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We have measured parity-violating asymmetries in elastic electron-proton scattering over the range of momentum transfers 0.12 < or =Q2 < or =1.0 GeV2. These asymmetries, arising from interference of the electromagnetic and neutral weak interactions, are sensitive to strange-quark contributions to the currents of the proton. The measurements were made at Jefferson Laboratory using a toroidal spectrometer to detect the recoiling protons from a liquid hydrogen target. The results indicate nonzero, Q2 dependent, strange-quark contributions and provide new information beyond that obtained in previous experiments.

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.

beta-Glucan synthetase activity in growing regions of pea (Pisum sativum L.) epicotyls was assayed by supplying UDP-glucose to particulate fractions of tissue homogenates or to thin tissue slices. Particulate fractions are less active in forming alkali-insoluble glucan than slices from the same tissue, although many kinetic characteristics (pH and Mg(2+) optimum, apparent K(m)) are similar for the two systems. Synthesis by tissue slices progresses linearly without lag period for at least an hour and is proportional to cut surface area. It is much more rapid from UDP-glucose than from glucose, glucose-1-P, or sucrose. Tests with plasmolyzing agents and trypsin support the conclusion that synthesis from UDP-glucose by slices occurs at accessible surfaces of cut cells. Analyses of glucan products by GLC of partially methylated and acetylated derivatives and by hydrolysis with various beta-glucanases all show that both beta-1,3 and beta-1,4 linkages are formed by particulate fractions and slices at substrate concentrations ranging from micro- to millimolar. beta-1,4 Linkages predominate at low substrate (5 mum) concentration. Kinetic data indicate that the capacity to synthesize beta-1,3-glucan is substrate-activated, and this product predominates in preparations supplied with high (5 mm) substrate.

Two endo-1,3,-beta-d-glucanases (I and II, EC 3.2.1.6) are present in etiolated peas at opposite ends of the stem. Glucanase I from subapical regions degrades substrates to a series of low molecular weight dextrins, and is most readily assayed reductometrically (e.g. as laminarinase). Glucanase II from basal regions preferentially hydrolyzes internal linkages of long chains, and is most sensitively assayed viscometrically (e.g. as carboxymethylpachymanase). The activity of glucanase II but not I increases greatly near the apex in response to treatment of the tissue with auxin, and ethylene gas suppresses endogenous activities and the auxin response, i.e. levels of these enzymes are under developmental controls which can be regulated. Different natural substrates for the two enzymes were identified primarily in tissue fractions soluble in hot water. Substrates for glucanase I are concentrated in apical regions, as is the enzyme itself, and those for glucanase II are in basal regions, implying that enzymes and substrates are normally in separate cellular compartments. Tissue sections stained with aniline blue for beta-glucan show enhanced fluorescence in cell walls, and most of this can be removed either by hot water or the appropriate purified beta-glucanase. The enzymes are not likely to function directly in promoting nutrition or growth in peas, but they could help, following secretion, to maintain channels for communication and translocation through cell walls.

beta-1, 4-Glucan (cellulose) synthetase activity (UDP-glucose: beta-1, 4-glucan-glucosyl transferase) present at cell surfaces of growing regions of Pisum sativum epicotyl was assayed by supplying UDP-(14)C-glucose directly to thin slices of tissue. Initial rates of glucosyl transfer under these conditions approached the rates of cellulose deposition observed in vivo in intact tissue at various stages of growth. Normal tissue homogenization procedures destroyed the high surface activity, although a small amount of residual activity (3-10% of total) could be detected in particulate fractions. In homogenates from elongating tissue, the residual activity was almost entirely associated with Golgi membrane. In homogenates of tissue which had ceased elongating, whether because of normal maturation or treatment with ethylene (or high levels of auxin), the activity was present in Golgi plus a membrane fraction rich in smooth endoplasmic reticulum vesicles. It is suggested that cellulose synthetase activity associated with these two organelles represents intracellular enzyme in transit to specific sites of cellulose synthesis and microfibrillar orientation at the cell surface.

beta-Glucan synthesis from uridine diphosphoglucose by pea epicotyl tissue slices is increased two- to threefold by preliminary, short-term treatment with cellulases purified from auxin-treated peas. We suggest that cellulases introduce chain ends in accessible regions of cellulose microfibrils which then act as primers for chain elongation.

In the subapical region of dark-grown pea epicotyls about 40% of the total polysomes are associated with membranes. The presence of poly(A) in polysomal mRNA was detected by hybridization of unlabeled RNA with (3)H-poly(U). Both free mRNA and messenger ribonucleoprotein particles in polysomes hybridize with (3)H-poly(U) quantitatively. The binding of (3)H-poly(U) to polysomes is increased by treatment with the detergent sodium dodecyl sulfate. Since detergent influenced the (3)H-poly(U) binding more in membrane-bound polysomes than in free, there may be more protein(s) associated with the poly(A) portion of the mRNA in membrane-bound polysomes. Analysis of the poly(A) segments isolated from the mRNA of these two classes of polysomes indicates that there are discrete classes of poly(A) and they appear to be differentially associated with free and membrane-bound polysomes. Mean size distribution of poly(A) in free polysomes is larger than in membrane-bound polysomes.Following treatment (2 days) with the plant growth hormone indoleacetic acid, there is a gradual decrease in the mean length of total poly(A), which appears to correspond to a decrease in the size of the polysomes and their associated mRNA.

Two cellulases purified from growing regions of auxin-treated peas (buffer-soluble and buffer-insoluble) hydrolyze cellulose powder, partially substituted carboxymethylcellulose (CM-cellulose), higher cellodextrins, and certain mixed linkage glucans (e.g. barley beta-glucan), at rates comparable to these reported for the most active fungal cellulases, and with kinetics and product formation characteristic of endohydrolase action. They are unable to cleave 1,3-linkages in beta-glucans, or 1,4-linkages in dextrins containing excessive substitution at C6, alpha configuration, alternating beta-1,3- and 1,4-linkages, or residues other than anhydroglucose. They are not active towards cellobiose or the 1,4-linkage adjacent to the reducing end of cellodextrin chains. It is concluded that buffer-soluble and buffer-insoluble cellulases are true beta-1,4-glucan 4-glucanohydrolases (EC 3.2.1.4). On a molar basis, Vmax values for buffer-insoluble are higher than buffer-soluble cellulase acting towards any of the substrates tested, but Km values towards CM-cellulose and cellohexaose are essentially identical. Both cellulases were inhibited by C12+, Hg2+, and sulfhydryl-binding reagents. Buffer-insoluble, but not buffer-soluble, cellulose was inactivated by reagents that bind serine and threonine, which reflects differences in their amino acid composition. No major qualitative differences have been detected in the mode of action of the two enzymes. Despite marked differences in their physical and immunological properties, close similarities between buffer-soluble and buffer-insoluble enzymic properties suggest that their active sites are the same.

Membrane preparations from growing regions of 8-day old Pisum sativum epicotyls contain multiple beta-1,4-glucan (cellulose) synthetase activities (UDP- or GDP-glucose: beta-1,4-glucan-glucosyl transferase), and the levels of some of these are influenced by treatments with the growth hormone, indoleacetic acid (IAA). When membranes from control epicotyl segments (zero time) are fractionated by isopycnic sedimentation in sucrose density gradients, all of the synthetase activities are associated mainly with Golgi membrane (density 1.55 g/cm3). After decapitation and treatment of epicotyls with IAA, synthetases also appear in a smooth vesicle fraction (density 1.11 g/cm3) which is rich in endoplasmic reticulum (ER) marker enzyme. Major fractions of these synthetases are not recovered in association with plasma membrane or washed cell walls. When [14-C]sucrose is supplied in vivo to segments +/- IAA, radioactive cellulose is deposited only in the wall. Cellulose or cellodextrin precursors do not accumulate in those membranes in which synthetase activities are recovered in vitro. In experiments where tissue slices containing intact cells are supplied with [14C]sugar nucleotide in vitro, alkali-insoluble beta-1,4-glucan is synthesized (presumably outside the protoplast) at rates which greatly exceeded (20-30 times) those obtained using isolated membrane preparations. Progressive disruption of cell structure results in increasing losses of this high activity. These results are consistent with the interpretation that Golgi and ER-associated synthetases are not themselves loci for cellulose synthesis in vivo, but represent enzymes in transit to sites of action at the wall:protoplast omterface. There they operate only if integrity of cellular organization is maintained.

Two forms of beta-1,4-glucan 4-glucanohydrolase (EC 3.2.1.4) were extracted from growing regions of Pisum sativum epicotyls which had been treated with the auxin, (2,4-dichlorophenoxy)acetic acid. One cellulase is buffer-soluble, the other buffer-insoluble but extractable with high salt concentrations. Both enzymes catalyze endohydrolysis of carboxymethylcellulose with the same pH optimum (5.5 to 6.0). They were purified with the use of DEAE-cellulose chromatography, Sephadex gel filtration, and ultrafiltration. They are distinct proteins as characterized by: electrofocusing and disc gel electrophoresis (pI values = 5.2 and 6.9, respectively); mobility in sodium dodecyl sulfate polyacrylamide gels, fractionation on Sephadex, and sedimentation in the ultracentrifuge (mol wt = approximately 20,000 and 70,000, S values 2.63 and 3.73); and immunological properties. The buffer-soluble enzyme tends to dimerize on purification. Amino acid analyses show that the buffer-soluble enzyme is relatively rich in glycine, alanine, and valine and deficient in cystine, tryosine, and phenylalanine compared to the buffer-insoluble enzyme. The two cellulase activities were generated in approximately equal amounts after auxin treatment. Within 5 days their levels had increased at least 100-fold and they constituted about 0.1% of total cellular protein. Present data indicate that one is not derived from the other.

Two forms of cellulase, buffer soluble (BS) and buffer insoluble (BI), are induced as a result of auxin treatment of dark-grown pea epicotyls. These two cellulases have been purified to homogeneity. Antibodies raised against the purified cellulases were conjugated with ferritin and were used to localize the two cellulases. Tissue sections were fixed in cold paraformaldehyde-glutaraldehyde and incubated for 1 h in the ferritin conjugates. The sections were washed with continuous shaking for 18 h and subsequently postfixed in osmium tetroxide. Tissue incubated in unconjugated ferritin was used as a control. A major part of BI cellulase is localized at the inner surface of the cell wall in close association with microfibrils. BS cellulase is localized mainly within the distended endoplasmic reticulum. Gogli complex and plasma membrane appear to be completely devoid of any cellulase activity. These observations are consistent with cytochemical localization and biochemical data on the distribution of these two cellulases among various cell and membrane fractions.

Two buffer-soluble endo-1,3-beta-D-glucanases (EC 3.2.1.6) have been purified to within 1% of electrophoretic homogeneity from etiolated Pisum sativum stem tissues. Purified glucanase I and II differ in physical properties, such as electrophoretic mobility in sodium dodecyl sulfate polyacrylamide gels (Mr values were 22 000 and 37 000, respectively) and isoelectric focusing, (pI values were 5.4 and 6.8, respectively). Although the enzymes have similar pH optima (5.5--6.0), Km values for various substrates (0.6--7.4 mg/ml) and thermal inactivation profiles, they are localized in different tissues and they differ markedly in the rates with which they attack the internal linkages of long- vs. short-chain substrates. Glucanase I is concentrated in apical regions of the stem and is most effectively assayed reductometrically (as laminarinase), while glucanase II is localized in mature regions and is relatively more active in viscometric assays (as carboxymethyl-pachymanase).

Two purified pea 1,3-beta-D-glucanases (EC 3.2.1.6) hydrolyse laminarin (degree of polymerization 20), laminaridextrins (degree of polymerization 3--7), and their reduced 3H-derivatives, 1,3-beta-D-glucans which are partially substituted (carboxymethyl-pachyman) or crystalline (curdlan), and mixed-linkage beta-glucans. Enzyme kinetics and product-formation indicate endo-hydrolase activity with weak transglycosylase capacity. The enzymes do not hydrolyse beta-glucosides, the 1,3 linkage adjacent to the reducing end of chains, or cellulose and its derivatives. They degrade mixed-linkage beta-glucans, in a manner similar to Rhizopus arrhizus endo-1,3-beta-D-glucanase, to form the products expected from hydrolysis of linkages adjacent to 1,3-beta linkages. With respect to action patterns, glucanase I (from apical growing tissue) differs from glucanase II (from basal maturing tissue) in several respects: (a) on a molar basis, I generates reducing groups from all substances more rapidly than II; (b) lower laminaridextrins are hydrolysed by I at the non-reducing terminal linkage, while II preferentially hydrolyses internal linkages; (c) laminarin is hydrolysed to lower laminaridextrins by I more rapidly than II, but I takes longer than II to completely degrade laminarin chains; (d) the enzymes are differentially sensitive to different classes of non-competitive inhibitors. It is concluded that these beta-glucanases differ in such a way that I preferentially continues to degrade fragments produced by endo-hydrolytic attack on long chains ('multiple attack' action pattern), while II hydrolyses internal linkages of the longest chains available ('multi-chain attack').

Membrane preparations from developing soybean (var. Prize) cotyledon tissue, at the time of synthesis of storage glycoproteins, catalyze the sequential assembly of lipid-linked oligosaccharides from uridine-5'-diphospho-N-acetyl-d-[6-(3)H] glucosamine and guanosine-5'diphospho-d-[U-(14)C]mannose. The maximum size of lipid-linked oligosaccharide that accumulates contains the equivalent of 10 saccharide units on the basis of Bio-Gel P-2 gel filtration studies. These lipid-linked oligosaccharides show similar characteristics to polyisoprenyl diphosphate derivatives on diethylaminoethyl-cellulose chromatography and are potential intermediates in glycoprotein biosynthesis in this tissue. These glycolipids do not appear to turn over in pulse-chase experiments and no completed storage glycoproteins were detected among the products of these incubations.Tissue slices from cotyledons at the same stage of development synthesize lipid-linked oligosaccharides from [(3)H]mannose and [(3)H]glucosamine with sizes equivalent to 1, 7, 10, and approximately 15 saccharide units. In pulse-chase experiments, the lipid-linked saccharides with the equivalent of 1 and 10 units rapidly turnover, whereas those with 7 and 15 units do not. Examination of the higher oligosaccharide peaks (10 and 15) by Bio-Gel P-4 gel filtration shows them to comprise 2 distinct subsets of oligosaccharides containing different proportions of glucosamine and mannose units. Tissue slices synthesize products which resemble the completed 7S storage glycoproteins as judged by similarity of molecular weight and precipitation with specific antisera. Analysis of the oligosaccharides obtained by hydrazinolysis of glycoproteins shows the presence of a similar size "high-mannose" type N-linked oligosaccharides as in other glycoproteins from animal and plant cells.

Homogenates of growing regions of the pea (Pisum sativum L.) epicotyl contain soluble factors (130,000g supernatant) which alter pea beta-glucan synthetase activity, as assayed using the substrate UDP-glucose and either particulate fractions or tissue slices as source of enzyme. A heat-stable dialyzable component is present which enhances as much as 3-fold the synthesis of alkali-soluble and -insoluble products from millimolar levels of substrate. A heat-labile nondialyzable component is also present which suppresses synthesis. This component dominates (the net effect of total crude extract) when low (mum) levels of substrate are employed. Methylation analysis shows that both components primarily affect the proportion of beta-1,4 rather than beta-1,3 linkages which are synthesized. The enhancing factor increases V(max) of the synthetase system and only activates in the presence of high levels of substrate. The suppressing factor appears to inactivate the synthetase, since losses of product or substrate are not significant during brief incubation with extract, the factor acts progressively with time with a pH optimum, and it destroys activity during preincubation with particles or slices. It co-precipitates with a protease (gelatinase) at between 20% and 40%-saturated (NH(4))(2)SO(4), and it co-fractionates with a major component of total protease on Sephadex gel columns (G-200) with an elution volume corresponding to molecular weight 65,000. The concentrations of these factors are such that they could be natural moderators of synthetase activity in vivo if the two were ever brought in contact, and the inactivator could account for the lability of beta1,4-glucan synthetase which occurs upon tissue homogenization.

Polysomal RNA was isolated from pea epicotyls treated with (2,4-dichlorophenoxy)acetic acid, and fractionated on oligo(dT)-CELLULOSE TO YIELD POLY(A)-containing RNA. This RNA fraction was translated in a wheat embryo cell-free system and found to have more than 90% of the messenger activity in total polysomal RNA. Immunoprecipitation of the translation product by monospecific antibodies to pea cellulases (beta-1,4-glucan 4-glucanohydrolase, EC 3.2.1.4) indicated that cellulase was synthesized in this system. The immunoprecipitate co-migrated with the buffer-soluble cellulase component in sodium dodecyl sulfate-gel electrophoresis. Buffer-insoluble cellulase was not detected in the in vitro translation products. Fractionation of mRNA from membrane-bound and free polysomes and their subsequent translation indicated preferential synthesis of buffer-soluble cellulase on membrane-bound polysomes. With the above techniques for assaying buffer-soluble cellulase mRNA, a 10-fold increase in the level of this messenger per tissue segment was observed within 48 hours following (2,4-dichlorophenoxy)acetic acid treatment. There was no evidence for pre-existing untranslated message for cellulase in control tissues. Since there was no delay in the appearance of mRNA for cellulase, compared to a 24-hour lag in the increase of cellulase activity, it is suggested that translational as well as transcriptional controls are exerted on the biosynthesis of cellulase in vivo. Analysis of the rates of peptide chain initiation and elongation, both in vivo and in vitro, indicated that peptide chain elongation may be rate-limiting during the lag phase of cellulase development.

A procedure is described for preparing cellulose nitrate from pea tissues (Pisum sativum L. var. Alaska) in quantitative yield, undegraded and uncontaminated by other polysaccharides. The average degree of polymerization of this product, estimated from viscosity measurements, increased during cell growth and development from a value of about 5000 glucose units in the apical meristem (plumule plus hook) to values near 8000 in fully grown maturing tissues (>20 mm from apex). The cellulose content per cell also increased (approximately 10-fold) during growth in these tissues, as did particulate glucan synthetase activity (3-fold rise). Since the yield of soluble cellulase activity is known to decrease from high values in the meristem to barely detectable amounts in mature tissues, it is suggested that the relative levels and properties of these hydrolytic and synthetic enzyme activities control the amount and degree of polymerization of cellulose formed during cell expansion in the pea epicotyl.Degree of polymerization distribution patterns showed that a low molecular weight component of cellulose (degree of polymerization < 500) was prominent in young tissues whereas high molecular weight components (degree of polymerization > 7000) predominated in mature tissues. Also, cellulose which was formed from radioactive sucrose during 30 minutes of incubation showed a remarkably similar degree of polymerization distribution to cellulose which was present in the tissue at the time of synthesis. It is concluded that new and old parts of the epicotyl cellulose framework are subject to constant modification and equilibration by cellulose-metabolizing enzymes.

Measurements were made over a 4-day period of the effect of added indoleacetic acid (IAA), puromycin, actinomycin D and 5-fluorodeoxyuridine (FUdR) on growth and the levels of total DNA, RNA, protein and cellulase in segments of tissue at the apex of decapitated etiolated epicotyls of Pisum sativum, L. var. Alaska.The hormone induced swelling of parenchyma cells and cell division. By 3 days after IAA application, the amounts of DNA and protein were approximately double, RNA triple and cellulase 12 to 16 times the levels in controls. All of these changes were prevented by both puromycin and actinomycin D. FUdR prevented DNA synthesis and cell division but not swelling or synthesis of RNA, protein and cellulase.It is concluded that IAA-induced RNA synthesis is required for cellulase synthesis and lateral cell expansion, whether or not cell division takes place.

RNase activity was assayed in subcellular fractions of apical regions of Pisum sativum L. var. Alaska epicotyls after seedling decapitation and treatments with various growth regulators. High concentrations of applied indoleacetic acid caused a marked increase to occur in the RNase activity level associated with "heavy" microsomes, e.g., a 20-fold rise per unit RNA or protein in 3 days. This rise could be abolished by treating with the cytokinin benzyladenine along with indoleacetic acid. Nevertheless, indoleacetic acid and benzyladenine acted synergistically in their abilities to evoke swelling and net synthesis of RNA and protein. Polysomal profiles prepared after treatment with indoleacetic acid plus benzyladenine showed less degradation than profiles from any other treatment. It is concluded that auxin generates and cytokinin suppresses the activity of a particular membrane-bound RNase which can control turnover of the auxin-evoked polysomes required for growth in peas. Synergism between the two hormones in this system may be explained by the action of one to increase RNA synthesis and the other to decrease RNA destruction.

Indoleacetic acid (IAA) and/or inhibitors of DNA, RNA or protein synthesis were added to the apex of decapitated seedlings of Pisum sativum L. var. Alaska. At various times up to 4 days, enzymic protein was extracted from a segment of epicotyl immediately below the apex and assayed for its ability to hydrolyse polysaccharides or their derivatives. With the exception of amylase, the total amounts per segment of all of the tested enzymes increased due to IAA treatment. The development of beta-1,4-glucanase (cellulase) activity per unit of protein or fresh weight proceeded according to a typical sigmoid induction curve. Pectinase was formed for about 2 days in control segments and IAA treatment resulted in continued synthesis for at least another 2 days provided cell division took place. beta-1,3-glucanase and pectinesterase activities were only enhanced by IAA to the extent that total protein levels increased. Reaction mechanisms for these effects and functions for the enzymes during growth are discussed.