[Show abstract][Hide abstract] ABSTRACT: Peptidyl proline hydroxylase inhibitors block the growth of cultured soybean (Glycine max) cells and bring about the disappearance of the major salt-extractable hydroxyproline-rich protein, the 33 kilodalton repetitive proline-rich protein (RPRP2). Three polypeptides of 28, 20, and 14 kilodalton that cross-react with an antibody to RPRP2 accumulate in the culture during steady-state growth. In the presence of the proline hydroxylase inhibitors, all of these repetitive proline-rich proteins disappear. These results indicate that the hydroxyproline-rich proteins play a role in cell growth, and that hydroxylation may regulate the steady-state level of at least one of these proteins by influencing its turnover.
[Show abstract][Hide abstract] ABSTRACT: We have resolved and analyzed two proline-rich proteins isolated from the walls of soybean cells in culture. The proteins are similar in amino acid content, containing 20% proline, 20% hydroxyproline, 20% lysine, 16% valine, 10% tyrosine, and 10% glutamate. The proteins undergo a rearrangement or a limited cleavage in dilute NaOH, but are otherwise remarkably stable to a high concentration of alkali. We have cloned and sequenced a cDNA from soybean axes germinated for 31 hours (1A10-2) coding for a protein that closely corresponds in its amino acid content to that of the proline-rich proteins. The cDNA sequence predicts a decameric repeat of Pro-Pro-Val-Tyr-Lys-Pro-Pro-Val-Glu-Lys. Consequently, this class of proteins is referred to as repetitive proline-rich proteins, i.e., RPRP2 and RPRP3. We have also analyzed RNA gel blots with probes that discriminate between the new cDNA clone and a related cDNA previously reported [SbPRP1; Hong, Nagao, and Key (1987). J. Biol. Chem. 262, 8367-8376]. Messenger RNAs from young seedlings and from soybean suspension cultures correspond primarily to the new RPRP clone (1A10-2), whereas the predominant mRNA accumulating later in the roots corresponds to SbPRP1.
The Plant Cell 10/1989; 1(9):945-52. DOI:10.1105/tpc.1.9.945 · 9.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A cDNA clone that hybridizes to an mRNA (1A10) that accumulates to a substantial level in the axis of the germinating soybean seed was sequenced. The amino acid sequence of the clone indicates an almost perfect repeat of Pro-Pro-Val-Tyr-Lys resulting in a protein containing 40% proline and lacking serine and histidine. On the likelihood that such a protein might be a hydroxyproline-rich cell-wall glycoprotein (HRGP), cell walls of a soybean cell culture were extracted by procedures used to obtain soluble basic cell-wall glycoproteins, and the proteins were fractionated and purified. A 33-kDa protein (and possibly a 28-kDa protein) was obtained that has an amino acid distribution similar to that of the cDNA clone. The protein lacks histidine and serine and contains 20% hydroxyproline and 20% proline. The HRGP is thus distinct both in its amino acid content and in its pentameric repeat of Pro-Pro-Val-Tyr-Lys, with half of the prolines being hydroxylated.
Proceedings of the National Academy of Sciences 03/1988; 85(4):1082-5. DOI:10.1073/pnas.85.4.1082 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Copy-DNA clones have been obtained that distinguish eight messenger mRNAs, moderately abundant in the axes of the germinating soybean (Glycine max (L.) Merr.) seedling. These clones have been used to characterize the size of the mRNAs and to anlyze the accumulation of the mRNAs at different time points and in different parts of the axis during germination and early seedling growth. Three of the mRNAs accumulate to a substantial level by 9 h, a time point before either the beginning of growth or the accumulation of polyribosomes. Four other mRNAs reach a substantial level only at 24 h, a period when rapid seedling growth is occurring. Those mRNAs whose accumulation begins at 24 h were found only in the top (hypocotyl) half of the 24-h seedlings, while the remaining mRNAs were present also in the bottom half of the seedlings in different amounts. By 44 h, the bottom 0.5 cm of the seedlings, i.e., the region of meristematic growth, had little or none of the mRNAs, with the exception of one mRNA. These temporal and spatial observations indicate that many of the mRNAs are not involved simply in the general maintenance of ongoing cell proliferation, but that they may be related to differentiation during early seedling formation. Further, the early accumulating mRNAs may be functioning in regulating the onset of seedling growth.
[Show abstract][Hide abstract] ABSTRACT: The requirement for the synthesis of RNA and DNA in early germination of wheat (Triticum aestivum var Newana) embryonic axes has been studied by incubating embryos in the presence of appropriate inhibitors and monitoring both embryo growth and the rates of specific metabolic processes. Experiments with 5-fluorouridine showed that both rRNA and DNA synthesis could be curtailed by 60 to 70% without affecting embryo growth to 24 hours. Similarly, the presence of mitomycin C and methotrexate inhibited DNA synthesis 70%, with only a small effect on growth. Experiments with a range of concentrations of cordycepin and alpha-amanitin indicated that mRNA synthesis could be curtailed by 30 to 40% within the first 8 hours of germination with only a small effect on embryo growth. Thus, at least the initial phases of seed embryo germination are not closely linked to the synthesis of mRNA, rRNA, or DNA. Maximal sensitivity of embryo growth was obtained with cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl propionamide, supporting the idea that protein synthesis is the macromolecular process most closely linked to early germination.
[Show abstract][Hide abstract] ABSTRACT: Wheat (Triticum aestivum L. var. Lew) embryonic axes take up externally supplied radioactive amino acid (from a solution greater than 2 millimolar) such that the specific radioactivity of the total internal amino acid rapidly reaches that of the external solution. Nevertheless, incorporation of radioactive amino acid into protein increases steadily as the concentration of external amino acid is increased, indicating that the amino acid that is precursor to protein synthesis is not in equilibrium with the total internal amino acid pool. When the external source of amino acid is removed, incorporation of radiolabeled amino acid into protein continues at a rate comparable to that of embryos maintained in the radioactive solution. In explanation of these data, it is suggested that there are two separate cytoplasmic pools of amino acids, one a protein synthesis precursor pool, and the second, an expandable pool into which exogenous radioactive amino acids are taken up. The protein synthesis pool is fed at a limited rate from the expandable pool and at a far greater rate from an endogenous source. As a consequence, the specific activity of the amino acid that is the precursor for protein synthesis is considerably below that of the total internal pool and is determined by the rate of movement into the protein synthesis pool from the expanded radioactive cytoplasmic pool.The rate of movement of amino acids from the expandable pool into the protein synthesis pool increases approximately 5-fold during the initial 4.5 hours of embryo germination. When this change is considered in analyzing the relative rates of protein synthesis, there is probably no more than a 2-fold increase in protein synthetic capacity between embryos germinated for 1.5 and 4.5 hours. The leveling off of the change in transport capacity after 4.5 hours suggests that the earlier increase in the rate of this process may be a necessary step before the embryos can begin to accelerate their growth rate.
[Show abstract][Hide abstract] ABSTRACT: Growth and the rate of protein synthesis in germinating wheat embryonic axes are inhibited by the analog 6-azauridine via a mechanism which is independent of the usual effect of this compound as an inhibitor of de novo synthesis of UTP. The effects on growth and protein synthesis can be separated from that on UTP biosynthesis by analyses of the kinetics by which each effect is maximized following a 1.5-h pulse with 6-azauridine, and by saturation of the responses at different doses of the analog. The inhibitions of growth and protein synthesis are apparently not mediated through the rate of poly A(+) RNA synthesis (reduced as little as 8%), but rather by an effect on translation. Since cordycepin reduces the azauridine inhibitions of growth and protein synthesis, it is suggested that these latter effects of 6-azauridine may depend upon the synthesis of an inhibitory azauridyl-RNA.
Archives of Biochemistry and Biophysics 04/1982; 215(1):140-7. DOI:10.1016/0003-9861(82)90288-0 · 3.02 Impact Factor