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91.
14-3-3 proteins are a family of conserved dimeric molecules that interact with a broad range of target proteins, most of which contain phosphoserine/threonine. The amphipathic groove of 14-3-3 is the main structural feature involved in mediating its associations. We have studied another domain of 14-3-3, the C-terminal loop, to determine what role it plays in ligand interaction. A truncated form of 14-3-3zeta lacking this C-terminal loop was generated and found to bind with higher affinity than the wild-type 14-3-3zeta protein to the ligands Raf-1 and Bad. Interestingly, the truncated 14-3-3zeta also showed increased association with the 14-3-3 binding-deficient Bad/S136A mutant. Taken together, these data support a role for the C-terminal loop as a general inhibitor of 14-3-3/ligand interactions. This may provide a mechanism by which inappropriate associations with 14-3-3 are prevented. 相似文献
92.
Giragossian C Stone S Papini AM Moroder L Mierke DF 《Biochemical and biophysical research communications》2002,293(3):1053-1059
Conformational features of the C-terminal carboxyamidated pentadecapeptide of CCK (S(19)HRISDRD[SO(4)]-YMGWMDF(33)-NH(2)) were determined by NMR spectroscopy in a zwitterionic membrane-mimetic solvent system, composed of DPC micelles. The C-terminal octapeptide consisted of a well-defined pseudohelix that was nearly identical to the structure previously reported for nonsulfated CCK-8 in the same solvent system. N-terminal amino acids of CCK-15 were highly disordered, with no clear conformational preference. Extensive NOE-restrained molecular dynamics simulations of the CCK-15/CCK(1)-R complex suggested that almost all the experimentally determined intermolecular contact points provided by NMR, site-directed mutagenesis, and photoaffinity labeling could be simultaneously satisfied, when the N-terminus of the ligand is placed in close spatial proximity to the N-terminus of the receptor. 相似文献
93.
94.
D'souza VM Swierczek SI Cosper NJ Meng L Ruebush S Copik AJ Scott RA Holz RC 《Biochemistry》2002,41(43):13096-13105
Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1). 相似文献
95.
Several studies with the nematode Caenorhabditis elegans have made the unexpected discovery that certain hypomorphic mutations in genes encoding mitochondrial proteins result in life span extension. These mutations appear to act independently of the other known pathway that regulates life span extension, the dauer-specifying insulin/IGF-1-like pathway. Here we present a hypothesis that unifies the effects of these two classes of genes on longevity. The central concept is that energy generation in C. elegans occurs by differential flux through two coexisting mitochondrial metabolic pathways-aerobic respiration and fermentative malate dismutation. In the latter process, fumarate is terminally reduced at complex II to succinate. We suggest that most, if not all, long-lived mutants in C. elegans utilize malate dismutation, a byproduct of which is the generation of fewer radical species. 相似文献
96.
Shane Michael W. de Vos Martin de Roock Sytze Cawthray Gregory R. Lambers Hans 《Plant and Soil》2003,248(1-2):209-219
The response of internal phosphorus concentration, cluster-root initiation, and growth and carboxylate exudation to different external P supplies was investigated in Hakea prostrata R.Br. using a split-root design. After removal of most of the taproot, equal amounts of laterals were allowed to grow in two separate pots fastened together at the top, so that the separate root halves could be exposed to different conditions. Plants were grown for 10 weeks in this system; one root half was supplied with 1 M P while the other halves were supplied with 0, 1, 25 or 75 M P. Higher concentrations of P supplied to one root half significantly increased the P concentration of those roots and in the shoots. The P concentrations in root halves supplied with 1 M P were invariably low, regardless of the P concentration supplied to the other root half. Cluster root initiation was completely suppressed on root halves supplied with 25 or 75 M P, whereas it continued on the other halves supplied with 1 M P indicating that cluster-root initiation was regulated by local root P concentration. Cluster-root growth (dry mass increment) on root halves supplied with 1 M P was significantly reduced when the other half was either deprived of P or supplied with 25 or 75 M P. Cluster-root growth was favoured by a low shoot P status at a root P supply that was adequate for increased growth of roots and shoots without increased tissue P concentrations. The differences in cluster-root growth on root halves with the same P supply suggest that decreased cluster-root growth was systemically regulated. Carboxylate-exudation rates from cluster roots on root halves supplied with 1 M P were the same, whether the other root half was supplied with 1, 25 or 75 M P, but were approximately 30 times faster when the other half was deprived of P. Estimates of root P-uptake rates suggest a rather limited capacity for down-regulating P uptake when phosphate was readily available. 相似文献
97.
Hardin SC Tang GQ Scholz A Holtgraewe D Winter H Huber SC 《The Plant journal : for cell and molecular biology》2003,35(5):588-603
Sequence analysis identified serine 170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an sucrose non-fermenting 1 (SNF1)-related protein kinase. Phosphorylation of the novel S170 and the known serine 15 (S15) site by these protein kinases was determined in peptide substrates and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein-kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild-type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was threefold lower than that for S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by early nodulin 40 (ENOD40) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: (i) were detected and possessed relatively high phosphorylated-S170 (pS170) stoichiometry; (ii) were spatially coincident with proteasome activity within developing leaves; and (iii) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves. 相似文献
98.
Gillian P. McMahon Shane J. O' Connor Desmond J. Fitzgerald Sylvie le Roy Mary T. Kelly 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,707(1-2)
A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of aspirin and salicylic acid in transdermal perfusates. The compounds were separated on a C8 Nucleosil column (5 μm, 250×4.6 mm) using a mobile phase containing a mixture of water–acetonitrile–orthophosphoric acid (650:350:2, v/v/v) and a flow-rate of 1 ml/min. The transdermal samples were in phosphate-buffered saline (PBS) and could be injected directly onto the HPLC system. The method was reproducible with inter-day R.S.D. values of no greater than 3.46 and 2.60% for aspirin and salicylic acid, respectively. The method was linear over the concentration range 0.2–5.0 μg/ml and had a limit of detection of 0.05 μg/ml for both compounds. For certain samples, it was necessary to ensure that no transmembrane leakage of the aspirin prodrugs had occurred. In these cases, a gradient was introduced by increasing the acetonitrile content of the mobile phase after the salicylic acid had eluted. The method has been applied to the determination of aspirin and salicylic acid in PBS following in vitro application of the compounds to mouse skin samples. 相似文献
99.
Charan Nirmal B.; Carvalho Paula; Johnson Shane R.; Thompson William H.; Lakshminarayan S. 《Journal of applied physiology》1998,85(2):432-436
We studiedthe effects of aerosolized as well as intravenous infusion ofacetylcholine on bronchial blood flow in six anesthetized sheep.Intravenous infusion of acetylcholine, at a dose of 2 µg/kg, increased bronchial blood flow from 45 ± 15 (SE) to 74 ± 30 ml/min, and vascular conductance increased by 76 ± 22%. In contrast, aerosolized acetylcholine at doses of 2 and 20 µg/kg decreased bronchial vascular conductance by ~10%. At anaerosolized dose of 200 µg/kg, the bronchial vascular conductanceincreased by ~15%, and there was no further increase in conductancewhen the aerosolized dose was increased to 2,000 µg/kg. Pretreatmentof animals with a nitric oxide synthase inhibitor,N-nitro-L-argininemethyl ester hydrochloride, partially blocked the vasodilatory effectsof intravenous acetylcholine and completely blocked the vasodilatoryeffects of high-dose aerosolized acetylcholine. These data suggest thataerosolized acetylcholine does not readily penetrate the vascular wallof bronchial circulatory system and, therefore, has minimalvasodilatory effects on the bronchial vasculature. 相似文献
100.
Pravin L. Kotian Raman Krishnan Scott Rowland Yahya El-Kattan Surendra K. Saini Ramanda Upshaw Shanta Bantia Shane Arnold Y. Sudhakar Babu Pooran Chand 《Bioorganic & medicinal chemistry》2009,17(11):3934-3958
Factor VIIa (FVIIa), a serine protease enzyme, coupled with tissue factor (TF) plays an important role in a number of thrombosis-related disorders. Inhibition of TF·FVIIa occurs early in the coagulation cascade and might provide some safety advantages over other related enzymes. We report here a novel series of substituted biphenyl derivatives that are highly potent and selective TF·FVIIa inhibitors. Parallel synthesis coupled with structure-based drug design allowed us to explore the S2 pocket of the enzyme active site. A number of compounds with IC50 value of <10 nM were synthesized. The X-ray crystal structures of some of these compounds complexed with TF·FVIIa were determined and results were applied to design the next round of inhibitors. All the potent inhibitors were tested for inhibition against a panel of related enzymes and selectivity of 17,600 over thrombin, 450 over trypsin, 685 over FXa, and 76 over plasmin was achieved. Two groups, vinyl 36b and 2-furan 36ab, were identified as the optimum binding substituents on the phenyl ring in the S2 pocket. Compounds with these two substituents are the most potent compounds in this series with good selectivity over related serine proteases. These compounds will be further explored for structure–activity relationship. 相似文献