The biosynthesis of elastin was examined in matrix-free cells isolated by enzymic digestion of aortas from 17-day old chick embryos. After the cells were incubated with [14C]proline and then were rapidly boiled in buffer containing high concentrations of protease inhibitors and sodiumdodecyl sulfate, about one-quarter of the intracellular 14C-labeled protein was recovered as an elastin component with an apparent molecular weight of about 72 000. Examination of the medium from the cell suspension indicated that the largest elastin component secreted by the cells also had an apparent molecular weight of about 72 000. Pulse-chase experiments with intact aortas demonstrated that about two-thirds of the 72 000-dalton component disappeared in 2 h, apparently because it was converted to cross-linked fibers. When cross-linking was inhibited with penicillamine, the 72 000-dalton component persisted in the tissue 5 h. When cross-linking was inhibited with beta-aminopropionitrile, the elastin component of 72 000 daltons persisted for about 2 h, but thereafter it was gradually degraded to small peptides which were recovered in the incubation medium. The results suggest that elastin is secreted by cells in chick aorta as a polypeptide of about 72 000 daltons and that the secreted protein is incorporated into elastin fibers without cleavage to a protein of considerably smaller size. 相似文献
In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many 32P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of [γ-32P]ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of [γ-32P] phosphate from [γ-32P]ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: [ATP] = [ATP]0 e?[cAMP]kt. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the protein kinase stimulation assay based on transfer of [32P] phosphate from [γ-32P]ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use. 相似文献
With the aid of a sulfur-specific flame photometric detector, an emission of volatile sulfur was detected from leaves of cucumber (Cucumis sativus L.), squash and pumpkin (Cucurbita pepo L.), cantaloupe (Cucumis melo L.), corn (Zea mays L.), soybean (Glycine max [L.] Merr.) and cotton (Gossypium hirsutum L.). The emission was studied in detail in squash and pumpkin. It occurred following treatment of the roots of plants with sulfate and was markedly higher from either detached leaves treated via the cut petiole, or whole plants treated via mechanically injured roots. Bisulfite elicited higher rates of emission than sulfate. The emission was completely light-dependent and increased with light intensity. The rate of emission rose to a maximum and then declined steadily toward zero in the course of a few hours. However, emission resumed after reinjury of roots, an increase in light intensity, an increase in sulfur anion concentration, or a dark period of several hours.
The emission was identified as H2S by the following criteria: it had the odor of H2S; it was not trapped by distilled H2O, but was trapped by acidic CdCl2 resulting in the formation of a yellow precipitate, CdS; it was also trapped by base and the contents of the trap formed methylene blue when reacted with N,N-dimethyl-p-phenylenediamine and Fe3+.
H2S emission is not the cause of leaf injury by SO2, since bisulfite produced SO2 injury symptoms in dim light when H2S emission was low, while sulfate did not produce injury symptoms in bright light when H2S emission was high.
The maximum rates of emission observed, about 8 nmol min−1 g fresh weight−1, are about the activity that would be expected for the sulfur assimilation pathway of a normal leaf. H2S emission may be a means by which the plant can rid itself of excess inorganic sulfur when HS− acceptors are not available in sufficient quantity.
Methodology is presented for the determination of growth yield (Y(g)) and maintenance coefficient (m) for carbon utilization of plant cells grown in suspension culture. Estimation of Y(g) and m requires measurements of specific growth rate (micro) and specific rate of substrate uptake (q) at different growth limiting substrate concentrations. Batch culture of tobacco cells did not permit evaluation of Y(g) and m because micro is constant and maximal during most of the growth cycle. In batch culture, the period of declining specific growth rate is extremely brief because of the rapid transition from logarithmic growth to stationary phase. This occurs because the K(m) for growth is relatively small compared to the initial sucrose concentration. Thus, when the substrate level reaches the K(m), the large mass of cells rapidly depletes the remaining substrate. In contrast, semicontinuous culture facilitates the determination of Y(g) and m because various steady-state growth rates can be achieved. Mathematical expressions were developed to determine the effective values of micro and q over the semicontinuous replacement interval. The validity of this approach was verified by conducting simulations using experimentally determined parameters. 相似文献
Summary Growth reduction or cessation is an initial response of Atriplex nummularia L. cells to NaCl. However, A. nummularia L. cells that are adapted to 342 and 428 mM NaCl are capable of sustained growth in the presence of salt. Cells that are adapted to NaCl exhibit a reduced rate of division compared to unadapted cells. Unlike salt adapted cells of the glycophyte Nicotiana tabacum L., A. nummularia L. cells do not exhibit reduced rate of cell expansion after adaptation. However, the cell expansion rate of unadapted A. nummularia L. cells is considerably slower than that of unadapted glycophyte cells and this normally low rate of cell expansion may contribute to the enhanced capacity of the halophyte to tolerate salt. Turgor of NaCl adapted cells was equivalent to unadapted cells indicating that the cells of the halophyte do not respond to salt by osmotic over adjustment as reported for the glycophyte tobacco (Binzel et al. 1985, Plant Physiol. 79:118–125). 相似文献
Cell lines of tomato (Lycopersicon esculentum Mill., cv. VFNT-Cherry) have been isolated, which are capable of growing in media containing polyethylene glycol (PEG) 6000 with water potentials as low as −15 or −22 bar. After prolonged exposure to media containing PEG, these cell populations have reduced abilities to grow in the absence of PEG. Upon resuspension in PEG-free medium, the cells swell and begin to release metabolites, including protein. Measurement by plasmometry of the osmotic potential of cells selected in medium with −22 bar water potential indicates that they maintain, at the end of the growth cycle, an osmotic potential of approximately −26 bar. This is compared to an osmotic potential of −9 bar for nonselected cells in medium without PEG, having an initial water potential of −4 bar. Thus, considerable osmotic adjustment occurs as a result of exposure to external low water potential. The results also indicate that PEG does not contribute significantly to osmotic adjustment of the cells. 相似文献
Endogenous levels of cyclic adenosine-3′:5′-monophosphate in coleoptile first leaf segments of oat (Avena sativa L.), potato (Solanum tuberosum L.) tubers, tobacco (Nicotiana tabacum L.) callus, and germinating seeds of lettuce (Lactuca sativa L.) were measured with a modified Gilman binding assay and a protein kinase activation assay. The incorporation of adenosine-8-14C into compounds with properties similar to those of cyclic AMP was also measured in studies with germinating lettuce seeds. The binding assay proved reliable for mouse and rat liver analyses, but was nonspecific for plant tissues. It responded to various components from lettuce and potato tissues chromatographically similar to but not identical with cyclic AMP. The protein kinase activation assay was much more specific, but it also exhibited positive responses in the presence of compounds not chromatographically identical to cyclic AMP. The concentrations of cyclic AMP in the plant tissues tested were at the lower limits of detection and characterization obtainable with these assays. The estimates of maximal levels were much lower than reported in many previous studies. 相似文献
Plants were regenerated from both the wild type and a stable NaCI-tolerant line of tobacco cells ( Nicotiana tabacum/gossii ). The regeneration process was much more difficult in the case of the NaCI-tolerant line and was only successful in the absence of NaCI. These plants differed morphologically from those regenerated from the wild type cell line, exhibiting abnormally short internodes, small leaves and reduced growth. Cell suspension cultures derived from plants regenerated from the stable NaCI-tolerant line retained a high level of tolerance to salt. The NaCI-concentration required to reduce fresh and dry weight gain by 50% was about twice that observed in the case of the cells obtained from wild type plants. The results presented here, together with those of Watad et al. (1985), indicate that resistance to salt is operating and stable at the cellular level before and after plant regeneration. When the regenerated plants were grown in increasing levels of salt their growth response was not clearly different from that of the plants regenerated from the wild type cell line. However, the survival of plants on high concentrations of NaCI tended to be higher in the case of plants regenerated from the NaCI-tolerant cell line. 相似文献