Measurements of GnE/GnM from the 2H(e-->,en-->)1H Reaction to Q2=1.45 (GeV/c)2.

We report new measurements of the ratio of the electric form factor to the magnetic form factor of the neutron, G(n)(E)/G(n)(M), obtained via recoil polarimetry from the quasielastic 2H(e-->,e(')n-->)1H reaction at Q2 values of 0.45, 1.13, and 1.45 (GeV/c)(2) with relative statistical uncertainties of 7.6% and 8.4% at the two higher Q2 points, which points have never been achieved in polarization measurements.

Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions.

Polypeptide assemblies cross-linked by S-S bonds (molecular mass>200 kDa) and single polypeptides folded with internal S-S cross-links (<41 kDa) have been detected by SDS/PAGE in particulate membranes and soluble extracts of developing cotyledons of nasturtium (Tropaeolum majus L.). When first prepared from fruit homogenates, these polypeptides were found to bind reversibly to UDP-Gal (labelled with [(14)C]Gal or [(3)H]uridine), and to co-precipitate specifically with added xyloglucan from solutions made with 67% ethanol. Initially, the bound UDP-[(14)C]Gal could be replaced (bumped) by adding excess UDP, or exchanged (chased) with UDP-Gal, -Glc, -Man or -Xyl. However, this capacity for turnover was lost during incubation in reaction media, or during SDS/PAGE under reducing conditions, even as the glycone moiety was conserved by autoglycosylation to form a stable 41 kDa polypeptide. Polyclonal antibodies raised to a similar product purified from Arabidopsis bound to all the labelled nasturtium polypeptides in immunoblotting tests. The antibodies also inhibited the binding of nasturtium polypeptides to UDP-Gal, the uptake of UDP-[(14)C]Gal into intact nasturtium membrane vesicles and the incorporation of [(14)C]Gal into nascent xyloglucan within these vesicles. This is the first direct evidence that these polypeptides facilitate the channelling of UDP-activated sugars from the cytoplasm through Golgi vesicle membranes to lumenal sites, where they can be used as substrates for glycosyltransferases to synthesize products such as xyloglucan.

Recurrence and survival after surgical management of rectal cancer.

BACKGROUND: Reported local recurrence rates for rectal cancer are significantly reduced using a combination of superior surgical technique, in the form of total mesorectal excision, and routine radiotherapy. In an attempt to determine the effectiveness of current local management strategies, a review of Vancouver Island Cancer Centre patients with rectal cancer was performed and the overall local recurrence rate was identified. METHODS: We retrospectively reviewed the charts of 272 rectal cancer patients from 1988 to 1998. Two hundred and twenty-nine patients met inclusion criteria. Analysis of patient factors included age, gender, type of surgery, and adjuvant therapy. Tumors were assessed for level, stage, and grade. Local recurrence and distant metastases were also documented. Variables influencing local recurrence in this group were identified and disease-free and actuarial survival determined. RESULTS: Of 229 patients analyzed, 12.7% (29) had local recurrences. Variables influencing local recurrence were number of positive lymph nodes, vascular invasion, and neural invasion. There was no significant difference in local recurrence between patients having anterior resection and those having abdominoperineal resection. None of the patients who received preoperative radiotherapy had a local recurrence. Actuarial disease-free survival was 87% at 5 years. CONCLUSIONS: Limiting local recurrence is one of the most important goals in the treatment of rectal cancer. It is essential to identify those patients with "high risk" tumors as identified by endorectal ultrasound or pathologic features. These patients comprise the group most likely to benefit from a routine mesorectal excision combined with adjuvant radiotherapy.

Fucosyltransferase and the biosynthesis of storage and structural xyloglucan in developing nasturtium fruits

Young, developing fruits of nasturtium (Tropaeolum majus L.) accumulate large deposits of nonfucosylated xyloglucan (XG) in periplasmic spaces of cotyledon cells. This "storage" XG can be fucosylated by a nasturtium transferase in vitro, but this does not happen in vivo, even as a transitory signal for secretion. The only XG that is clearly fucosylated in these fruits is the structural fraction (approximately 1% total) that is bound to cellulose in growing primary walls. The two fucosylated subunits that are formed in vitro are identical to those found in structural XG in vivo. The yield of XG-fucosyltransferase activity from membrane fractions is highest per unit fresh weight in the youngest fruits, especially in dissected cotyledons, but declines when storage XG is forming. A block appears to develop in the secretory machinery of young cotyledon cells between sites that galactosylate and those that fucosylate nascent XG. After extensive galactosylation, XG traffic is diverted to the periplasm without fucosylation. The primary walls buried beneath accretions of storage XG eventually swell and lose cohesion, probably because they continue to extend without incorporating components such as fucosylated XG that are needed to maintain wall integrity.

Xyloglucan galactosyl- and fucosyltransferase activities from pea epicotyl microsomes.

Microsomal membranes from growing tissue of pea (Pisum sativum L.) epicotyls were incubated with the substrate UDP-[14C]galactose (Gal) with or without tamarind seed xyloglucan (XG) as a potential galactosyl acceptor. Added tamarind seed XG enhanced incorporation of [14C]Gal into high-molecular-weight products (eluted from columns of Sepharose CL-6B in the void volume) that were trichloroacetic acid-soluble but insoluble in 67% ethanol. These products were hydrolyzed by cellulase to fragments comparable in size to XG subunit oligosaccharides. XG-dependent galactosyltransferase activity could be solubilized, along with XG fucosyltransferase, by the detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. When this enzyme was incubated with tamarind (Tamarindus indica L.) seed XG or nasturtium (Tropaeolum majus L.) seed XG that had been partially degalactosylated with an XG-specific beta-galactosidase, the rates of Gal transfer increased and fucose transfer decreased compared with controls with native XG. The reaction products were hydrolyzed by cellulase to 14C fragments that were analyzed by gel-filtration and high-performance liquid chromatography fractionation with pulsed amperometric detection. The major components were XG subunits, namely one of the two possible monogalactosyl octasaccharides (-XXLG-) and digalactosyl nonasaccharide (-XLLG-), whether the predominant octasaccharide in the acceptor was XXLG (as in tamarind seed XG) or XLXG (as in nasturtium seed XG). It is concluded that the first xylosylglucose from the reducing end of the subunits was the Gal acceptor locus preferred by the solubilized pea transferase. These observations are incorporated into a model for the biosynthesis of cell wall XGs.

Xyloglucan sidechains modulate binding to cellulose during in vitro binding assays as predicted by conformational dynamics simulations.

Cross-links between cellulose microfibrils and xyloglucan (XG) molecules play a major role in defining the structural properties of plant cell walls and the regulation of growth and development of dicotyledonous plants. How these cross-links are established and how they are regulated has yet to be determined. In a previous study, preliminary data were presented which suggested that the different sidechains of XG may play a role in controlling cellulose microfibril-XG interactions. In this study, this question is addressed directly by analyzing to what extent the different sidechains of pea cell wall XG and nasturtium seed storage XG affect their binding to cellulose microfibrils. Of particular importance to this study are the chemical data indicating that pea XG possesses a trisaccharide sidechain, which is not found in nasturtium XG. To this end, conformational dynamic simulations have been used to predict whether oligosaccharides representative of pea and nasturtium XG can adopt a hypothesized cellulose-binding conformation and which of these XGs exhibits a preferential ability to bind cellulose. Extensive analysis of the conformational forms populated during 300 K and high-temperature Monte Carlo simulations established that a planar, sterically accessible, glucan backbone is essential for optimal cellulose-binding. For the trisaccharide sidechain-containing oligosaccharide as found in pea XG, sidechain orientation appeared to regulate the gradual acquisition of this hypothesized cellulose binding conformation. Thus, conformational forms were identified that included the twisted backbone (non-planar) putative solution form of XG, forms in which the trisaccharide sidechain orientation enables increased backbone planarity and steric accessibility, and finally a planar, sterically accessible, backbone. By applying these conformational requirements for cellulose binding, it has been determined that pea XG possesses a two- to threefold occurrence of the cellulose binding conformation than nasturtium XG. Based on this finding, it was predicted that pea XG would bind to cellulose at a higher rate than nasturtium XG. In vitro binding assays showed that pea XG-avicel binding does indeed occur at a twofold higher rate than nasturtium XG-avicel binding. The enhanced ability of pea cell wall XG over nasturtium seed storage XG to associate with cellulose is consistent with a structural role of the former during epicotyl growth where efficient association with cellulose is a requirement. In contrast, the relatively low ability of nasturtium XG to bind cellulose is consistent with the need to enhance the accessibility of this polymer to glycanases during germination. These findings suggest potential roles for XG sidechain substitution, enabling XG to function in a variety of different biological contexts.

Endo-1,4-[beta]-Glucanase, Xyloglucanase, and Xyloglucan Endo-Transglycosylase Activities Versus Potential Substrates in Ripening Tomatoes.

In ripening fruits of tomato (Lycopersicon esculentum L. var 83-G-38), the amounts of cellulose and xyloglucan (XG) remained constant during tissue softening, but the relative molecular weight (Mr) of XG decreased markedly and the Mr of cellulose declined slightly. These changes could have been due to activities of non-specific endo-1,4-[beta]-glucanases and/or buffer-soluble XG endo-transglycosylase, both of which increased when tissue firmness declined most rapidly. Tomato extracts also reduced the viscosity of XG solutions, especially in the presence of added XG oligosac-charides. This depolymerizing (XGase) capacity differed from [beta]-glucanase and XG transglycosylase activity (a) by being almost entirely buffer insoluble, and (b) by declining precipitously during fruit softening. Although it disappeared from ripe fruit, XGase may have functioned in promoting wall loosening at earlier stages of fruit development when its activity was highest. By contrast, during aging of fruit in the ripening-inhibited mutant rin there was no change in Mr of XG or cellulose, and activities of [beta]-glucanases and XG transglycosylase were lower than in wild-type tomato. Nevertheless, some softening of the fruit did take place over time and XG amounts declined, possibly because high XGase activity was maintained in the mutant, unlike in wild-type fruit.

Cleavage of xyloglucan by nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunit oligosaccharides.

Oligosaccharide subunits were prepared from xyloglucan (XG) by partial hydrolysis with cellulase and added back at micro- to millimolar concentrations to XG in the presence of nasturtium seed xyloglucanase (XG-ase). The oligosaccharides (0.2 mM) stimulated the capacity of this XG-ase to reduce the viscosity of XG solutions by 10- to 20-fold. Purification and fractionation of seed XG-ase activity by gel permeation fast protein liquid chromatography produced a single peak that was much more active in the presence than absence of added XG oligosaccharide. [14C]Fucose-labeled XG nonasaccharide was synthesized by pea fucosyltransferase and shown to be incorporated into polymeric XG in the presence of seed XG-ase without the net production of new reducing chain ends, even while the loss of XG viscosity and XG depolymerization were enhanced. It is concluded that in vitro seed XG-ase can transfer cleavage products of XG to XG oligosaccharides via endotransglycosylation reactions, thereby reducing XG M(r) without hydrolysis. Since this is the only XG-cleaving enzyme that develops in nasturtium seeds during germination, it may be that its transglycosylase and hydrolase capacities are both necessary to account for the rapid and complete depolymerization of XG that takes place.

Acceptor requirements for GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase activity solubilized from pea epicotyl membranes.

GDP-fucose:xyloglucan (XG) fucosyltransferase from growing Pisum epicotyl tissue was solubilized in detergent and used to examine the capacity of intact XG from Tamarindus seeds, and its partial hydrolysis products, to act as fucose acceptors with GDP-[14C]fucose as donor. Native seed XG (Mr greater than 10(6) Da) was partially depolymerized by incubation with Trichoderma cellulase for various periods of time. Cellulase was inactivated and reaction mixtures were incubated with GDP-[14C]fucose plus solubilized pea fucosyltransferase and then fractionated on columns of Sepharose CL-6B or Bio-Gel P4. Specific activities (Bq/microgram carbohydrate) of fragments with Mr ranging from 10(6) to 10(4) Da were constant throughout the size ranges, indicating that all stretches of the XG chains were available for fucosylation. More complete cellulase hydrolysis yielded subunit oligosaccharides that chromatographed in a cluster of hepta-, octa-, and nonasaccharides, none of which acted as fucosyl acceptors when incubated with pea fucosyltransferase. However, a substantial amount (up to half of hydrolysate) of larger transient oligosaccharides was also formed with a size equivalent to three of the oligosaccharide subunits. Octasaccharide subunits in this trimer were readily fucosylated. This fucosyltransfer was inhibited by uncombined (free) subunit oligosaccharides, which implies that the latter could bind to the transferase and displace at least part of the trimer, even though they could not themselves be fucosylated. Reduction of the trimer oligosaccharide with NaB3H4, followed by further hydrolysis with cellulase, resulted in tritiated nonasaccharide and unlabeled octasaccharide in a concentration ratio of 1:2. The tamarind XG trimer which accepts fucose is therefore composed mainly of the subunit sequence: octa-octa-nonasaccharide (reducing). One of the terminal oligosaccharide subunits in this trimer, probably the nonasaccharide, appears to be required as a recognition (binding) site in fucosyltransferase in order for adjacent octasaccharide(s) to be fucosylated by the active (catalytic) enzyme site.

Solubilization and properties of GDP-fucose: xyloglucan 1,2-alpha-L-fucosyltransferase from pea epicotyl membranes.

GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase from pea (Pisum sativum) epicotyl microsomal membranes was readily solubilized by extraction with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). When using GDP-[14C]fucose as fucosyl donor and tamarind xyloglucan (XG) as acceptor, maximum activation was observed at 0.3% (w/v) Chaps and the highest yield of solubilized activity at 0.4%. The reaction product was hydrolyzed by Trichoderma cellulase to yield labeled oligosaccharides that peaked on gel permeation chromatography at the same elution volume as pea XG nona- and decasaccharide subunits. The apparent Km for fucosyl transfer to tamarind XG by the membrane-bound or solubilized enzyme was about 80 microM GDP-fucose. This was 10 times the apparent Km for fucosyl transfer to endogenous pea nascent XG. Optimum activity was between pH 6 and 7, and the isoelectric point was close to pH 4.8. The solubilized enzyme showed no requirement for, or stimulation by, added cations or phospholipids, and was stable for several months at -70 degrees C. Solubilization and gel permeation chromatography on columns of Sepharose CL-6B enriched the specific activity of the enzyme by about 20-fold relative to microsomes. Activity fractionated on columns of CL-6B with an apparent molecular weight of 150 kDa. The solubilized fucosyltransferase was electrophoresed on nondenaturing polyacrylamide slab gels containing 0.02% (w/v) tamarind XG, and its activity located by incubation in GDP-[14C]fucose, washing, and autoradiographing the gel. A single band of labeled reaction product appeared with an apparent molecular weight of 150 kDa.

Xyloglucan oligosaccharide alpha-L-fucosidase activity from growing pea stems and germinating nasturtium seeds.

[14C]Fucose-labelled xyloglucan (XG) was synthesized from tamarind seed XG by incubating it with GDP-[14C]fucose plus solubilized pea fucosyltransferase, and [14C]fucose-labelled XG nonasaccharide was prepared from the parent hemicellulose by partial hydrolysis with fungal cellulase. alpha-L-Fucosidase activity was readily detected in crude enzyme extracts of growing regions of etiolated pea stems (Pisum sativum) and in cotyledons of germinating nasturtium seedlings (Tropaeolum majus) using the fucosylated XG-nonasaccharide as substrate. Both enzymes showed little activity against intact fucosylated XG and they were totally inactive against p-nitrophenyl-alpha-L-fucoside. Auxin treatment of pea stems, which greatly increased the activity of endo-1,4-beta-glucanases that hydrolyse XG in apical growing regions, failed to result in a similar increase in XG-nonasaccharide alpha-fucosidase activity. However, germination of nasturtium seed, which resulted in a large increase in endo-1,4-beta-glucanase (XG-ase) activity in the cotyledons, was accompanied by comparable increases in XG-alpha-fucosidase activity.

Auxin-dependent breakdown of xyloglucan in cotyledons of germinating nasturtium seeds.

Rapid mobilisation of storage products, including xyloglucan, in cotyledons of germinating nasturtium (Tropaeolum majus L.) normally starts about 7-8 d after imbibition and growth of the seedling at 20-25° C. Levels of activity of endo-1,4-ß-glucanase (EC 3.2.1.4) in cotyledons, as assayed viscometrically with xyloglucan as substrate, varied in parallel with the rate of breakdown of xyloglucan. When cotyledons were excised from the seedling axis and incubated on moist filter paper at any point before 7 d, the catabolic reactions which normally occurred in the intact seedling were suspended. If, however, cotyledons excised at 8 d were incubated in 10(-6) M 2,4-dichlorophenoxyacetic acid, a rise in endo-1,4-ß-glucanase (xyloglucanase) activity was observed and a sharp decrease in fresh and dry weight as well as xyloglucan levels ensued at rates comparable to those observed in cotyledons attached to the seedling. Neither gibberellin nor kinetin treatments promoted xyloglucan breakdown or enhanced xyloglucanase activity. Addition of auxin to excised cotyledons before 7 d did not evoke premature breakdown, indicating that the tissue became receptive to auxin only at this time. The triggering process took place in darkness and was unaffected by various light-dark cycles. It is concluded that the sudden degradation of xyloglucan which occurs in nasturtium seeds about a week after germination begins is the result of enhanced activity of a depolymerizing xyloglucanase, this activity being evoked by auxin originating in the emerging seedling axis.

Influence of cyclosporin A and alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis, on two rodent T-cell cancers in vivo.

We have examined the influence of cyclosporin A (CsA), administered together with the polyamine antimetabolite, alpha-difluoromethylornithine (DFMO), on growth of the Roser acute T-cell leukaemia in PVG rats and on growth of the EL4 lymphoma in C57BL/6 mice. CsA or DFMO alone, administered from the time of tumour injection, markedly reduced numbers of circulating lymphoblasts in leukaemic rats, although survival was prolonged only in those animals given DFMO. Drug combination further reduced blood-borne tumour cells, but had no additional effects on tumour growth within organs or on host survival, compared to that achieved with DFMO treatment alone. Neither CsA nor DFMO, administered from the time of tumour-cell injection, nor both drugs in combination, affected peritoneal growth of the EL4 lymphoma or organ infiltration. Host survival was prolonged by DFMO. As anticipated, DFMO inhibited polyamine synthesis in vivo, but the observed anti-tumour effect of CsA was not accompanied by an alteration in polyamine biosynthesis. By reducing polyamine synthesis, however, DFMO may enhance the vulnerability of those malignant T cells which are susceptible to the as yet unexplained selective inhibitory action of CsA in vivo.

Incorporation of UDP-[C]Glucose into Xyloglucan by Pea Membranes.

The water-insoluble 1,4-beta-linked products formed from UDP-[(14)C]glucose by pea membranes were dissolved in hot dimethyl-sulfoxide/paraformaldehyde and fractionated on columns of controlled pore glass beads calibrated with dextran standards. The products eluted with a peak size close to 70 kilodaltons in dextran equivalents. Similar elution profiles were obtained for products formed in brief or extended incubations and at high or low substrate concentrations. Methylation analysis indicated that only a few [(14)C]glucose units had been added to an endogenous acceptor to form this product. In the presence of UDP-xylose at concentrations equal to or less than UDP-[(14)C]glucose, incorporation from the latter was enhanced and the products elongated with time to a size range where the major components eluted between dextran 264 and 500 kilodaltons. Treatment with endo-1,4-beta-glucanase resulted in a mixture of oligosaccharides, including the xyloglucan subunit Glc(4)Xyl(3), which were hydrolyzed further by mixed glycosidases to labeled glucose and isoprimeverose (xylosyl-1,6-alpha-d-glucose). In pulse-chase experiments, the low molecular weight product formed from UDP-[(14)C]glucose alone was clearly a precursor for high molecular weight products formed subsequently in the presence of both UDP-glucose and UDP-xylose. It is concluded that the 1,4-beta-transglucosylation activity detected in these tests was due to an enzyme that is required for biosynthesis of the backbone of xyloglucan.

Phosphatases and phosphodiesterases interfere with 1,3-beta-D-glucan synthase activity in pea epicotyl membrane preparations.

Soluble and membrane-bound phosphatase and phosphodiesterase activities are present in preparations of 1,3-beta-D-glucan synthase from pea epicotyls. UDP-glucose phosphodiesterase and non-specific alkaline phosphatase could be partially inhibited by N-ethylmaleimide or iodoacetamide and partially removed from membranes by washing. Such treatments helped to prolong 1,3-beta-glucan synthase activity. Nevertheless, the 1,3-beta-D-glucan synthase activity in washed membranes still gradually decreased during incubation in buffer at 30 degrees C. The rate of decay was reduced by adding more specific phosphatase inhibitors, e.g. molybdate, vanadate or fluoride, or by addition of nucleotides, and much of the loss of 1,3-beta-D-glucan synthase activity during preincubation could be restored by addition of phosphatidylethanolamine to the assay mixtures. It is concluded that membrane phospholipid is an essential part of the environment of 1,3-beta-glucan synthase and must be maintained intact in order for the enzyme to remain fully active.

Fucosylation of exogenous xyloglucans by pea microsomal membranes.

Microsomal membrane preparations from growing regions of etiolated pea stems catalyzed the transfer of [14C]fucosyl units from GDP-[U-14C]-L-fucose into exogenously added xyloglucan acceptors, as well as into endogenous xyloglucan. The transfer was more effective using nonfucosylated tamarind seed xyloglucan than with pea wall xyloglucan in which almost all galactose units are already fucosylated. Hydrolysis of products by endo-1,4-beta-D-glucanase yielded in each case radioactive nonasaccharide as the main fucosylated product. UDP-galactose enhanced the fucosylation of endogenous primer but it had little effect on fucosyl transfer to exogenously added xyloglucans. Low-molecular-weight nonfucosylated oligosaccharide fragments up to the octasaccharide Glc4Xyl3Gal (obtained by endoglucanase action on tamarind seed xyloglucan) were ineffectual as fucosyl acceptors but inhibited the fucosylation of endogenous as well as of added xyloglucan. With octasaccharide, the inhibition was competitive in relation to the xyloglucan acceptor (Ki = 70 microM) and noncompetitive in relation to the donor GDP-fucose (Ki = 210 microM). It is concluded that fucosyltransferase acts independently and in a noncoordinated manner from other glycosyltransferases that are required to synthesize xyloglucan. Its active site recognizes a fragment longer than the galactosylated octasaccharide unit before transfucosylation will ensue.

Modulation of Pea Membrane beta-Glucan Synthase Activity by Calcium, Polycation, Endogenous Protease, and Protease Inhibitor.

beta-Glucan synthase activity in plant membranes can be markedly altered by a multiplicity of apparently unrelated factors. In pea epicotyl membranes it is enhanced by low and inhibited by high concentrations of added Ca(2+), trypsin or soluble pea protease. Ca(2+) stimulates preexisting synthase activity, particularly in the presence of polycations (spermidine), but protease treatments activate and, with time, inactivate synthase zymogen. Endogenous pea protease activity is also associated with washed pea membrane and appears to be responsible for the decay observed with time in the beta-glucan synthase activity. Endogenous pea protease activity is inhibited by thiol inhibitors, e.g. iodoacetamide and Hg(2+), and by a heat-stable peptide, molecular weight approximately 10,000, that is found in supernatants of pea extracts. These protease inhibitors have the capacity to protect beta-glucan synthase activity from denaturation or its zymogen from activation due to endogenous or added protease activity. Evidence is described which supports the proposal that 1,4-beta-glucan synthase is destroyed and possibly converted to 1,3-beta-glucan synthase activity by protease action, and that the latter may then be greatly enhanced by Ca(2+) and polycations.

Glycosyltransfer by pea membranes from sugar nucleotides to added prenyl phosphates.

Pea membranes supplied with GDP-[14C]mannose, UDP-N-[14C]acetylglucosamine or UDP-[14C]glucose catalyze the transfer of 14C-labeled sugars or sugar phosphates to endogenous lipid acceptors as well as to exogenously added dolichyl phosphates. Fully unsaturated polyprenyl phosphates were not used as effective acceptors by this system. Mannosyl-P-dolichol was formed most rapidly in the presence of long-chained dolichyl-P while mannosyl-PP-, glucosyl-PP- and GlcNAc-PP-dolichol were preferentially formed from relatively short-chained dolichyl phosphate acceptors. Glucosyl-PP- and mannosyl-PP-dolichol accumulated in the preparation without further metabolism, but GlcNAc-PP-dolichol was lengthened by addition of a second GlcNAc plus several [14C]mannose units to form an oligosaccharide fraction susceptible to the action of endoglycosidase H. This lipid-linked oligosaccharide could then be glycosylated in the presence of UDP-[14C]glucose to form a longer oligosaccharide. It is concluded that levels of endogenous dolichyl phosphates in pea membranes are rate-limiting for several of the key glycosyltransferases required for oligosaccharide assembly.