeds had considerable acid and alkaline lipase activity and this only increased two- to three-fold during the incubation period. 3. Incubation of the seeds with gibberellic acid accelerated the rate of appearance of isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase. It also increased the total activity attained. However, the application of hormone had, in comparison, little effect on the development of lipase activity. 4. The removal of the embryo had little influence on the development of enzyme activity in the endosperm tissue; only with isocitrate lyase was a decrease in activity observed in the absence of the embryo.

1. Discs of tobacco leaf lamina were floated on media containing D-[U-14C]glucose. Glucose uptake was mainly through the cut edges, and diffusion through the veins and intracellular spaces was slow. 2. Radioactivity was detected in all polysaccharide fractions extracted from the discs, including those associated with the wall. 3. Plasmolysis in sorbitol or KCL decreased the incorporation of radioactive material into all fractions. 4. Incorporation of arabinose into pectins was increased or unaffected by plasmolysis, but the incorporations of other sugars were decreased. Removal of the lower epidermis did not affect this result. 5. Seperate mechanisms for arabinan and polygalacturonorhamman syntheses must exist, and these must differ in their responses to the physiochemical, structural and organizational changes that accompany plasmolysis.

1. Lipid extracts were obtained from castor-bean endosperm tissue at various times during germination and, after purification, the total lipid content was determined. Quantitative measurements of the triglyceride and phospholipid content together with the fatty acid composition were made. 2. The total lipid content of the endosperm rapidly decreased during germination; after 10 days less than 20% of the original weight of lipid remained. In contrast, the phospholipid content (initially less than 0.5% of the total lipid) increased slightly during this time. The fatty acid composition and the relative proportions of the triglyceride species of the total lipid extract remained constant during 10 days of germination. 3. Gibberellic acid (0.3 mM) markedly stimulated the rate of lipid breakdown but did not alter either the fatty acid composition or the relative proportion of triglyceride species. 4. The embryo had little effect on lipid metabolism in the endosperm tissue; only after 6 days of germination were differences observed in the rate of fat utilization in the presence and absence of the embryo.

Membrane fractions were obtained from peas roots by using a method that permitted the isolation of a fraction rich in relatively intact dictyosome stacks. No chemical fixatives were used. The method involved incubation of the roots with cellulase, followed by gentle homogenization and sucrose-density-gradient fractionation of the homogenate. The fractions were characterized by electron microscopy. All fractions were enzymically active in incorporating glucose from UDP-glucose into water-insoluble glycolipids containing both single glucose residues and glucose oligosaccharides. Some or all of the linkages of glucose to lipid were through phosphate esters. A substance containing glucose oligosaccharides attached to or very strongly adsorbed on to protein was also formed. The membrane fractions also incorporated glucose from UDP-glucose into alkali-soluble and alkali-insoluble β-glucans, which like the oligosaccharides contained β(1leads to 3) and β-(1leads to4) linkages. The distribution of the enzymic activities and the chemical properties of the lipid-linked and protein-linked oligosaccharides suggest that they may be intermediates in β-glucan synthesis. The synthetic activity is associated with smooth-membrane vesicles which may be derived from the plasma membrane.

1. Maize seedling roots were incubated in vivo with d -[U- 14 C]glucose for 2, 5, 10, 15, 30 and 45min. The total incorporation of radioactivity into polysaccharide components in isolated fractions was investigated, and the pattern of incorporation into different polysaccharide components within the rough endoplasmic reticulum, Golgi apparatus and exported material was analysed. 2. The membrane compartments reached a saturation value of radioactivity in polysaccharide components by 30min incubation. Radioactivity in exported polysaccharide continued to increase after that time. The latter was formed and maintained by a steady-state turnover of polysaccharide synthesis and transport from the membrane system. 3. If the only access of the slime polysaccharide to the cell surface is via dictyosome-derived vesicles, the amount of slime components in the Golgi apparatus would have to be displaced every 0.3min in order to maintain the observed rates of increase in slime. This is in contrast with a displacement time of about 2.5min that is necessary for polysaccharide components in the Golgi apparatus to produce the observed increase in cell-wall material. The activity of the membrane system in the production of maize root slime is 8 times as great as that of the membrane system involved in cell-wall synthesis. 4. If the amount of polysaccharide material in the Golgi apparatus is maintained only by inflow of polymeric material from the rough endoplasmic reticulum the total amount of slime components in the rough endoplasmic reticulum would have to be displaced every 7min to maintain a constant amount in the Golgi apparatus. If the endoplasmic reticulum contributed directly to the cell surface in the synthesis of cell-wall material, displacement times necessary to maintain the observed rate of polymer production would be very slow.

1. The patterns of incorporation of radioactivity from d -[U- 14 C]glucose into the pectic components of sections of sycamore roots changed so that sections nearer the tip incorporated relatively more label into arabinose and galactose compared with uronic acid. 2. Radioactive maize root-cap slime was prepared and found to contain three water-soluble component polymers which were electrophoretically (i) neutral, (ii) weakly acidic and (iii) strongly acidic at pH6.5. The neutral component was a glucan. The other components, which could be degraded by trans -elimination, consisted of an acidic backbone chain composed of galacturonic acid and glucose, attached to which were different proportions of neutral sugars. Arabinose, galactose and fucose, the main neutral sugars of the weakly and strongly acidic materials, were absent from the neutral fraction. 3. Fucose was a major sugar in maize-root slime and in a slime of similar composition synthesized by a maize callus of shoot origin. Only trace amounts were found in sycamore, pea and wheat root tips, and in pectin prepared from maize roots and coleoptiles. A high proportion of fucose is therefore a chemical characteristic of maize slime, and slime synthesis indicated a state of differentiation of the tissue. 4. The similarity between the slime and pectin is discussed; slime is a form of pectin modified in such a way as to provide a hydrated protective coating around the root tip.

Degradation of the sycamore cell wall by hydrazinolysis yielded soluble glycopeptides. The peptide portion of the predominant glycopeptide was shown to be cyclic (4- trans -hydroxy- l -prolyl)-4- trans -hydroxy- l -proline. From this structure and the composition of the major glycopeptide it was deduced that only short oligosaccharides are attached to the hydroxyprolyl residues of the wall glycoprotein. Detailed evidence for the structure of the peptide is given in a supplementary paper that has been deposited as Supplementary Publication SUP 50020, at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131 , 5.

1. Subcellular fractionation of maize roots resulted in the isolation of the following enriched fractions: cell wall, dictyosome, smooth-membrane and rough-microsomal fractions. In addition, extracellular polysaccharide of the root slime was isolated. 2. Maizeseedling roots were incubated in vivo with d -[U- 14 C]glucose, and the pattern of incorporation of radioactivity into the polysaccharides of each fraction was investigated. 3. The differentiation of maize-root cells with respect to the synthesis of specific extracellular polysaccharide directly relates to the polysaccharide synthesized and transported within the membrane system of the cell. A fucose-containing polysaccharide, characteristic only of root slime, was present only in the membrane system of the root-tip region of the root. Regions of typical secondary wall development within the root were characterized by an increased incorporation of radioactivity into xylose of polysaccharide within the membrane system. 4. The incorporation of radioactivity into glucan polymers in the membrane fractions was very low in all regions of the root. Since in regions of secondary wall development greater than 60% of all radioactive incorporation was into a glucan polymer, it can be inferred that this polymer, most probably cellulose, is not synthesized or transported within the compartments of the membrane system. It is suggested that synthesis of cellulose occurs at the surface of the plasmalemma. 5. Maize-root cells contained 40 times more rough endoplasmic reticulum than dictyosome membrane. The relative specific radioactivities of each fraction indicated that polysaccharide was concentrated in the region of the Golgi apparatus, which showed a 100% increase in specific radioactivity compared with the rough endoplasmic reticulum. The Golgi apparatus can thus be regarded as a localized focal point on the synthetic and transport system of polysaccharide by the intracellular membrane compartments.

1. A glycoprotein containing a large amount of hydroxyproline is present in the cell walls of sycamore callus cells. This protein is insoluble and remained in the α-cellulose when a mild separation procedure was used to obtain the polysaccharide fractions of the wall. The glycoprotein contained a high proportion of arabinose and galactose. 2. Soluble glycopeptides were prepared from the α-cellulose fraction when peptide bonds were broken by hydrazinolysis. The soluble material was fractionated by gel filtration and one glycopeptide was further purified by electrophoresis; it had a composition of 10% hydroxyproline, 35% arabinose and 55% galactose, and each hydroxyproline residue carried a glycosyl radical so that the oligosaccharides on the glycopeptide had an average degree of polymerization of 9. 3. The extraction of the glycopeptides was achieved without cleavage of glycosyl bonds, so that the glycoprotein cannot act as a covalent cross-link between the major polysaccharides of the wall. 4. The wall protein approximates in conformation to polyhydroxyproline and therefore it probably has similar physicochemical properties to polyhydroxyproline. This is discussed in relation to the function of the glycoprotein and its effect on the physical and chemical nature of the wall.

1. A study of caffeine biosynthesis has been made by following the incorporation of radioactive carbon dioxide and methionine into the methylated purines produced by tea callus tissue. 2. The uptake of the radioactive labels into nucleic acid and caffeine was followed over a period of approximately 9h. 3. The distribution of the radioactive labels in both nucleic acid and caffeine was determined after incorporation and subsequent incubation of the tissue in a non-radioactive medium. 4. The results of the experiments indicated that the caffeine arose from purines released from the breakdown of nucleic acids rather than that it was formed directly from a purine pool. 5. A metabolic scheme to show the production of caffeine from the nucleotides of the nucleic acid is discussed.

1. A study of wall synthesis has been made by following the incorporation of radioactive glucose and threonine into the cytoplasm and wall of yeast. 2. Both glucose and threonine are incorporated into a mannan glycopeptide. The glucose is also synthesized into a structural glucan of the wall. 3. The mannan glycopeptide contains high-molecular-weight mannan and low-molecular-weight mannose and oligosaccharide units composed of mannose. Both types of carbohydrate are attached to the peptide. The extent of radioactive incorporation into these different carbohydrate constituents of the glycopeptide remained constant during a pulse-chase experiment. No evidence of a sequential synthesis of oligosaccharides and high-molecular-weight mannan was obtained. 4. Cycloheximide inhibits the incorporation of threonine into the wall but only partially inhibits the incorporation of glucose. Thus not all the polysaccharide deposited into the wall is dependent on a simultaneous peptide synthesis and incorporation. 5. Protoplasts grown in an iso-osmotic medium secreted a mannan polymer that was probably a glycopeptide.

1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a β-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the β-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O -mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the β-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O -mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia . This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate–amino acid linkages found in the glycoprotein.

A method is described for the detection and estimation of radioactive polysaccharides separated by zone electrophoresis on glass-fibre paper.

1. d -Glucose and l -arabinose serve as precursors of the pectic polysaccharides of sycamore suspension-callus tissue. 2. The rates and characteristics of the incorporation of radioactive sucrose, glucose and meso inositol by sycamore callus tissue have been compared and shown to be different. 3. The time-course of the incorporation of radioactive glucose into the major fractions within the cells has been determined. Approx. 7–10% of the radioactivity incorporated is present in the whole pectin of the cells. 4. A study of the continuous incorporation of radioactive glucose showed that the neutral arabinan–galactan fraction of the pectin quickly became saturated with the radioactive label. During the incorporation of radioactivity from a pulse of radioactive glucose the neutral fraction became progressively less labelled, with a corresponding increase in the radioactivity of the weakly acidic pectinic acid, which is known to contain neutral sugars. 5. When the cells were exposed to a pulse of radioactive l -arabinose, the label accumulated first in the neutral fraction and then after 4hr. it passed to the weakly acidic pectinic acid with a corresponding decrease in the radioactivity of the neutral fraction. 6. The product that was initially labelled during the first hour of exposure of the cells in the stationary phase to radioactive glucose was identified as an incompletely methylated galacturonan in which the radioactivity was present in the anhydrogalacturonide residues. This polysaccharide probably acts as the precursor of the polyuronide portions of both the strongly acidic and weakly acidic pectinic acids. 7. The observations are discussed in relation to the structure of the pectic substances and their function in cell growth and development. A tentative model for their metabolic relationship is put forward.