Activation and inhibition of rabbit muscle pyruvate kinase by transition-metal ions.
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ABSTRACT:
SUBMITTER: Ainsworth S
PROVIDER: S-EPMC1165187 | biostudies-other | 1975 Jan
REPOSITORIES: biostudies-other
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Project description:1. Pyruvate kinase (ATP-pyruvate phosphotransferase, EC 2.7.1.40) from Ehrlich ascites-tumour cells was purified approximately fivefold by chromatography on DEAE-cellulose. The enzyme was shown to have an absolute requirement for one univalent and for one bivalent metal ion. 2. The univalent metal ion requirements were satisfied by K(+), Rb(+) or NH(4) (+); Na(+) and Cs(+) were weak activators but Li(+) was inactive. 3. Ca(2+) exhibited ;non-competitive' and ;apparent competitive' effects in relation to the K(+) activation. 4. The bivalent metal ion requirements were satisfied by Mg(2+), Mn(2+) or Co(2+); Ba(2+), Sr(2+), Ca(2+), Ni(2+), Be(2+) and Cu(2+) were inactive. Mn(2+) and Co(2+) were better activators than Mg(2+). 5. The bivalent metal ion requirements of purified pyruvate kinase from rabbit muscle were satisfied by Mg(2+), Mn(2+), Co(2+) and to a smaller extent by Ni(2+). Mn(2+) and Co(2+) were better activators than Mg(2+). 6. Ca(2+) competitively inhibited the activation by Mg(2+), Mn(2+) and Co(2+) for both the tumour and rabbit enzymes. 7. It is concluded that there are no significant differences in metal ion specificity between the tumour and rabbit enzymes. 8. The possible role of metal ions in regulating enzymic and metabolic activities is considered further.
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Project description:The refolding of rabbit muscle pyruvate kinase after denaturation by guanidine hydrochloride was studied. On dilution of the denaturing agent, enzyme activity is only partially regained. The extent of regain of activity is dependent on protein concentration, showing a marked decrease at higher concentrations. The failure to regain complete activity appears to be related to the formation of inactive aggregates, which can be separated from active enzyme by gel filtration. Insoluble aggregates can be partially re-activated after solubilization in guanidine hydrochloride. Changes in the circular-dichroism and fluorescence spectra during refolding suggest that a partially folded, inactive species is formed rapidly; this differs from native enzyme in being more susceptible to proteolysis by trypsin.
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Project description:The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg(2+) catalysed by rabbit muscle pyruvate kinase. The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg(2+), pyruvate and MgATP, and that dead-end complexes, between pyruvate, ADP and Mg(2+), form randomly and exist in equilibrium with themselves and other substrate complexes. Values were determined for the Michaelis, dissociation and inhibition constants of the reaction and are compared with values ascertained by previous workers.
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Project description:Organic-inorganic perovskite materials have become star materials for future wide band gap optoelectronics due to their excellent optical and electrical properties. However, the lead ions inside perovskites have become a crucial environmental issue in the commercialization of wide band gap perovskite devices . This research tries to find the structure and properties of lead-free perovskite materials by screening Sn2+ and transition-metal ions to replace Pb2+ within the methylammonium (MA)-based chloride perovskite and find out a new two-dimensional structure of MA-based transition-metal ion chlorides. Overall, MAZnCl3 may be a potential ultraviolet-C luminescent material with a stable two-dimensional structure with a wide band gap of 5.64 eV, which is suitable for ultraviolet-C luminescence applications.
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Project description:Photoreceptor cell death is the ultimate cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. Similar to cancer cells, photoreceptors maintain pyruvate kinase muscle isoform 2 (PKM2) expression, which is a critical regulator in aerobic glycolysis. Unlike PKM1, which has constitutively high catalytic activity, PKM2 is under complex regulation. Recently, we demonstrated that genetically reprogramming photoreceptor metabolism via PKM2-to-PKM1 substitution is a promising neuroprotective strategy. Here, we explored the neuroprotective effects of pharmacologically activating PKM2 via ML-265, a small molecule activator of PKM2, during acute outer retinal stress. We found that ML-265 increased PKM2 activity in 661?W cells and in vivo in rat eyes without affecting the expression of genes involved in glucose metabolism. ML-265 treatment did, however, alter metabolic intermediates of glucose metabolism and those necessary for biosynthesis in cultured cells. Long-term exposure to ML-265 did not result in decreased photoreceptor function or survival under baseline conditions. Notably, though, ML-265-treatment did reduce entrance into the apoptotic cascade in in vitro and in vivo models of outer retinal stress. These data suggest that reprogramming metabolism via activation of PKM2 is a novel, and promising, therapeutic strategy for photoreceptor neuroprotection.
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Project description:Polypyrrole (PPy) nanoparticles (NPs) possess high near-infrared absorption and good biosafety, showing the potentials as photothermal therapeutic materials. However, the single function and the weak diagnostic function limit the further combination with other functional units to achieve theranostics. In this work, polyaminopyrrole (PPy-NH2) is demonstrated as the alternative of PPy for preparing NPs. Because of the amino group, metal ions, such as Cu(II) and Fe(III) can be loaded in PPy-NH2 NPs, which extends the applications in multimodal theranostics. Systematical studies reveal that the contribution of Cu(II) in multimodal theranostics is greater than Fe(III). Cu can enhance T1 response signal for magnetic resonance imaging (MRI) and be released controllably in the organism, leading to the effect of chemotherapy. Therefore, Cu(II) and Fe(III) co-loaded PPy-NH2 NPs are defined as CuPPy-NH2 NPs. Experimental results indicate that the optimal size of CuPPy-NH2 NPs is 50.2 nm. The photothermal transduction efficiency is 76.4%. After thermochemotherapy, a complete ablation of human oral epithelial carcinoma tumors is observed. No tumor recurrence is found. Methods: Cu(II) and Fe(III) co-loaded PPy-NH2 NPs are prepared through oxidation polymerization by mixing Py-NH2, CuCl2, and FeCl3 in water under stirring at room temperature, which are defined as CuPPy-NH2 NPs. The as-prepared CuPPy-NH2 NPs are tested with a variety of cell and animal experiments for tumor theranostics. Results: CuPPy-NH2 NPs have good light stability, photothermal stability, biosafety and low toxicity. The optimal size of theranostic CuPPy-NH2 NPs is 50.2 nm, which present a photothermal transduction efficiency of 76.4%. The doped Cu(II) ions also show chemotherapeutic behavior. After thermochemotherapy, a complete ablation of human oral epithelial carcinoma tumors is observed. No tumor recurrence is found. Because of the unpaired electron in Cu atomic orbits, CuPPy-NH2 NPs also show T1-weighted magnetic resonance imaging. Conclusions: This kind of transition metal-doped polymer gives a competitive approach for designing and fabricating multimodal theranostic nanodevices, which shows the potential in tumor treatment.
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Project description:Mass spectrometry has been used to determine the number of exchangeable backbone amide protons and the associated rate constants that are altered when rabbit muscle pyruvate kinase (rM1-PYK) binds either the allosteric inhibitor (phenylalanine) or a nonallosteric analogue of the inhibitor. Alanine is used as the nonallosteric analogue because it binds competitively with phenylalanine but elicits a negligible allosteric inhibition, i.e., a negligible reduction in the affinity of rM1-PYK for the substrate, phosphoenolpyruvate. This experimental design is expected to distinguish changes in the protein caused by effector binding (i.e., those changes common upon the addition of alanine vs phenylalanine) from changes associated with allosteric regulation (i.e., those elicited by the addition of phenylalanine binding, but not alanine binding). High-quality peptic fragments covering 98% of the protein were identified. Changes in both the number of exchangeable protons per peptide and in the rate constant associated with exchange highlight regions of the protein with allosteric roles. The set of allosterically relevant peptides identified by this technique includes residues previously identified by mutagenesis to have roles in allosteric regulation by phenylalanine.
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Project description:The regulatory behavior of rabbit pyruvate kinase has been studied as a function of pH. The initial velocity of the enzyme-catalysed reaction as a function of ADP concentration was analysed with the exponential model for a regulatory enzyme. The analysis of the exponential model parameters as functions of pH provided pK values of 6.6 and 8.08 for the free enzyme in its fully ADP-bound conformation. By contrast, the binding of ADP to the ADP-free conformation of the free enzyme did not involve groups that ionize within the pH range (6.2-8.5) of these experiments. The results suggest that homotropic allosteric interactions actually alter the mode of ADP binding. The pK values of 6.63 and 9.00 determined from the analysis of V as a function of pH are readily interpreted in terms of a direct phosphoryl-transfer mechanism in which the beta-phosphoryl group of ADP (pK 6.63) acts as the nucleophile and a lysine epsilon-amino group (pK 9.0) acts as the proton donor in the pyruvate kinase reaction.
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Project description:The large values of spin relaxation enhancement (RE) for PC spin-labels in the phospholipid membrane induced by paramagnetic metal salts dissolved in the aqueous phase can be explained by Heisenberg spin exchange due to conformational fluctuations of the nitroxide group as a result of membrane fluidity, flexibility of lipid chains, and, possibly, amphiphilic nature of the nitroxide label. Whether the magnetic interaction occurs predominantly via Heisenberg spin exchange (Ni) or by the dipole-dipole (Gd) mechanism, it is essential for the paramagnetic ion to get into close proximity to the nitroxide moiety for efficient RE. For different salts of Ni the RE in phosphatidylcholine membranes follows the anionic Hofmeister series and reflects anion adsorption followed by anion-driven attraction of paramagnetic cations on the choline groups. This adsorption is higher for chaotropic ions, e.g., perchlorate. (A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules.) However, there is no anionic dependence of RE for model membranes made from negatively charged lipids devoid of choline groups. We used Ni-induced RE to study the thermodynamics and electrostatics of ion/membrane interactions. We also studied the effect of membrane composition and the phase state on the RE values. In membranes with cholesterol a significant difference is observed between PC labels with nitroxide tethers long enough vs not long enough to reach deep into the membrane hydrophobic core behind the area of fused cholesterol rings. This study indicates one must be cautious in interpreting data obtained by PC labels in fluid membranes in terms of probing membrane properties at different immersion depths when it can be affected by paramagnetic species at the membrane surface.
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Project description:Murine norovirus (MNV) undergoes extremely large conformational changes in response to the environment. The T = 3 icosahedral capsid is composed of 180 copies of ~58-kDa VP1 comprised of N-terminus (N), shell (S), and C-terminal protruding (P) domains. At neutral pH, the P domains are loosely tethered to the shell and float ~15 Å above the surface. At low pH or in the presence of bile salts, the P domain drops onto the shell and this movement is accompanied by conformational changes within the P domain that enhance receptor interactions while blocking antibody binding. While previous crystallographic studies identified metal binding sites in the isolated P domain, the ~2.7-Å cryo-electron microscopy structures of MNV in the presence of Mg2+ or Ca2+ presented here show that metal ions can recapitulate the contraction observed at low pH or in the presence of bile. Further, we show that these conformational changes are reversed by dialysis against EDTA. As observed in the P domain crystal structures, metal ions bind to and contract the G'H' loop. This movement is correlated with the lifting of the C'D' loop and rotation of the P domain dimers about each other, exposing the bile salt binding pocket. Isothermal titration calorimetry experiments presented here demonstrate that the activation signals (bile salts, low pH, and metal ions) act in a synergistic manner that, individually, all result in the same activated structure. We present a model whereby these reversible conformational changes represent a uniquely dynamic and tissue-specific structural adaptation to the in vivo environment.IMPORTANCEThe highly mobile protruding domains on the calicivirus capsids are recognized by cell receptor(s) and antibodies. At neutral pH, they float ~15 Å above the shell but at low pH or in the presence of bile salts, they contract onto the surface. Concomitantly, changes within the P domain block antibody binding while enhancing receptor binding. While we previously demonstrated that metals also block antibody binding, it was unknown whether they might also cause similar conformational changes in the virion. Here, we present the near atomic cryo-electron microscopy structures of infectious murine norovirus (MNV) in the presence of calcium or magnesium ions. The metal ions reversibly induce the same P domain contraction as low pH and bile salts and act in a synergistic manner with the other stimuli. We propose that, unlike most other viruses, MNV facilely changes conformations as a unique means to escape immune surveillance as it moves through various tissues.
| S-EPMC10878237 | biostudies-literature