Fructosyl Transfer between 1-Kestose and Sucrose in Wheat Leaves.

The labeling pattern of the sugar moieties of 1-kestose after in vivo pulse labeling with (14)CO(2) was not the same as that after in vitro labeling with (14)C-sucrose. The two fructosyl residues of 1-kestose had similar specific radioactivities after in vitro synthesis, but after in vivo radiolabeling the specific radioactivity of the terminal fructosyl moiety was significantly less than the internal fructosyl moiety. Evidence is presented that the uneven specific radioactivity of in vivo radiolabeling results from enzymatic transfer of terminal fructosyl residue from 1-kestose to sucrose.

Chemical structure of the cell walls of dwarf maize and changes mediated by gibberellin.

Dwarf maize (Zea mays L.), a mutant deficient in gibberellin synthesis, provides an excellent model to study the influence of gibberellin on biochemical processes related to plant development. Alterations in the chemical structure of the cell wall mediated by gibberellin were examined in seedlings of this mutant. The composition of the walls of roots, mesocotyl, coleoptile, and primary leaves of dwarf maize was similar to that of normal maize and other cereal grasses. Glucuronoarabinoxylans constituted the principal hemicelluloses, but walls also contained substantial amounts of xyloglucan and mixed-linkage beta-d-glucan. Root growth in dwarf maize was essentially normal, but growth of mesocotyl and primary leaves was severely retarded. Injection of the gibberellin into the cavity of the coleoptile resulted in a marked increase in elongation of the primary leaves. This elongation was accompanied by increases in total wall mass, but the proportion of beta-d-glucan decreased from 20% to 15% of the hemicellulosic polysaccharide. During leaf expansion, the proportion decreased further to only 10%. Through 4 days incubation, the proportion of beta-d-glucan in leaves of control seedlings without gibberellin was nearly constant. Extraction of exo- and endo-beta-d-glucan hydrolases from purified cell walls and assay against a purified oat bran beta-d-glucan demonstrated that gibberellin increased the activity of the endo-beta-d-glucan hydrolase. These and other data support the hypothesis that beta-d-glucan metabolism is central to control of cell expansion in cereal grasses.

Extraction of starch by dimethyl sulfoxide and quantitation by enzymatic assay.

A method for the rapid, sensitive, and specific determination of starch in plant tissues is described. Starch from a variety of plant tissues is solubilized by stirring for 24 h or by sonication for 40 min in dimethyl sulfoxide. Dilution of this extract to less than 20% dimethyl sulfoxide permits a nearly complete hydrolysis of the starch in less than 3 h with glucoamylase from Rhizopus niveus. Quantitation of liberated glucose by a coupled hexokinase and glucose-6-phosphate dehydrogenase method provides an additional degree of specificity.

Carbon assimilation in carrot cells in liquid culture.

Assimilation of carbohydrates by carrot (Daucus carota L. cv Danvers) cells in liquid culture was studied to delineate the major metabolic pathways used in transformation of external carbohydrates to UDP-glucose. The cells grown on either sucrose or glucose for several years proved equally capable of utilizing each of these sugars. Sucrose was rapidly hydrolyzed extracellularly to glucose and fructose, and glucose was preferentially taken up. Uptake of fructose was slower and delayed until glucose was nearly depleted from the medium. Concentrations of cellular sugars, mainly glucose and sucrose, increased during late logarithmic phase of growth and decreased during the plateau phase. Continuous labeling of the cells with d-[(14)C]glucose resulted in rapid accumulation of radioactivity in glucose-6-phosphate and UDP-glucose. Because there was virtually no uptake of sucrose, UDP-glucose was likely derived from glucose-1-phosphate in a reaction catalyzed by UDP-glucose pyrophosphorylase and not directly from sucrose. Concentrations of major nucleotides and nucleotide sugars were maximal during the early logarithmic phase of growth and decreased several-fold in the stationary phase. A modified ;energy charge' for adenylates calculated with the omission of AMP decreased steadily from 0.9 to 0.8 during the course of culture cycle. An analogous uracil nucleotide ratio was considerably lower (0.85) during early culture, decreased to about 0.7 for the entire logarithmic phase, and returned to initial values as cells entered stationary phase. The uracil nucleotide ratio may provide a useful index to assess the coupling between the energy available in phosphoanhydride bond in adenine nucleotides and the demand for sugar for polysaccharide synthesis through uridine diphosphate-sugar pools.

Heat Shock Proteins in Tobacco Cell Suspension during Growth Cycle.

Tobacco (Nicotiana tabacum L. cv Wisconsin 38) cells grown in suspension culture at 26 degrees C produce heat shock proteins (HSPs) when exposed to elevated temperature of 34 to 42 degrees C. At 34 and 38 degrees C, synthesis of normal proteins is maintained while HSPs are expressed within 30 minutes after initiation of the shock. At 42 degrees C, HSPs are still expressed but normal proteins are made at a reduced rate or not at all. Exposure of cells to 38 degrees C allows for a full expression of HSPs without inhibition of the synthesis of normal proteins. Induced synthesis of HSPs at 38 degrees C is maximal 1 to 2 hours after elevation of temperature and diminishes thereafter through at least 6 hours. Cells growing asynchronously in the logarithmic phase of growth produce HSPs at a much higher rate than those in the stationary phase. The ability to synthesize HSPs disappears about one generation time before the cells reach a growth plateau.

Properties of human chorionic gonadotropin produced in vitro by ovarian carcinoma cells.

The present study was designed to compare the immunological, physical, and biological properties of native hCG with an hCG molecule secreted ectopically in vitro by an ovarian adenocarcinoma cell line maintained in long term tissue culture. The hCG produced by the cell line was concentrated by ultrafiltration of the tissue culture medium. The inhibition curves generated by serial dilutions of the culture medium concentrates were parallel to those obtained with purified urinary hCG in the beta-hCG RIA and the rat Leydig cell radioreceptor assay (RRA). The ectopic hCG also reacted with an antibody generated against the carboxyl-terminal peptide (109-145) of beta-hCG. The immunoreactive material cochromatographed with urinary hCG on a Sephadex G-100 column, as determined by the beta-hCG RIA and RRA. Neither free alpha nor free beta subunits were found in the tissue culture medium. The tissue culture gonadotropin was adsorbed onto a Concanavalin A-Sepharose column and could be eluted with alpha-D-methylglucoside. The biological activity of the ectopic hCG was 9289 IU/mg, as determined by the ventral prostate weight (VPW) method in hypophysectomized immature male rats. The biological to immunological ratios by the ventral prostate weight method and RRA were 1.79 and 2.17, respectively. The in vivo disappearance rate of ectopic hCG after injection into immature female rats was significantly faster than that of placental or urinary hCG, but was considerably slower than the disappearance rate of human LH. These studies demonstrate that the immunoreactive and biologically active portions of the hCG produced by the ovarian adenocarcinoma cell line and native hCG are similar or identical. The faster disappearance rate of the ectopic hCG in the rat model may be due to incomplete sialylation of the oligosaccharide moiety of the hCG molecule.

Kinetics of growth and ectopic production of human chorionic gonadotropin by an ovarian cystadenocarcinoma cell line maintained in vitro.

An established ovarian papillary cystadenocarcinoma cell line, designated 163, released immunoreactive human chorionic gonadotropin (HCG) in vitro into the culture medium. The maximal rate of this ectopic activity (10 to 15 ng/day/10(6) cells) occurred at the onset of the logarithmic phase of cell growth and greatly exceeded the intracellular HCG content of cells approaching confluency (0.037 ng/10(6) cells). The HCG production was not detected during the plateau phase of growth. The rate of cellular proliferation and HCG release depended upon the frequency of media change in a manner suggesting that the two processes are interrelated and may be affected by such environmental factors as cell density, nutrient availability, and the accumulation of waste products. The addition of purified HCG to the cultures had no appreciable effect on either the cell growth or the accumulation of HCG. The release of HCG into the medium was greatly stimulated in the presence of sodium butyrate at concentrations from 1 to 10 mM, despite the fact that these concentrations of butyrate resulted in a marked decrease in the cell number/culture in comparison with control media. Equivalent amounts of sodium acetate had no effect on either cell growth or the release of HCG.

Isolation of an organ-specific leucyl-tRNA synthetase from soybean seedling.

The leucyl-tRNA synthetase activity from cotyledons of 4-day-old soybean seedlings has been fractionated into three components. One of these exclusively acylates two of the six tRNA(Leu) species present in this tissue. The remaining two enzyme fractions charge the other four tRNA(Leu) species equally well. Soybean hypocotyls appear to contain only the last two enzyme fractions.