The effect of hydrostatic pressure on the stability of tetrameric rabbit muscle pyruvate kinase was investigated by enzyme activity measurements, size-exclusion chromatography, circular dichroism and fluorescence spectroscopies. Under nonreducing conditions, enzyme activity was irreversibly inhibited by increasing pressure and was completely abolished at 350 MPa. Inhibition was dependent on the concentration of pyruvate kinase, indicating that it was related to pressure-induced subunit dissociation. Size-exclusion chromatography of pressurized samples confirmed a decrease in the proportion of tetramers and an increase in monomers relative to native samples. Addition of dithiothreitol immediately following pressure release led to full recovery of both enzyme activity and of native tetramers. Furthermore, no irreversible inhibition of pyruvate kinase was observed if pressure treatment was carried out in the presence of dithiothreitol. These data suggest that pressure-dissociated monomers undergo conformational changes leading to oxidation of sulfhydryl groups, which prevents correct refolding of native tetramers on decompression. These conformational changes are relatively subtle, as indicated by the lack of significant changes in far-UV circular dichroism and intrinsic fluorescence emission spectra of previously pressurized samples. The effects of various physiological ligands on the pressure stability of pyruvate kinase were also investigated. A slight protection against inhibition was observed in the simultaneous presence of K + , Mg 2+ and ADP. Both phosphoenolpyruvate and the allosteric inhibitor, phenylalanine, caused marked stabilization against pressure, suggesting significant energy coupling between binding of these ligands and stabilization of the tetramer.Keywords: pyruvate kinase; hydrostatic pressure; ligand binding; subunit dissociation; inactivation.Pyruvate kinase is an important regulatory glycolytic enzyme that catalyses the transfer of the phosphoryl group from phosphoenolpyruvate (PEP) to ADP, generating ATP and pyruvate. Pyruvate kinase requires both K and Mg 2 for activity [1±6]. In mammals, four different isozymes of pyruvate kinase that have different kinetic properties exist. The muscle isozyme (M1) is a homotetramer of 59 kDa subunits and does not exhibit cooperative behavior [7]. The kinetic mechanisms of pyruvate kinase from various sources and organisms have been investigated in detail [8±13]. The three-dimensional structure of rabbit muscle pyruvate kinase complexed with K + , Mn 2+ and pyruvate has recently been determined [14].The unfolding and refolding of pyruvate kinase by denaturing agents such as guanidine hydrochloride and urea have been investigated and were shown to be only partially reversible [15±20]. Hydrostatic pressure has been increasingly used to study protein subunit interactions and stability (reviewed in [21±23]). Protein oligomers ranging from dimers to large aggregates such as viral particles have been shown to undergo pressure-induced subunit dissociation. Press...
Palm seeds are known for displaying dormancy and a thick endocarp that delays germination and embryo growth; but water treatments may accelerate their germination process. Additionally, ex-situ conservation can cause seed viability loss over time. Data on seed dormancy and storage have been neglected for many native palm species in Brazil. We investigated the effect of water treatments and one-year storage on the germination of Copernicia alba Morong, a palm tree from the Brazilian Pantanal wetland. To address our aim, fresh and stored pyrenes were immersed in water (at room temperature for 24, 48, and 72h) and in hot water (~75°C for 5- and 10-min). Fresh pyrenes germinated up to 84% in control, reaching 100% after water immersion for 48 and 72h. In contrast, one-year storage reduced germination by almost 50%, but water immersion slightly increased the germination of stored pyrenes. On the contrary, hot water decreased germination for both fresh and stored pyrenes. Seeds of C. alba may be classified as orthodox seed storage behavior. Taken all together, water treatments at room temperature improved the germination of the pyrenes. In contrast, long-term seed storage and hot-water treatments may jeopardize germination.
Emergence and early seedling growth of Dilodendron bipinnatum Radlk in different substratesEmergência e crescimento inicial de mudas de Dilodendron bipinnatum Radlk em diferentes substratos
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