Deletion of Ala144-Lys145 in Thermus thermophilus inorganic pyrophosphatase suppresses thermal aggregation.
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The regions contributing to the thermostability of inorganic pyrophosphatase (PPase, EC 3.6.1.1) from Thermus thermophilus (Tth) were deduced in our previous study by random chimeragenesis, one of them being estimated to be Ala144-Lys145 [Satoh, T., Takahashi, Y., Oshida, N., Shimizu, A., Shinoda, H., Watanabe, M., and Samejima, T. (1999) Biochemistry 38, 1531-1536]. Therefore, we investigated the contributions of these two residues in Tth by preparing a deletion mutant (del.144-145 mutant) of Tth PPase. We examined its thermostability in terms of the CD and fluorescence spectra, and the thermal change in the enzymatic activity. The thermostability of the enzymatic activity of the del.144-145 mutant was similar to that of the wild type Tth PPase, whereas this mutant was more stable against heating. Furthermore, we compared the thermal aggregation of the wild type with that of the del.144-145 mutant. We found that the thermal aggregation of the mutant was reduced relative to that of the wild type. Moreover, the molecular weight of the mutant after heating at 90 degrees C was higher than that of the unheated one, whereas the wild type aggregated under the same conditions. Therefore, we can conclude that although the Ala144-Lys145 residues in Tth PPase may partly cause thermal aggregation, the deletion of these residues may stabilize the Tth PPase molecule structurally against heating and suppress thermal aggregation. (+info)
Stimulation of Na+-alanine cotransport activates a voltage-dependent conductance in single proximal tubule cells isolated from frog kidney.
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1. The swelling induced by Na+-alanine cotransport in proximal tubule cells of the frog kidney is followed by regulatory volume decrease (RVD). This RVD is inhibited by gadolinium (Gd3+), an inhibitor of stretch-activated channels, but is independent of extracellular Ca2+. 2. In this study, the whole cell patch clamp technique was utilized to examine the effect of Na+-alanine cotransport on two previously identified volume- and Gd3+-sensitive conductances. One conductance is voltage dependent and anion selective (GVD) whilst the other is voltage independent and cation selective (GVI). 3. Addition of 5 mM L-alanine to the bathing solution increased the whole cell conductance and gave a positive (depolarizing) shift in the reversal potential (Vrev, equivalent to the membrane potential in current-clamped cells) consistent with activation of Na+-alanine cotransport. Vrev shifted from -36 +/- 4.9 to +12.9 +/- 4.2 mV (n = 15). 4. In the presence of alanine, the total whole cell conductance had several components including the cotransporter conductance and GVD and GVI. These conductances were separated using Gd3+, which inhibits both GVD and GVI, and the time dependency of GVD. Of these two volume-sensitive conductances, L-alanine elicited a specific increase in GVD, whereas GVI was unaffected. 5. The L-alanine-induced activation of GVD was significantly reduced when cells were incubated in a hypertonic bathing solution. 6. In summary, in single proximal tubule cells isolated from frog kidney, on stimulation of Na+-alanine cotransport GVD is activated, while GVI is unaffected. Taken with other evidence, this suggests that GVD is activated by cell swelling, consequent upon alanine entry, and may play a role as an anion efflux pathway during alanine-induced volume regulation. (+info)
gamma-L-glutamyl-L-DOPA inhibits Na(+)-phosphate cotransport across renal brush border membranes and increases renal excretion of phosphate.
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BACKGROUND: For treatment of phosphate (Pi) overload in various pathophysiological states, an agent that selectively increases renal Pi excretion would be of major value. Previously, we have shown that dopamine (DA) inhibits Na(+)-Pi cotransport in renal epithelia. However, the administration of DA or its immediate precursor L-DOPA increases DA in multiple tissues. Synthetic dipeptide gamma-L-glutamyl-L-DOPA (gludopa) can serve as an inactive precursor (pro-pro-drug) of DA. This study tested the hypothesis that, because of the unique colocalization of gamma-glutamyltransferase (gamma-GT), aromatic amino acid decarboxylase, Na(+)-Pi cotransporter, and Na(+)-L-DOPA cotransporter in brush border membrane (BBM) of proximal tubular cells, gludopa may elicit phosphaturia by action of DA generated within the kidney. METHODS: Thyroparathyrectomized rats were given placebo, or gludopa, or gludopa + gamma-GT inhibitor acivicin. Urinary excretion of Pi, Ca2+, Na+, K+, DA, cAMP, and cGMP was determined, and Na(+)-Pi cotransport was measured in BBM prepared from kidneys of rats at the end of the experiment. RESULTS: The administration of gludopa resulted in: (a) an inhibition of Na(+)-Pi cotransport, but not cotransport of Na(+)-proline and Na(+)-alanine in BBM; (b) an increase (+300%) of fractional excretion (FE) of Pi and a drop (-35%) of plasma Pi, whereas the plasma levels and FEs of Ca2+, Na+, and K+ were unchanged; (c) an increase in urinary excretion of cAMP. but not cGMP; (d) a 1000-fold increase of urinary excretion of DA, without a change in excretion of norepinephrine; and (e) an incubation of gludopa with BBM in vitro, which caused a release of L-DOPA, and the in vivo administration of acivicin, which blocked actions of gludopa to inhibit Na(+)-Pi cotransport and to increase urinary excretions of Pi and DA. CONCLUSIONS: We conclude that colocalization of enzymes of biotransformation, BBM transporters, and the autocrine/paracrine DA system in cells of proximal tubules constitutes a cellular basis for the potent and specific phosphaturic action of gludopa. (+info)
Impairment of the proapoptotic activity of Bax by missense mutations found in gastrointestinal cancers.
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We have reported previously that codon 169 of the proapoptotic gene BAX is a mutational hot spot in gastrointestinal cancer. Two different mutations were found in this codon, replacing the wild-type threonine by alanine or methionine. To compare the proapoptotic activity of these Bax mutants with wild-type Bax, we established an ecdysone (muristerone A)-inducible system in cultured human embryonal kidney 293 cells. Addition of muristerone A induced a dose-dependent decrease in the viability of cells transfected with wild-type BAX, but this loss of viability was inhibited in cells transfected with BAX mutants. Furthermore, muristerone A induced morphological changes characteristic of apoptosis, including cell shrinkage, rounding, formation of apoptotic bodies, detachment and nuclear condensation and fragmentation, in cells transfected with wild-type BAX. These hallmarks of apoptosis were clearly diminished in cells transfected with BAX mutants. Mutation of threonine 169 did not affect the binding of Bax to Bax, Bcl-2, or Bcl-X(L). These results demonstrate that missense mutations at codon 169 of BAX are functional because they inhibit its apoptotic activity. This is the first report of the functional significance of missense mutations in BAX, or any other proapoptotic member of the Bcl-2 family, in primary human tumors. (+info)
Hydration-coupled dynamics in proteins studied by neutron scattering and NMR: the case of the typical EF-hand calcium-binding parvalbumin.
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The influence of hydration on the internal dynamics of a typical EF-hand calciprotein, parvalbumin, was investigated by incoherent quasi-elastic neutron scattering (IQNS) and solid-state 13C-NMR spectroscopy using the powdered protein at different hydration levels. Both approaches establish an increase in protein dynamics upon progressive hydration above a threshold that only corresponds to partial coverage of the protein surface by the water molecules. Selective motions are apparent by NMR in the 10-ns time scale at the level of the polar lysyl side chains (externally located), as well as of more internally located side chains (from Ala and Ile), whereas IQNS monitors diffusive motions of hydrogen atoms in the protein at time scales up to 20 ps. Hydration-induced dynamics at the level of the abundant lysyl residues mainly involve the ammonium extremity of the side chain, as shown by NMR. The combined results suggest that peripheral water-protein interactions influence the protein dynamics in a global manner. There is a progressive induction of mobility at increasing hydration from the periphery toward the protein interior. This study gives a microscopic view of the structural and dynamic events following the hydration of a globular protein. (+info)
A mutational analysis of the Abetaz/Aalphad major histocompatibility complex class II molecule that restricts autoreactive T cells in (NZBxNZW)F1 mice. The critical influence of alanine at position 69 in the Aalphad chain.
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Autoimmune symptoms of (NZBxNZW)F1 (H-2d/z) mice are reported to be critically related to the heterozygosity at the H-2 complex of the murine major histocompatibility complex (MHC). We previously showed that several Abetaz/Aalphad MHC class II molecule-restricted autoreactive T-cell clones from B/WF1 mice were pathogenic upon transfer to preautoimmune B/WF1 mice. In this study, to identify the crucial amino acid residues in Abetaz/Aalphad molecules for T-cell activation, we generated a panel of transfectant cell lines. These transfectant cell lines express the Abetaz/Aalphad MHC molecules with a mutation at each residue alpha11, alpha28, alpha57, alpha69, alpha70, alpha76 of Aalphad chain and beta86 of Abetaz chain. Replacing alpha69 alanine with threonine, valine or serine completely eliminated the ability to stimulate autoreactive T-cell clones without affecting the ability to present foreign antigen keyhole limpet haemocyanin (KLH) or L-plastin peptide to specific T-cell clones. Replacing beta86 valine with aspartic acid resulted in a decrease in the stimulation for antigen-reactive as well as autoreactive T-cell clones. Substitutions at other residues had minimal or no effect on the stimulation of either auto- or antigen-reactive T-cell clones. These results suggest that alanine at residue 69 of the Aalphad chain is critical for the activation of autoreactive Abetaz/Aalphad-restricted T-cell clones. Possible explanations for this are discussed. (+info)
Specificity and genetic restrictions of the guinea-pig immune response to dinitrophenyl-lysyl-alanyl octapeptides.
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A series of 2,4-dinitrophenyl (DNP) octapeptides containing L-lysine and L-alanine were prepared to examine the specificity and genetic restrictions of both cellular and humoral immune responses in inbred guinea-pigs. Strains 2 and 13 guinea-pigs were therefore immunized with Lys4-Ala3-Lys(DNP), Lys3Ala4-Lys(DNP), Lys2-Ala5-Lys(DNP) and Lys-Ala6-Lys(DNP). Only Lys4-Ala3-Lys(DNP) was under Ir gene control and could induce both antibody and T-cell responses in strain 2 guinea-pigs. In contrast, Lys4-Ala3-Lys(DNP) injected in strain 13 guinea-pigs and the other DNP-octapeptides injected in strain 2 or 13 guinea-pigs elicited only antibody formation and no specific T-cell mediated response. Antibody formed in the absence of specific T-cell responsiveness in either strain 2 or 13 was hapten specific and lacked the capacity to discriminate the immunizing antigen from closely related DNP-peptides. Antibody produced by animals with specific T-cell responses, on the other hand, was exquisitely specific for the immunizing peptide and could discriminate it from closely related peptides. (+info)
Suppressor T-cell activity in responder X nonresponder (C57BL/10 X DBA/1)F1 spleen cells responsive to L-glutamic acid60-L-alanine30-L-tyrosine10.
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The ability of spleen cells from (responder X nonresponder)F(1) mice immunized with various GAT-Mphi, GAT-MBSA, and soluble GAT to develop IgG GAT-specific PFC responses in vitro after stimulation with responder and nonresponder parental and F(1) GAT-Mphi, was investigated. F(1) spleen cells from mice immunized with F(1) GAT-Mphi or GAT-MBSA developed secondary responses to responder and nonresponder parental and F(1) GAT- Mphi, but not to unrelated third party GAT-Mphi. Spleen cells from F(1) mice immunized with either parental GAT-Mphi developed secondary responses to F(1) GAT-Mphi and only the parental GAT-Mphi used for immunization in vivo. Soluble GAT-primed F(1) spleen cells responded to F(1) and responder parental, but not nonresponder parental, GAT-Mphi. Simultaneous immunization in vivo with the various GAT-Mphi or GAT-MBSA plus soluble GAT modulated the response pattern of these F(1) spleen cells such that they developed secondary responses only to F(1) and parental responder GAT-Mphi regardless of the response pattern observed after immunization with the various GAT-Mphi or GAT-MBSA alone. These observations demonstrate the critical importance of the physical state of the GAT used for immunization in determining the subsequent response pattern of immune F(1) spleen cells to the parental and F(1) GAT-Mphi. Further, suppressor T cells, capable of inhibiting primary responses to GAT by virgin F(1) spleen cells stimulated by nonresponder parental GAT-Mphi, were demonstrated in spleens of F(1) mice immunized with soluble GAT, but not those primed with F(1) GAT-Mphi. Because responder parental mice develop both helper and suppressor T cells after immunization with GAT-Mphi, and soluble GAT preferentially stimulates suppressor T cells whereas GAT-Mphi stimulate helper T cells in nonresponder parental mice, these observations suggest that distinct subsets of T cells exist in F(1) mice which behave phenotypically as responder and nonresponder parental T cells after immunization with soluble GAT and GAT- Mphi. (+info)