Secondary structure determination by FTIR of an archaeal ubiquitin-like polypeptide from Natrialba magadii.

The ubiquitin protein belongs to the ß-grasp fold family, characterized by four or five ß-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 ß-sheet structures than in α-helix. These findings suggest that P400 is a structural homologue of the ubiquitin family proteins.

Two dimensional non equilibrium pH gel electrophoresis mapping of cytosolic protein changes caused by dietary protein depletion in mouse liver.

Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate the effects of dietary protein depletion on the protein composition of mouse liver cytosol. Analysing the cytosol from both normal and protein depleted liver, the position in gels of more than three hundred protein spots was determined. After 5 days of protein depletion, about 20% of the spots either increased or decreased more than 2 fold. Five spots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recognised by specific antibodies. The glutathione S-transferase (GSTs) subunits Ybl, Yc and Yf were identified by the simultaneous analysis of both glutathione-binding cytosolic proteins and the corresponding standards. As estimated by internal optical density (IOD) of spots, the changes caused by protein depletion in GAPDH and GST subunit contents were similar to those obtained by other methods. By means of mass spectrometric analysis of tryptic peptides generated from spots and/or comparison of two-dimensional gel electrophoretic patterns, carbonic anhydrase III (CAIII), Cu, Zn superoxide dismutase (CuZnSOD) and a cytochrome P450 cytosolic protein (cyt P450) were identified. These three proteins, as well as GSTs, are related with intracellular detoxification and free radical scavenging systems. Their contents were regulated by dietary protein restriction in a manner indicative of diminished liver defence against oxidising agents.

Contribution of breakdown in the regulation of mouse liver glutathione S-transferase subunits during protein depletion and re-feeding.

The in vivo radioactivity decay of glutathione S-transferase (GST) was observed in livers of normal-fed (N), protein-depleted (D), and re-fed mice (R), labelled with [35S]methionine. Half-lives in days at N, D and R, respectively, were: total GST, 1.7, 1.4, 4.3; Yb1-subunit, 2.0, 1.8, 3.3; Yc-subunit, 2.4, 1.0, 4.2; Yf-subunit, 1. 1, 2.1, 5.0. These findings, together with the fact that the proportions of GST-subunits depend on dietary protein, show that breakdown contributes differentially to control the content of each GST subunit. Data also indicate that GSTs are long-life proteins.

Protein depletion and refeeding change the proportion of mouse liver glutathione S-transferase subunits.

The effect of protein depletion followed by refeeding with a normal diet on the content of mouse liver cytosolic proteins was studied. By peptide-mass fingerprinting and N-terminal sequencing, three polypeptides whose contents changed with dietary protein level were identified as glutathione S-transferases (GST) Yb1, Yc and Yf subunits. Five days of depletion caused the increase of Yb1 and Yf (21.6% and 78.5%, respectively) and the decrease of Yc (31.2%). After two days of refeeding, Yb1 and Yc were practically restored, while the neoplastic marker Yf remained higher (63.4%). None of the nutritional conditions tested induced new GSTs. While protein depletion-refeeding altered the ratios between the constitutive GST subunits, total liver GST content and activity were unaffected by depletion and slightly increased by refeeding. The increased amounts of Yb1 and Yf, and the maintenance of total GST content, indicate that during protein depletion, the GST subunits levels are controlled by mechanisms different from the majority of cytosolic proteins.

Influence of protein nutrition on calpain activity of mouse kidney: modulation by calpastatin.

The effect of protein depletion and refeeding with a normal diet on calpain activity was examined in mouse kidney soluble homogenate. In terms of units per gram of protein, it increased 2.9 times with depletion and decreased upon refeeding. After a DEAE-Sephacel chromatography, the homogenate yielded three enzymatic activities. Their sum, assessed as total calpain activity, was higher than the activity measured before fractionation and did not appreciably change during protein depletion and refeeding. Because the proportion of total activity displayed by the complete homogenate increased with depletion and decreased with refeeding, a low calpastatin content in depleted kidney was envisaged. This was confirmed by direct estimations: depleted kidney had 6 times less calpastatin compared to both normal and 16 h refed tissue. We concluded that a decrease in calpastatin content contributes to an increased calpain activity related to degradable protein in protein depleted kidney. In view of this, it seems not unlikely that the in vivo rate of protein breakdown depicted by kidney during protein depletion and refeeding is in part effected through modulation of the calpain proteolytic system.

Accumulation of cytosolic glyceraldehyde-3-phosphate dehydrogenase RNA under biological stress conditions and elicitor treatments in potato.

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.

Effect of dietary level of protein on the activity of mouse liver calpain.

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.

Influence of protein nutrition on proteolytic activity and histone content in isolated mouse liver nuclei.

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.

Dietary level of protein regulates glyceraldehyde-3-phosphate dehydrogenase content and synthesis rate in mouse liver cytosol.

The content of liver cytosolic proteins was studied in mice subjected to protein depletion followed by refeeding with a normal diet. Depletion elicited either the accumulation or the decrease of several polypeptides, being the early increase of a M(r) 36,000 polypeptide the most pronounced change observed. The refeeding with a normal diet for 2 days caused a return of the cytosol protein composition to that of normally fed animals. The M(r) 36,000 polypeptide was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Its molecular weight, the sequence of its first twenty amino acid residues, its amino acid composition and its antigenic properties were found to be similar with those of GAPDH from different mammalian cells. During the first 2 days of protein depletion, both the GAPDH polypeptide content and activity increased. Thereafter, the enzymatic activity of GAPDH decreased, whereas GAPDH protein mass decreased in a lesser extent. The accumulation of GAPDH and other particular polypeptides in the cytosols of protein depleted mice was associated with an increased synthesis. The refeeding with a normal diet caused an immediate return to the synthesis pattern of normal livers.

Effect of dietary level of protein on the metabolism of mouse liver nuclear proteins.

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.

Proteolytic activities in kidney and liver during refeeding of protein depleted mice.

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.

Proteolytic activities in kidney and liver during refeeding of protein depleted mice.

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.

Estimation of rRNA synthesis and degradation rates in senescing wheat leaves.

Changes in cytoplasmic and chloroplast rRNA content and rates of rRNA synthesis and degradation of detached wheat leaves were determined. It was found that rRNA loss is proportionally higher in chloroplasts than in cytoplasm. Rates of synthesis were measured by incorporation of large amounts of [3H]orotic acid into rRNA. This approach overcame size differences between pyrimidine pools of cells under different physiological status. Furthermore, these pools reached nearly the same specific radioactivity as that of the administered solution. Rates of degradation were estimated either as the difference between synthesis and net variation of rRNA or by disappearance of radioactivity from 32P-labeled rRNA. Results indicated a decrease in the net rRNA synthesis capacity of leaves after 48 h of detachment. However, the fractional rates of rRNA synthesis were maintained in both cytoplasm and chloroplasts. Ribosomal RNA degradation rates were 2.5-fold higher in chloroplast than in cytoplasm. The observed chloroplast rRNA loss is due to an increased degradation rate which is 15-fold higher than the synthesis rate 48 h after detachment.

Quantification of the kinetin effect on protein synthesis and degradation in senescing wheat leaves.

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.

Increased formation and keeping of ribosomes during dietary recovery of mouse kidney.

In the kidney of 5-day protein-depleted mice there is a decrease of 23% in the rRNA mass. When these animals are fed with a complete diet, rRNA content is restored to its normal value after 24 h of refeeding. The mechanisms that underlie this phenomenon were studied. It was found that the activity of rRNA polymerase I in the nuclei of kidneys from refed mice showed an increase of about twofold compared with the activity in normal and protein-depleted nuclei. The in vivo incorporation of a large dose (nontrace) of [14C]orotic acid into rRNA was also twofold enhanced in kidneys from refed mice. Ribosome degradation (measured by the disappearance of radioactivity from either ribosomal proteins or rRNA previously labeled by the injection of NaH14CO3 and [14C]orotic acid to the mice, respectively) stopped during the 1st day after refeeding. The estimation of the difference between the rRNA synthesis rate and the net rRNA increase also demonstrated a decrease in the rRNA degradation rate in refed mice.

Protein metabolism in senescing wheat leaves : determination of synthesis and degradation rates and their effects on protein loss.

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.

Breakdown of proteins from mouse liver subcellular fractions. Effect of nutritional changes.

The rapid restoration of liver protein mass observed in protein-depleted mice when they are fed with an adequate diet is quantitatively explained by a large decrease in the average rate of breakdown of total liver proteins. This study was performed in order to know whether this inhibition of breakdown affects in the same way all the protein constituents of the tissue, or only affects a group of these proteins belonging to a particular subcellular fraction. Subcellular fractions were obtained by differential centrifugation. The relative rates of breakdown of their proteins were estimated by the conservation of radioactivity in these proteins previously labelled by the administration of NaH14CO3 to mice. The results obtained indicated: 1) a general decrease in the rate of breakdown of proteins of subcellular fractions from re-fed livers compared with livers of protein-depleted mice; 2) a decrease of breakdown of proteins from cytosol in re-fed mice which is higher as lower is the molecular weight of the proteins subunits.

Breakdown of proteins from mouse liver subcellular fractions. Effect of nutritional changes.

The rapid restoration of liver protein mass observed in protein-depleted mice when they are fed with an adequate diet is quantitatively explained by a large decrease in the average rate of breakdown of total liver proteins. This study was performed in order to know whether this inhibition of breakdown affects in the same way all the protein constituents of the tissue, or only affects a group of these proteins belonging to a particular subcellular fraction. Subcellular fractions were obtained by differential centrifugation. The relative rates of breakdown of their proteins were estimated by the conservation of radioactivity in these proteins previously labelled by the administration of NaH14CO3 to mice. The results obtained indicated: 1) a general decrease in the rate of breakdown of proteins of subcellular fractions from re-fed livers compared with livers of protein-depleted mice; 2) a decrease of breakdown of proteins from cytosol in re-fed mice which is higher as lower is the molecular weight of the proteins subunits.