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The effect of a high-performance retarding additive in oil well cements was investigated under elevated temperature (165°C) and pressure (1000 psi) conditions via in situ synchrotron-based X-ray diffraction (XRD) and quasielastic neutron scattering (QENS) techniques. Under these temperature and pressure conditions, crystalline calcium silicate hydrates (C–S–H) are formed through the cement hydration process. From in situ XRD experiments, the retardation effect was observed by a change in the rate of the appearance of 11 Å tobermorites as well as a change in the rate of the α-C2SH generation and depletion. QENS analysis revealed that the retardation effect was related to the non-conversion of free water to chemical and constrained water components. A high presence of free water components was attributed to a decrease in 11 Å tobermorites along with slower consumption of the quartz and portlandite phases. Furthermore, QENS results infer that the water molecules experienced confinement in the restricted pore spaces. The retarder inhibited this initial water confinement by slowing the bulk diffusion of free water in the confined region.

Powerful synergies between phosphonate, zinc oxide, and acrylamido-tert-butyl-sulfonate (ATBS) copolymer chemical additives render superior performance in a high-temperature retarder system for oil well grade Portland cement. The phosphonate retarder and ATBS-based retarders establish a two-tiered strength development where amorphous C-S-H converts to crystalline dicalcium silicate hydrate (C2SH) in the first (low compressive strength) tier prior to the reaction of Portlandite with quartz. The three additive retarder system can be tuned with nanosilica to eliminate the two-tiered strength development effect leading to a smooth transition from the cement in the slurry form directly to its highest compressive strength.

Conjugates between proteins and small molecules enable access to a vast chemical space that is not achievable with either type of molecule alone; however, the paucity of specific reactions capable of functionalizing proteins and natural products presents a formidable challenge for preparing conjugates. Here we report a strategy for conjugating electron-rich (hetero)arenes to polypeptides and proteins. Our bioconjugation technique exploits the electrophilic reactivity of an oxidized selenocysteine residue in polypeptides and proteins, and the electron-rich character of certain small molecules to provide bioconjugates in excellent yields under mild conditions. This conjugation chemistry enabled the synthesis of peptide–vancomycin conjugates without the prefunctionalization of vancomycin. These conjugates have an enhanced in vitro potency for resistant Gram-positive and Gram-negative pathogens. Additionally, we show that a 6 kDa affibody protein and a 150 kDa immunoglobulin-G antibody could be modified without diminishing bioactivity. The preparation of conjugates between proteins and small molecules is often challenging and requires several synthetic steps to functionalize each component for conjugation. Now, a conjugation methodology that leverages an electrophilic Se–S bond of selenocysteine to create bioconjugates between polypeptides and complex small molecules has been described.

To combat antimicrobial infections, new active molecules are needed. Antimicrobial peptides, ever abundant in nature, are a fertile starting point to develop new antimicrobial agents but suffer from low stability, low specificity, and off-target toxicity. These drawbacks have limited their development. To overcome some of these limitations, we developed antibody-bactericidal macrocyclic peptide conjugates (ABCs), in which the antibody directs the bioactive macrocyclic peptide to the targeted Gram-negative bacteria. We used cysteine SN Ar chemistry to synthesize and systematically study a library of large (>30-mer) macrocyclic antimicrobial peptides (mAMPs) to discover variants with extended proteolytic stability in human serum and low hemolytic activity while maintaining bioactivity. We then conjugated, by using sortase A, these bioactive variants onto an Escherichia coli targeted monoclonal antibody. We found that these ABCs had minimized hemolytic activity and were able to kill E. coli at nanomolar concentrations. Our findings suggest macrocyclic peptides if fused to antibodies may facilitate the discovery of new agents to treat bacterial infections.

The respiratory syncytial virus (RSV) L protein is a viral RNA-dependent RNA polymerase that contains multiple enzyme activities required for RSV replication. The RSV L inhibitors described in literature are limited by their cytotoxicity or the lack of RSV B subtype coverage. Here, we characterize a new RSV L inhibitor with strong antiviral activity against both RSV A and B subtypes and no detectable cytotoxicity. This compound, AZ-27, was equally active against RSV live viruses and subgenomic replicons and demonstrated advantages over other classes of RSV inhibitors in time-of-addition and cell line dependency studies. Resistance studies identified a dominant mutation in the putative capping enzyme domain of L protein, which conferred strong resistance to the AZ-27 series but not other classes of RSV inhibitors, supporting RSV L protein as the direct target for AZ-27. This novel and broad-spectrum RSV L polymerase inhibitor may pave the way toward an efficacious RSV therapeutic and provide a new tool for interrogation of the L protein function.

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.

Toward a complete LC-MS mapping system for peptides and proteins, we have coupled a precision-flow microbore HPLC system to an electrospray single quadrupole mass spectrometer. The HPLC system allows fast separation of protein digests with UV detection at the low pmol level. A 2 microliters/min portion (1:25) of the effluent is passed into a high-sensitivity electrospray MS system. The electrospray source allows for molecular mass associated ions (MH+, MH2(2+), MH3(3+), etc.) to be generated as well as collision induced dissociation of these ions before MS analysis. After LC-MS runs, with or without partial fragmentation, the data generated are largely interpreted by identification of predicted peptides, incompletely digested peptides, unusual peptide cleavages, and so on, using appropriate integrated software (PEPMAP, PEPMATCH). Examples of peptide mapping at the low pmol level using this integrated system will be shown (e.g., of the protein human growth hormone and of the glycoprotein, tissue plasminogen activator).

DNA ligases are indispensable enzymes playing a critical role in DNA replication, recombination, and repair in all living organisms. Bacterial NAD + -dependent DNA ligase (LigA) was evaluated for its potential as a broad-spectrum antibacterial target. A novel class of substituted adenosine analogs was discovered by target-based high-throughput screening (HTS), and these compounds were optimized to render them more effective and selective inhibitors of LigA. The adenosine analogs inhibited the LigA activities of Escherichia coli , Haemophilus influenzae , Mycoplasma pneumoniae , Streptococcus pneumoniae , and Staphylococcus aureus , with inhibitory activities in the nanomolar range. They were selective for bacterial NAD + -dependent DNA ligases, showing no inhibitory activity against ATP-dependent human DNA ligase 1 or bacteriophage T4 ligase. Enzyme kinetic measurements demonstrated that the compounds bind competitively with NAD + . X-ray crystallography demonstrated that the adenosine analogs bind in the AMP-binding pocket of the LigA adenylation domain. Antibacterial activity was observed against pathogenic Gram-positive and atypical bacteria, such as S. aureus , S. pneumoniae , Streptococcus pyogenes , and M. pneumoniae , as well as against Gram-negative pathogens, such as H. influenzae and Moraxella catarrhalis . The mode of action was verified using recombinant strains with altered LigA expression, an Okazaki fragment accumulation assay, and the isolation of resistant strains with ligA mutations. In vivo efficacy was demonstrated in a murine S. aureus thigh infection model and a murine S. pneumoniae lung infection model. Treatment with the adenosine analogs reduced the bacterial burden (expressed in CFU) in the corresponding infected organ tissue as much as 1,000-fold, thus validating LigA as a target for antibacterial therapy.

The efficacy of a bone-graft substitute (bovine-derived bone protein in a carrier of natural coral) in the healing of a segmental defect of a weight-bearing long bone was evaluated. Twenty dogs, divided into two groups, underwent bilateral radial osteotomies with creation of a 2.5 cm defect. On one side of each dog, the defect was filled with autogenous cancellous bone graft. Contralateral defects received, in a blinded randomized fashion, cylindrical implants consisting of natural coral (calcium carbonate) or calcium carbonate enhanced with a standard dose of bovine-derived bone protein (3.0 mg/implant; 0.68 mg bone protein/cm3). The limbs were stabilized with external fixators, and all animals underwent monthly radiographs. They were killed at 12 (group 1) or 24 (group 2) weeks, and regenerated bone was studied by biomechanical testing and histology. Radiographic union developed in all 20 radii with autogenous cancellous bone grafts and in all 10 of the radii with the composite implants. None of the radii with implants of calcium carbonate alone showed radiographic evidence of union. This represented a statistically significant difference between implant types. In addition, calcium carbonate implants both with and without bone protein demonstrated radiographic evidence of near total resorption of the radiodense carrier by 12 weeks. This resorption facilitated radiographic evaluation of healing. Mean values for biomechanical parameters of radii with the composite implants exceeded those for the contralateral controls at 12 and 24 weeks; the difference was statistically significant at 12 weeks. Histology revealed scant residual calcium carbonate carrier at either time in the defects with calcium carbonate implants; however, a moderate amount was present in defects with the composite implants. In these specimens, the residual carrier was completely surrounded by newly formed bone that may have insulated the calcium carbonate from further degradation. The present study used a carrier of granular calcium carbonate reconstituted with bovine type-I collagen to deliver an osteoinductive protein to the defect site. This carrier is of nonhuman origin (eliminating the risk of disease transmission or antigenicity) and resorbs rapidly. In this model, bovine-derived bone protein in a natural coral carrier performed consistently better than the gold standard autogenous cancellous bone graft in terms of the amount of bone formation and strength of the healed defect. This may have implications for removal of hardware or resumption of weight-bearing in certain clinical situations. These data also indicate that coralline calcium carbonate alone represents a poor option as a bone-graft substitute in this critical-sized segmental defect model.

Three porous ceramic bone graft materials were compared with regard to their ability to heal a 2.5 cm defect created surgically in a bilateral canine radius model. The ceramic materials were analyzed at 12 and 24 weeks after surgery and included tricalcium phosphate, hydroxyapatite, and collagen hydroxyapatite, which contained a mixture of 35% tricalcium phosphate and 65% hydroxyapatite with added collagen. Each material was evaluated alone and with added bone marrow aspirate. All the implants were compared with a graft of autogenous cancellous bone in the contralateral radius. Biomechanical testing and radiographic evaluation revealed that the addition of bone marrow aspirate was essential for tricalcium phosphate and hydroxyapatite to achieve results comparable with those of cancellous bone. Collagen hydroxyapatite performed well without the addition of bone marrow, although the addition of marrow did have a positive effect. Further qualitative radiographic and histological analysis demonstrated that tricalcium phosphate was the only ceramic that showed any sign of degradation at 24 weeks. This observed degradation proved to be an important factor in evaluating radiographs because the radiodensity of collagen hydroxyapatite and hydroxyapatite interfered with the determination of radiographic union. At 24 weeks, tricalcium phosphate with bone marrow was the material that performed most like cancellous bone. In this study, the biomechanical and radiographic parameters of tricalcium phosphate with bone marrow were roughly comparable with those of cancellous bone at 12 and 24 weeks. Tricalcium phosphate was the only implant that showed significant evidence of degradation at 24 weeks by both histological and radiographic evaluations, and this degradation took place only after a degree of mechanical competence necessary for weight-bearing was achieved.

Many bacterial capsular polysaccharides are regularly repeating units of oligosaccharides. Bacterial oligosaccharides have been used in neoglycoconjugate vaccines and as reagents in the study of specific antibody binding. Unfortunately, separation methods have not been adequate for the purification of preparative quantities of bacterial oligosaccharides. Here we describe a size-exclusion procedure that resulted in the resolution of group B Streptococcus type III oligosaccharides composed of 4-25 sugars.

The functional characterization of numerous transport systems across the vacuolar membrane (tonoplast) has provided a better understanding of the versatile role played by the vacuole in plant cells. The tonoplast H+-ATPase and the H+-pyrophosphatase pump protons into the vacuole making this organelle inside positive and acidic (Hedrich et al., 1989; Taiz, 1992). Maintaining an electrochemical gradient of protons, in addition to its role in cytoplasmic pH regulation, energizes various transport processes across the tonoplast. Several types of ions channels have been identified by the patch clamp technique, but the selectivity of most of them and hence their physiological role remain elusive (Hedrich and Schroeder, 1989). They probably represent major transport pathways for ions such as K+ and Cl-, the main determinants of cell turgor, and NO3- which can be stored in and mobilized from the vacuole. Calcium-permeable channels as well as a Ca++/H+ antiporter occur in plant vacuoles (Alexandre et al., 1990; Blackfordet al., 1990). They reflect the role that the vacuole as a major calcium store plays in signal transduction in plants. The tonoplast also carries Na+/H+ exchangers and Cd+ transporters that can account for the detoxifying role of the vacuole during saline and heavy metal stress, respectively (Taiz, 1992).

Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [(32)P]orthophosphate and in vitro by incubating purified tonoplasts with gamma-labeled [(32)P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca(2+)-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases.

The tonoplast mediates the transport of various ions and metabolites between the vacuole and cytosol by mechanisms that remain to be elucidated at the molecular level. The primary structure of only one tonoplast protein, the H(+)-ATPase, has been reported to date. Here we report the primary structure of tonoplast intrinsic protein (TIP), a 27-kilodalton intrinsic membrane protein that occurs widely in the tonoplasts of the protein storage vacuoles (protein bodies) of seeds [Johnson, K.D., et al. (1989). Plant Physiol. 91, 1006-1013]. Hydropathy plots and secondary structure analysis of the polypeptide predict six membrane-spanning domains connected by short loops and hydrophilic, cytoplasmically oriented N- and C-terminal regions. TIP displays significant homology with several other membrane proteins from diverse sources: major intrinsic polypeptide from bovine lens fiber plasma membrane; NOD 26, a peribacteroid membrane protein in the nitrogen-fixing root nodules of soybean; and interestingly, GIpF, the glycerol facilitator transport protein in the cytoplasmic membrane of Escherichia coli. Based on the homology between TIP and GIpF and the knowledge that the protein storage vacuolar membrane and the peribacteroid membrane are active in solute transport, we propose that TIP transports small metabolites between the storage vacuoles and cytoplasm of seed storage tissues.

The short-term effects of auxin (indole-3-acetic acid) and fusicoccin (FC) on Rb(+) uptake and malate accumulation in Avena sativa L. coleoptile sections have been investigated. FC stimulates (86)Rb(+) uptake within 1 min while auxin-enhanced uptake begins after a 15-20-min lag period. Auxin has little or no effect on (86)Rb(+) uptake at external pHs of 6.0 or less, but substantial auxin effects can be observed in the range of pH 6.5 to 7.5. Competition studies indicate that the uptake mechanism is specific for Rb(+) and K(+). After 3 h of auxin treatment the total amount of malate in the coleoptile sections is doubled compared to control sections. FC causes a doubling of malate levels within 60 min of treatment. Auxin-induced malate accumulation exhibits a sensitivity to inhibitors and pH which is similar to that observed for the H(+)-extrusion and Rb(+)-uptake responses. Both auxin- and FC-enhanced malate accumulation are stimulated by monovalent cations but this effect is not specific for K(+).

We have isolated the membranes of the protein storage vacuoles (protein bodies) from Phaseolus vulgaris cotyledons and purified an integral membrane protein with Mr 25,000 (TP 25). Antiserum to TP 25 recognizes an abundant polypeptide in the total cell extracts of many different seeds (monocots, dicots, and a gymnosperm), and specifically labels the vacuolar membranes of thin-sectioned soybean embryonic axes and cotyledons. TP 25 was not found in the starchy endosperm of barley and wheat or the seed coats of bean but was present in all seed parts examined that consist of living cells at seed maturity. The abundance of TP 25 was not correlated with the amount of storage protein in seed tissue, and the protein was not found in leaves that accumulate leaf storage protein. On the basis of its abundance, evolutionary conservation, and distribution in the plant, we propose that TP 25 may play a role in maintaining the integrity of the tonoplast during the dehydration/rehydration sequence of seeds.

As part of their posttranslational maturation process, newly synthesized glycoproteins that contain N-linked oligosaccharide side chains pass through the Golgi apparatus, where some of their oligosaccharides become modified by carbohydrate processing reactions. In this paper, we report the presence of Golgi-localized enzymes in plant cells (Phaseolus vulgaris cotyledons) that transfer GlcNAc, fucosyl, and xylosyl residues to the oligosaccharide side chains of glycoproteins. All three enzyme activities are involved in the transformation of high mannose side chains into complex glycans. As judged by acceptor specificity studies, at least two GlcNAc residues can be added to the nonreducing side of high mannose oligosaccharides, which have been trimmed by α-mannosidase(s). A Man5(GlcNAc)2-peptide serves as the acceptor for the first GlcNAc added. The second GlcNAc can be added only after the prior removal of two additional mannose residues, ultimately yielding (GlcNAc)2Man3(GlcNAc)2-peptide. Fucosyltransferase can transfer fucose to GlcNAcMan5(GlcNAc)2Asn, GlcNAcMan3(GlcNAc)2Asn, and (GlcNAc)2Man3(GlcNAc)2Asn; xylosyltransferase exhibits significant activity toward the latter two substrates only. These results suggest an overlapping sequence of oligosaccharide modification in the Golgi apparatus that, in regard to GlcNAc and fucose additions, is analogous to pathways of oligosaccharide processing reported for animal cells. To our knowledge, this is the first report characterizing a xylosyltransferase involved in N-linked oligosaccharide modification, an activity that is apparently absent in most animal cells.

Glycoproteins present in the soluble and organelle fractions of developing bean (Phaseolus vulgaris) cotyledons were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, affinoblotting, fractionation on immobilized concanavalin A (ConA), and digestion of the oligosaccharide side chains with specific glycosidases before and after protein denaturation. These studies led to the following observations. (a) Bean cotyledons contain a large variety of glycoproteins that bind to ConA. Binding to ConA can be eliminated by prior digestion of denatured proteins with alpha-mannosidase or endoglycosidase H, indicating that binding to ConA is mediated by high-mannose oligosaccharide side chains. (b) Bean cotyledons contain a large variety of fucosylated glycoproteins which bind to ConA. Because fucose-containing oligosaccharide side chains do not bind to ConA, such proteins must have both high-mannose and modified oligosaccharides. (c) For all the glycoproteins examined except one, the high-mannose oligosaccharides on the undenatured proteins are accessible to ConA and partially accessible to jack bean alpha-mannosidase. (d) Treatment of the native proteins with alpha-mannosidase removes only 1 or 2 mannose residues from the high-mannose oligosaccharides. Similar treatments of sodium dodecyl sulfate-denatured or pronase-digested glycoproteins removes all alpha-mannose residues. The results support the following conclusions: certain side chains remain unmodified as high-mannose oligosaccharides even though the proteins to which they are attached pass through the Golgi apparatus, where other oligosaccharide chains are modified. The chains remain unmodified because they are not accessible to processing enzymes such as the Golgilocalized alpha-mannosidase.

In plants, glycoproteins with asparagine-linked glycans (oligosaccharides) are found in vacuoles, in the extracellular space or matrix, and associated with the endo-membrane system (endoplasmic reticulum, Golgi apparatus, plasma membrane, tonoplast). These glycans are of the high-mannose type, with a structure identical to that found in other organisms (mammals, yeast), or of the complex type with a β1–2 linked xylosyl residue not found in mammalian complex glycans. Asparagine-linked glycans play multiple roles by modifying the physicochemical properties of the polypeptides to which they are attached.

Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc(3)Man(9)(GlcNAc)(2), was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associated with the Golgi. These include mannosidase I (removes 1-2 mannose residues from Man(6-9)[GlcNAc](2)), mannosidase II (removes mannose residues from GlcNAcMan(5)[GlcNAc](2)), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). We have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltransferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.

Several cell wall-bound glycosidases present in Avena sativa coleoptiles were assayed by following the hydrolysis of p-nitrophenyl-glycosides. Particular emphasis was placed on characterizing some parameters affecting the activity of beta-galactosidase. The pH optimum of this enzyme is 4.5 to 5.5; it is sensitive to copper ions and p-chloromercuribenzoate treatment and apparently has an exceptionally low turnover rate. Indoleacetic acid treatment enhanced in vivo beta-galactosidase activity of coleoptile segments by 36% over control after 60 minutes. This enhancement was prevented by abscisic acid and cycloheximide. High buffer strengths and low pH reduced the indoleacetic acid-enhanced increase in enzyme activity. These data lend support to the following proposed model of indoleacetic acid action. Indoleacetic acid enhances the release of hydrogen ions into the cell wall which promote the activities of cell wall glycosidases, some of which may participate in the cell extension process.

1. 1. The average cholesterol content of the coronary arteries in a group of patients who died from an acute coronary artery thrombosis was four times as great as the average cholesterol content of the coronary arteries in a comparable group of control patients. 2. 2. Hypercholesterolemia was found in most of the patients who died of acute coronary artery thrombosis, as compared to a normal blood cholesterol average in the comparable control group. 3. 3. These findings suggest that a disturbance in lipid metabolism is a factor in the pathogenesis of atherosclerosis.

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A(7) (GA(7)), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA(7) also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA(7) solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment.Age of the seedling tissue had a profound influence on the chromatin characteristics. With progression of development from the 2-day to the 4-day stage, the endogenous chromatin activity declined while the DNA template activity increased.

The enzymes of the cytidine diphosphate-choline pathway, which is involved in lecithin biosynthesis, are present in imbibed barley aleurone cells. The first enzyme, choline kinase (EC 2.7.1.32), is found in the soluble protein fraction. Its activity is not affected by prior treatment of aleurone layers with gibberellin. The second and third enzymes of the pathway, phosphorylcholine-cytidyl (EC 2.7.7.15) and phosphorylcholine-glyceride (EC 2.7.8.2) transferases, are associated with the particulate fractions. Their activities are greatly increased by gibberellin treatment during the lag phase (0-8 hr) of gibberellin-effected α-amylase synthesis. The hormonal effects are evident two hours after gibberellin treatment. Inhibitors that block gibberellin-effected α-amylase formation also inhibit the stimulation of these membrane-bound enzymes by the hormone.

Antibodies were raised against carrot (Daucus carota) cell wall beta-fructosidase that was either in a native configuration (this serum is called anti-betaF(1)) or chemically deglycosylated (anti-betaF(2)). The two antisera had completely different specificities when tested by immunoblotting. The anti-betaF(1) serum reacted with beta-fructosidase and many other carrot cell wall proteins as well as with many proteins in extracts of bean (Phaseolus vulgaris) cotyledons and tobacco (Nicotiana tabacum) seeds. It did not react with chemically deglycosylated beta-fructosidase. The anti-betaF(1) serum also reacted with the bean vacuolar protein, phytohemagglutinin, but not with deglycosylated phytohemagglutinin. The anti-betaF(2) serum reacted with both normal and deglycosylated beta-fructosidase but not with other proteins. These results indicate that the betaF(2) antibodies recognize the beta-fructosidase polypeptide, while the betaF(1) antibodies recognize glycan sidechains common to many glycoproteins. We used immunoadsorption on glycoprotein-Sepharose columns and hapten inhibition of immunoblot reactions to characterize the nature of the antigenic site. Antibody binding activity was found to be associated with Man(3)(Xyl)(GIcNAc)(2)Fuc, Man(3)(Xyl)(GIcNAc)(2), and Man(Xyl) (GIcNAc)(2) glycans, but not with Man(3)(GIcNAc)(2). Treatment of phytohemagglutinin, a glycoprotein with a Man(3)(Xyl)(GIcNAc)(2)Fuc glycan, with Charonia lampas beta-xylosidase (after treatment with jack-bean alpha-mannosidase) greatly diminished the binding between the antibodies and phytohemagglutinin. We conclude, therefore, that the antibodies bind primarily to the xylosebeta, 1--2mannose structure commonly found in the complex glycans of plant glycoproteins.

Treatment of isolated barley aleurone layers with gibberellic acid (GA3) resulted in a progressive inhibition of cell-wall synthesis after a 4-h lag period. The incorporation of both [(14)C]arabinose and [(14)C]glucose into the cell wall was inhibited by GA3, but analysis of the labelled sugars in the polymerized product showed that the process most affected by the hormone treatment was pentosan biosynthesis. Labelling kinetics and pulse-chase analysis indicated that the pentosans were synthesized in the cytoplasm and subsequently transferred to the cell wall; GA3 did not significantly affect the latter step. The GA3-inhibited labelling of the cell-wall pentosans cannot be explained on the basis of an effect on uptake of radioactive cell-wall precursor, expansion of the free pentose pool, or degradation of newly-formed pentosan. GA3 inhibited the activity of a membrane-bound arabinosyl transferase present in the aleurone layers. This inhibition may explain the inhibition of cell-wall pentosan synthesis by GA3.

Aleurone layers of barley contain large amounts of a soluble oligosaccharide which was identified as sucrose (30–40 μg/mg fresh weight). Treatment of the layers with gibberellic acid (GA3) causes the release of sucrose from the cells. This release requires the participation of metabolic processes, including protein synthesis. When embryoless half-seeds are incubated sucrose accumulates in the aleurone layers, but when seeds are germinated the sucrose content of the aleurone layers declines. Labeling experiments with radioactive glucose and fructose show that aleurone layers continuously synthesize sucrose and that the release, but not the synthesis of sucrose is enhanced by GA3.