Pyrophosphate Dependent Phosphofructokinase of Citrullus lanatus: Molecular Forms and Expression of Subunits

During germination and seedling establishment, the total pyrophosphate-dependent phosphofructokinase (PFP) activity in the cotyledons increases. Two types of subunits with molecular weights of 68 (α-subunit) and 65 (β-subunit) kilodaltons are present. The increase in activity coincides with an approximately 10-fold increase in β-subunit and twofold increase in α-subunit content. Different isoforms of PFP are present at all stages of incubation, but the ratio between the isoforms significantly changes. A linear relationship exists between the ratio of the two PFP subunits and the ratio of the two isoforms of the enzyme. The more anionic (peak 2) isoform of the enzyme apparently is favored by a high ratio of total β-subunit to α-subunit content. The β- to α-subunit ratio of the peak 2 isoform is also approximately fivefold higher than that of the peak 1 (less anionic) isoform. It is evident that the two subunits are not coordinately expressed and the level of expression of each subunit appears to be the primary factor determining the molecular form in which the enzyme is present. In some tissues, only the 65 kilodalton polypeptide is expressed in large amounts. The peak 1 isoform has a higher affinity for pyrophosphate than the peak 2 isoform, while the affinity for fructose-6-phosphate is similar. Both molecular forms are activated by fructose-2,6-bisphosphate.     

Changes in Wheat Leaf Polysomal Messenger RNA Populations during the Early Stages of Rust Infection: EFFECTS OF CHLORAMPHENICOL AND LINCOMYCIN ON CELL-FREE TRANSLATION BY POLYSOMES FROM HEALTHY AND INFECTED LEAVES

Polysomes isolated from a susceptible variety of wheat leaves (cultivar W2691) and those inoculated with the wheat stem rust fungus (f. sp. tritici, race 126-ANZ-6, 7) were incubated in a cell-free protein-synthesizing system. Under these conditions, different size classes of polypeptides, ranging in molecular weight from 10,000 to 80,000, are radiolabeled. Using double-isotope labeling technique, we show that some discrete size classes of polypeptides are synthesized in significantly greater quantitites by polysomes from inoculated leaves compared to the corresponding size classes synthesized by polysomes from healthy leaves. These results confirm our previous observation that there are significant changes in the wheat leaf polysomal messenger RNA populations at 3 days after inoculation with the rust fungus.The effects of the organelle-specific inhibitors of protein synthesis, chloramphenicol and lincomycin, on in vitro polysomal messenger RNA translation were investigated. The polypeptides synthesized by polysomes from healthy and inoculated leaves in the presence of chloramphenicol were compared. The results show that, even in the presence of this antibiotic, the polysomes from inoculated leaves synthesize greater quantities of some size classes of polypeptides. These data indicate that changes in polysomal messenger RNA populations involve, at least in part, cytoplasmic messenger RNA.     

Studies on Pea Ribosomal Proteins: Conformational and Biological Activity Changes of Ribosomal Subunits Derived by NH(4)Cl Dissociation

Ribosomal subunits prepared by NH(4)Cl dissociation (0.5 m) of the monomeric ribosomes were much less active in in vitro protein synthesis than those prepared by KCl dissociation. The decrease in activity correlated with a detachment of some proteins (L(2) and L(9) as shown by gel electrophoresis) within the 60S ribosomal subunits. Subunits prepared with 0.3 m NH(4)Cl retained L(2) and L(9), but the activity remained low. Incubation of these 60S subunits in TKM buffer (50 mm tris [pH 7.5], 20 mm KCl, and 5 mm MgCl(2)) for 20 min at 37 C restored the activity almost to the level of those obtained by KCl dissociation. Treatment of the 0.3 m NH(4)Cl-derived 60S subunits with a protein reagent, Procion brilliant blue, prior to extraction of the ribosomal proteins resulted in the loss of L(2) and L(9), showing that these proteins were made accessible for dye binding. These observations suggest that a considerable degree of unfolding of the 60S subunit occurs at 0.3 m NH(4)Cl (this apparently leads to a preferential detachment of L(2) and L(9) at 0.5 m NH(4)Cl) and that the activity of the purified subunits depends not only on the presence of L(2) and L(9) but also on the organization of these proteins within the 60S subunits.     

Development of Mitochondrial Activities in Pea Cotyledons: INFLUENCE OF DESICCATION DURING AND FOLLOWING GERMINATION OF THE AXIS

Mitochondria in 4-hour imbibed and desiccated pea cotyledons develop in a similar manner upon rehydration to those in cotyledons hydrated only once. As a consequence of desiccation during imbibition, mitochondria revert to their original state as in the mature dry cotyledon, although limited damage occurs. This damage is more evident when the initial imbibition time before desiccation is longer. The presence of the axis does not protect cotyledonary mitochondria from damage, nor does it influence mitochondrial development upon rehydration of desiccated cotyledons. During the early hours after the start of imbibition mitochondrial development is dependent both upon hydration levels of the cotyledon and upon other metabolic processes, as shown by sensitivity to conditions that prevent ATP synthesis.     

Glutathionylation of the Aquaporin-2 Water Channel: A NOVEL POST-TRANSLATIONAL MODIFICATION MODULATED BY THE OXIDATIVE STRESS*

Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and urine concentration. AQP2 undergoes different regulated post-translational modifications, including phosphorylation and ubiquitylation, which are fundamental for controlling AQP2 cellular localization, stability, and function. The relationship between AQP2 and S-glutathionylation is of potential interest because reactive oxygen species (ROS), produced under renal failure or nephrotoxic drugs, may influence renal function as well as the expression and the activity of different transporters and channels, including aquaporins. Here, we show for the first time that AQP2 is subjected to S-glutathionylation in kidney and in HEK-293 cells stably expressing AQP2. S-Glutathionylation is a redox-dependent post-translational modification controlling several signal transduction pathways and displaying an acute effect on free cytosolic calcium concentration. Interestingly, we found that in fresh kidney slices, the increased AQP2 S-glutathionylation correlated with tert-butyl hydroperoxide-induced ROS generation. Moreover, we also found that cells expressing wild-type human calcium-sensing receptor (hCaSR-wt) and its gain of function (hCaSR-R990G; hCaSR-N124K) had a significant decrease in AQP2 S-glutathionylation secondary to reduced ROS levels and reduced basal intracellular calcium concentration compared with mock cells. Together, these new findings provide fundamental insight into cell biological aspects of AQP2 function and may be relevant to better understand and explain pathological states characterized by an oxidative stress and AQP2-dependent water reabsorption disturbs.     

In Vitro Synthesis and Processing of Wheat alpha-Amylase : TRANSLATION OF GIBBERELLIC ACID-INDUCED WHEAT ALEURONE LAYER RNA BY WHEAT GERM AND XENOPUS LAEVIS OOCYTE SYSTEMS

Wheat (Triticum aestivum) RNA was used to program synthesis of the α-amylase protein by Xenopus laevis oocytes. A 41,500-dalton protein was made which was identified as α-amylase by immunoprecipitation with rabbit anti-α-amylase antiserum raised against the purified wheat protein and by its co-migration with authentic α-amylase on sodium dodecyl sulfate polyacrylamide gels. Synthesis of α-amylase was dependent upon injection of RNA extracted from gibberellic acid-induced aleurone layers from wheat. The amount of α-amylase produced was proportional to the amount of RNA injected and reached a plateau within 4 hours after injection. When the same RNA was translated in a wheat germ cell-free translation system, a 43,000-dalton protein was produced. Addition of dog pancreas microsomal membranes to the wheat germ translation system resulted in processing of the α-amylase protein to a form which co-migrated with authentic α-amylase purified from malted wheat and with the protein synthesized in oocytes.     

Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions: CHARACTERIZATION BY HYDROGEN EXCHANGE MASS SPECTROMETRY AND NMR*

The susceptibility of the cellular prion protein (PrP^C) to convert to an alternative misfolded conformation (PrP^Sc), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23–231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a β-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4.