- [Show abstract] [Hide abstract] ABSTRACT: The ubiquitin protein belongs to the β-grasp fold family, characterized by four or five β-sheets with a single α-helical middle region. Ubiquitin-like proteins (Ubls) are structural homologues with low sequence identity to ubiquitin and are widespread among both eukaryotes and prokaryotes. We previously demonstrated by bioinformatics that P400, a polypeptide from the haloalkaliphilic archaeon Natrialba magadii, has structural homology with both ubiquitin and Ubls. This work examines the secondary structure of P400 by Fourier transform infrared spectroscopy (FTIR). After expression in Escherichia coli, recombinant P400 (rP400) was separated by PAGE and eluted pure from zinc-imidazole reversely stained gels. The requirement of high salt concentration of this polypeptide to be folded was corroborated by intrinsic fluorescence spectrum. Our results show that fluorescence spectra of rP400 in 1.5 M KCl buffer shifts and decreases after thermal denaturation as well as after chemical treatment. rP400 was lyophilized and rehydrated in buffer containing 1.5 M KCl before both immunochemical and FTIR tests were performed. It was found that rP400 reacts with anti-ubiquitin antibody after rehydration in the presence of high salt concentrations. On the other hand, like ubiquitin and Ubls, the amide I' band for rP400 shows 10% more of its sequence to be involved in β-sheet structures than in α-helix. These findings suggest that P400 is a structural homologue of the ubiquitin family proteins.
- [Show abstract] [Hide abstract] ABSTRACT: The proteolytic activity of the leaf extracellular space of wheat cultivars Pigüé and Isla Verde was estimated after inoculation of either detached leaves or plants with the fungus Septoria tritici. Pigüé is resistant, whereas Isla Verde is susceptible to the disease caused by S. tritici. Viable conidiospores of the fungus caused similar increases in both hydrogen peroxide production and chitinase activity of the cultivars studied. In contrast, they caused a decrease in the extracellular serine proteinase activity of Isla Verde and a significant increase in that of Pigüé. Independently of the cultivar from which it was extracted, the extracellular serine proteinase inhibited the germination of Septoria tritici conidiospores. These results suggest that the proteolytic activity of the leaf extracellular space can participate in the defence of wheat plants against Septoria tritici. Its regulation may be controlled by specific defence components of each cultivar.
- [Show abstract] [Hide abstract] ABSTRACT: The effect of low temperature on the protein metabolism of wheat primary leaves was examined. In seedlings transferred from 25 to 5 C, total soluble protein accumulation, in vivo protein synthesis and breakdown, in vitro protein breakdown, and SDS-PAGE profiles of proteinases in gelatine-containing gels were analysed. Leaf protein content increased within a 7-d period (70 % over the initial value) in plants exposed to 5 C. The fast protein accumulation observed on days 0 – 2 was mainly attributed to a decreased breakdown. In further days, parallelly to a slowdown in the rate of protein accumulation, the leaf proteolytic activity increased. The incubation temperature also had an influence on the proteolytic activity: Q 10 values for the 15 – 5 C range were 80 – 200 % higher than those observed for the 25 – 15 C range. On the other hand, the in vivo protein synthesis capacity, at either 25 or 55 C, was not significantly modified in cold-treated plants. In addition to the enhanced activities of two serine-proteinases (previously found in control plants by SDS-PAGE analysis), cold-treated plants displayed a new proteinase, which had not been detected so far.
- [Show abstract] [Hide abstract] ABSTRACT: Wheat leaves(Triticum aestivum L. cv. San Agustin INTA) were detached at the moment they had reached maximum expansion, put in tubes containing water and left in darkness. Under these conditions, leaf protein content decreased mainly as consequence of an increased rate of breakdown. In the range of 0 to 72 h after detachment, western blot analysis of leaf protein extracts displayed both similar proportions of total protein and quality of ubiquitin conjugates. Northern blot analysis of leaf RNA extracts revealed a 1.6 kb ubiquitin mRNA transcript which increased 3.5-fold after 48 h of treatment. Thus wheat leaves maintain both their ability for the ubiquitination of proteins and the transcription of ubiquitin mRNA at stages of senescence in which rates of protein breakdown are increased. The results suggest that the ubiquitin-dependent proteolytic pathway contributes to leaf protein breakdown during senescence
- [Show abstract] [Hide abstract] ABSTRACT: Plants respond to pathogen infection and environmental stress by regulating the coordinate expression of many stress-related genes. In plants, the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced under environmental stress. This work was aimed at investigating whither the expression pattern of cytosolic GAPDH is also modulated upon infection of potato plants (Solanum tuberosum L.) with the late blight fungal agent Phytophthora infestans. Northern blot analysis showed the accumulation of the GAPDH gene transcripts in leaves and stems of inoculated potato plants. When tuber discs were treated with eicosapentaenoic acid (EPA), an elicitor found in P. infestans, GAPDH gene transcripts level increased. The increase was parallel to that of the hydroxymethyl glutharyl coenzyme A reductase (HMGR), an enzyme involved in pathogen defense reactions. Glucans obtained from P. infestans cell wall acts synergistically with EPA on GAPDH and HMGR gene induction. Salicylic acid, an endogenous signal for inducing systemic acquired resistance, was also effective in stimulating the GAPDH transcript accumulation in potato leaves. These experiments suggest that related multi-component factors, which are part of both primary and secondary metabolism, are probably regulated by similar signal transduction pathways when they are induced under biotic or abiotic stress conditions.
- [Show abstract] [Hide abstract] ABSTRACT: The effect of protein depletion and refeeding with a normal diet on mouse liver soluble homogenate calpain activity was studied. It was unchanged when expressed in terms of whole liver (units/liver). However, when expressed in terms of degradable protein (units/mg protein) it increased with depletion and decreased with refeeding. DEAE Sephacel chromatography of soluble homogenate yielded three calpain activities which were eluted at 0.04, 0.16 and 0.23 M NaCl, respectively. On the basis of whole liver, they decreased with depletion and increased with refeeding. Immunochemical analysis revealed similar changes in the mass of the calpain eluted with 0.16 M NaCl. The sum of these three activities (total liver calpain activity) was higher than the activity displayed by the soluble homogenate, indicating that they were separated from calpastatin. Furthermore, the percentage of total calpain activity displayed by soluble homogenate increased with depletion and decreased with refeeding, suggesting that depleted liver had the lowest calpastatin content. This was confirmed by direct measurements which indicated that depleted homogenate had in average 5.5 and 3.1 times less calpastatin compared to normal and 16 hours refed liver, respectively. It is concluded that a remarkable decrease in calpastatin content maintained unchanged whole liver soluble homogenate calpain activity during protein depletion and refeeding and contributes to an increased calpain activity related to degradable protein in depleted livers. This increase is in accordance with the high in vivo rate of protein breakdown depicted by these livers.
- [Show abstract] [Hide abstract] ABSTRACT: The proteolytic activity in isolated liver nuclei from mice subjected to different conditions of protein nutrition and its relationship with histones metabolism was studied. After five days of protein depletion, the nuclear azocaseinolytic activity increases concomitantly with a decrease in the concentration of histones. This activity resembles, in localization, optimum pH and inhibition behavior to rat liver chromatin neutral proteinase that degrades histones. Moreover, these proteins were identified as its main endogenous substrates. Refeeding of the protein depleted mice for 16h with a normal diet was unable to either diminish proteolytic activity or recover the normal histone level. Activity of the multicatalytic proteinase complex (proteasome) was not detected in nuclei. Furthermore, treatments known to activate this enzyme were ineffective. Taken together, these results suggest that nuclear proteases are mainly involved in the regulation of histone levels.
- [Show abstract] [Hide abstract] ABSTRACT: The effect of protein depletion and refeeding on the metabolism of mouse liver nuclear proteins was studied. Five days protein depletion caused a 35% decrease in total nuclear protein. A fast recovery of the lost proteins, except histones, was induced when depleted mice were refed with a normal diet. Depletion caused a decrease in total nuclear protein synthesis, whereas refeeding quickly restored its normal value. The rates of total nuclear protein breakdown were estimated either as the difference between synthesis and protein gain or from the decay of radioactivity in protein labeled by the administration of both sodium [14C]bicarbonate and [35S]methionine. By these procedures, it was found that refeeding caused a slowdown in total nuclear protein breakdown. Hence, the recovery of the protein content observed during refeeding is due to both a restoration of synthesis and a decrease of breakdown. The [14C]bicarbonate procedure did not permit to obtain a high efficiency of label and, therefore, it was unsatisfactory for the measurement of the breakdown of fractionated nuclear proteins. A labeling procedure using [35S]methionine was designed for adequate measures of the decay of radioactivity in these proteins. This allows us to find that a slow down in breakdown affects similarly during refeeding to histones, to non histones, and to a fraction which contains ribonucleoproteins and soluble proteins.
- [Show abstract] [Hide abstract] ABSTRACT: Proteolytic activities at pH 5.0 and pH 7.4 in both kidney and liver during refeeding of protein-depleted mice were determined. Under this nutritional condition, the in vivo rate of protein breakdown is inhibited. Protein depletion caused in both kidney and liver a loss of pH 5.0 proteolytic activity. It was restored to normal values after 12 hours of refeeding with a complete diet. The pH 7.4 proteolytic activity also decreased as a consequence of protein depletion, but remained low during refeeding. Both re-fed kidney and liver lysosomes showed an increased stability to osmotic shock and, therefore, a decreased autophagic activity. The results indicate that both kidney and liver exhibit a similar behavior. The decreased activity of the lysosomal-vacuolar system, and the low pH 7.4 proteolytic activity may account for the observed in vivo protein breakdown inhibition during refeeding of protein-depleted mice.
- [Show abstract] [Hide abstract] ABSTRACT: Wheat leaves (Triticum aestivum L. cv San Agustin INTA) were detached when they reached maximum expansion, put individually in tubes containing water and left in darkness. After 3 days the protein content had decreased to 46% of the initial value. When the leaves were placed in 1 micromolar kinetin, they retained 60% of the initial protein content for the same period. This effect was observed only when leaves were treated with kinetin within the first 24 hours after detachment. The action of kinetin on both protein synthesis and degradation was quantitatively measured. Synthesis was estimated by the incorporation of l-[(3)H]leucine into proteins. It was higher in kinetin treated than in non treated leaves. It contributed to about 14 micrograms of protein retention per leaf in 3 days. Measurement of protein degradation, evaluated by the decay of radioactivity in leaf proteins previously labeled with l-[(3)H] leucine or as the difference between rates of protein synthesis and protein content, showed that kinetin decreased protein breakdown rates. It accounted for about 186 micrograms of protein retention per leaf in 3 days. Hence, kinetin action on protein breakdown was 13-fold average higher than its action on synthesis for the conservation of leaf protein. This difference is higher in early stages of the process.
- [Show abstract] [Hide abstract] ABSTRACT: Wheat leaves (Triticum aestivum L.) at the moment of their maximum expansion were detached and put in darkness. Their protein, RNA and DNA contents, as well as their rates of protein synthesis and degradation, were measured at different times from 0 to 5 days after detachment. Rates of protein synthesis were measured by incorporation into proteins of large amounts of [(3)H]leucine. Fractional rates of protein degradation were estimated either from the difference between the rates of synthesis and the net protein change or by the disappearance of radioactivity from proteins previously labeled with [(3)H]leucine or [(14)C]proline.Protein loss reached a value of 20% during the first 48 hours of the process. RNA loss paralleled that of protein, whereas DNA content proved to be almost constant during the first 3 days and decreased dramatically thereafter.Measurements of protein synthesis and degradation indicate that, in spite of a slowdown in rate of protein synthesis, an increased rate of protein breakdown is mainly responsible for the observed rapid protein loss.
Universidad Nacional de Mar del Plata
Mar de Plata, Buenos Aires, Argentina
- Biological Research Institute (IIB)