Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis. To analyze the function of domains 2 [middle (M) domain] and 3 [C-terminal (C) domain], EF-Tu(DeltaM) and EF-Tu(DeltaC) were engineered as GST-fused products and purified. Circular dichroism and thermostability showed that both constructs have conserved organized structures. Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities. Therefore, like the isolated N-terminal domain, they had lost a typical feature of EF-Tu, the >100 times stronger affinity for GDP than for GTP. EF-Tu(DeltaM) and EF-Tu(DeltaC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu. Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(DeltaC), to a level, however, much lower than that of the intact molecule. The interaction with aa-tRNA of both mutants was >90% reduced. As a major result, their GDP-bound form could efficiently respond to EF-Ts. All four EF-Tu-specific antibiotics [kirromycin, pulvomycin, GE2270 A (=MDL 62 879), and enacyloxin IIa] retarded significantly the dissociation of EF-Tu(DeltaC).GTP, showing the same kind of effect as on EF-Tu.GTP, but they were little active on EF-Tu(DeltaM). GTP. Like EF-Tu(DeltaC).GTP, EF-Tu(DeltaM).GTP was, however, able to bind efficiently kirromycin and enacyloxin IIa, as determined via competition with EF-Ts. Together, these results enlight selective functions of domains 2 and 3, particularly toward the interaction with EF-Ts and antibiotics, and emphasize their functional cooperativity for an efficient interaction of EF-Tu with ribosomes and aa-tRNA and for maintaining the differential affinity for GTP and GDP.
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PMID:Functional role of the noncatalytic domains of elongation factor Tu in the interactions with ligands. 942 69

Activation of phospholipase D1 (PLD1) by Arf has been implicated in vesicle transport and membrane trafficking. PLD1 has also been shown to be associated with the small GTPase RalA, which functions downstream from Ras in a Ras-RalA GTPase cascade that facilitates intracellular signal transduction. Although PLD1 associates directly with RalA, RalA has no effect upon the activity of PLD1. However, PLD1 precipitated from cell lysates with immobilized glutathione S-transferase-RalA fusion protein is active. This suggests the presence of an additional activating factor in the active RalA-PLD1 complexes. Because Arf stimulates PLD1, we looked for the presence of Arf in the active RalA-PLD1 complexes isolated from v-Src- and v-Ras-transformed cell lysates. Low levels of Arf protein were detected in RalA-PLD1 complexes; however, if guanosine 5'-[gamma-thio]triphosphate was added to activate Arf and stimulate translocation to the membrane, high levels of Arf were precipitated by RalA from cell lysates. Interestingly, deletion of 11 amino-terminal amino acids unique to Ral GTPases, which abolished the ability of RalA to precipitate PLD activity, prevented the association between RalA and Arf. Brefeldin A, which inhibits Arf GDP-GTP exchange, inhibited PLD activity in v-Src- and v-Ras-transformed cells but not in the nontransformed cells, suggesting that the association of Arf with RalA is required for the increased PLD activity induced by v-Src and v-Ras. These data implicate Arf in the transduction of intracellular signals activated by v-Src and mediated by the Ras-RalA GTPase cascade. Because both Arf and PLD1 stimulate vesicle formation in the Golgi, these data raise the possibility that vesicle formation and trafficking may play a role in the transduction of intracellular signals.
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PMID:Functional association between Arf and RalA in active phospholipase D complex. 952 Apr 17

Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor resulted in a time-dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 was associated with an insulin-stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there was an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1-Raf1 binding, we observed a rapid insulin-stimulated dissociation of the CrkII-C3G complex which occurred in a Ras-independent manner. The dissociation of the CrkII-C3G was recapitulated in vitro using a GST-C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST-C3G with CrkII was also dose dependent and demonstrated that insulin reduced the affinity of CrkII for C3G without any effect on CrkII protein levels. Furthermore, the reduction in CrkII binding affinity was reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de-repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade.
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PMID:Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes. 956 38

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize Gi-family alpha subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Galpha subunit Gpa1(sst) that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780-5784), site-directed mutagenesis of alphao and alphai1 was done. G184S alphao and G183S alphai1 show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G --> S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nM, respectively, for aluminum fluoride-activated wild type alphao and alphai1 to compete with fluorescein isothiocyanate-alphao binding to glutathione S-transferase-RGS4. The G --> S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in alphao and alphai1 that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Gi function.
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PMID:A point mutation in Galphao and Galphai1 blocks interaction with regulator of G protein signaling proteins. 958 6

There are several recently reported examples of inositol phospholipids binding to pleckstrin homology (PH) domains of proteins. The PH domain of SOS, a guanine nucleotide exchange factor for Ras, binds to phosphatidylinositol 4,5 bisphosphate (PtdIns4,5P2). We found that binding of PtdIns4,5P2 to 6-his-tagged recombinant mSOS in vitro inhibits the ability of SOS to catalyze the association of GTP on p21RAS. This inhibition was specific for PtdIns4,5P2: a number of other phosphatidylinositols and phosphatidylserine failed to inhibit Ras GTP-association. We confirmed that the specificity of binding of PtdIns's to recombinant GST-SOS-PH domain is the same as the specificity of PtdIns's for inhibition of SOS activity: namely, that only PtdIns4,5P2 binds significantly to the SOS-PH domain. In addition, the inhibition of Ras GTP-binding is not blocked by excess free inositols suggesting that SOS binds to PtdIns4,5P2 with higher affinity than it binds to free inositols. Addition of SOS-PH domain protein prevented the inhibition of SOS by PtdIns4,5P2 as did addition of the high affinity PtdIns4,5P2-binding drug neomycin. This confirmed that SOS inhibition is mediated by the SOS-PH domain binding to the inositol moiety of PtdIns4,5P2. Binding of Grb2 to SOS did not prevent the inhibition of SOS by PtdIns4,5P2 suggesting that there must be another mechanism for regulating this inhibition. These findings show that the phospholipid PtdIns4,5P2 can suppress the activity of an enzyme involved in signal transduction and suggest that this inhibitory effect must be relieved when SOS is activated.
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PMID:Inhibition of mSOS-activity by binding of phosphatidylinositol 4,5-P2 to the mSOS pleckstrin homology domain. 962 May 47

The full-length dengue virus NS3 protein has been successfully expressed as a 94-kDa GST fusion protein in Escherichia coli. Treatment of the purified fusion protein with thrombin released a 68-kDa protein which is the expected molecular mass for the DEN1 NS3 protein. The identity of this protein was confirmed by Western blotting using dengue virus antisera. Two related activities of the recombinant NS3 protein were characterized, which were the binding of the protein to the 3'-noncoding region of the dengue virus RNA genome and NTPase activity. We demonstrated using a band shift assay that the DEN1 NS3 protein could form a complex with the stem-loop structure in the 3'-noncoding region (3'-NCR), although sites outside the stem-loop may also participate in binding. Using various unlabeled homopolymeric and heteropolymeric RNAs as competitors for binding, it was further shown that the DEN1 NS3 protein exhibits preferential binding to a 94-nt RNA transcript from the 3'-NCR of the dengue virus. The NTPase activity of the recombinant DEN1 NS3 protein was characterized using a thin-layer chromatography assay. We found that the DEN1 NS3 protein possesses some aspects of NTPase activity, which are distinct from those found in other flaviviruses. Although the NS3 protein was able to utilize all four ribonucleoside triphosphates as its substrates, the NS3 protein showed a distinct preference for purine triphosphates (i.e., ATP and GTP). The addition of poly(U) did not stimulate NTPase activity in DEN1 NS3 protein, which contrasts with the reports for other flaviviral NS3 proteins. However, NTPase activity was specifically stimulated by the viral NS5 protein, which was manifested by a more than twofold increase in the rate of ATP hydrolysis and a 25% increase in the yield of ADP at the end of a 120-min reaction. These data suggest that the NTPase activity of the NS3 protein may be regulated by the viral NS5 protein during virus replication.
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PMID:Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein. 965 59

Mixed-lineage kinase 2 (MLK2) is a cytoplasmic protein kinase expressed at high levels in mammalian brain. The MLK2 structure is composed of a Src homology 3 (SH3) domain, two leucine zippers, a basic motif, a Cdc42/Rac interactive binding motif and a large C-terminal domain rich in proline, serine and threonine residues. To begin to define the role of MLK2 in mammalian brain, we used an MLK2-SH3 domain-glutathione S-transferase fusion protein (GST-MLK2-SH3) to isolate MLK2-binding proteins from rat brain extract. This analysis revealed that the major MLK2-SH3-domain-binding protein in rat brain is the GTPase dynamin. By using two different forms of the dynamin proline-rich domain as affinity ligands, the binding site for MLK2-SH3 was mapped to the C-terminal region of dynamin between residues 832 and 864. In GTPase assays, the addition of MLK2-SH3 stimulated the activity of purified dynamin I by 3-fold over the basal level, whereas the addition of a known dynamin activator, phosphatidylserine (PtdSer), stimulated a 6-fold increase. When MLK2-SH3 was added to the assay together with PtdSer, however, dynamin GTPase activity accelerated by more than 23-fold over basal level. An MLK2 mutant (MLK2-W59A-SH3), with alanine replacing a conserved tryptophan residue in the SH3 domain consensus motif, had no effect on dynamin activity, either alone or in the presence of PtdSer. In the same assay the SH3 domain from the regulatory subunit of phosphatidylinositol 3'-kinase stimulated a similar synergistic acceleration of dynamin GTPase activity in the presence of PtdSer. These results suggest that synergy between phospholipid and SH3 domain binding might be a general mechanism for the regulation of GTP hydrolysis by dynamin.
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PMID:Mixed-lineage kinase 2-SH3 domain binds dynamin and greatly enhances activation of GTPase by phospholipid. 974 20

Using part of the dnaK gene from Bacillus subtilis as a probe, a 4. 4-kbp SacI-BglII fragment of chromosomal DNA of Bacillus brevis, a protein-hypersecreting bacterium, was cloned. Nucleotide sequencing revealed 3 open reading frames in the order of grpE-dnaK-dnaJ homologues. We purified DnaK protein to homogeneity from B. brevis HPD31 harboring a multi-copy dnaK expression plasmid. Purified DnaK showed ATPase activity which was synergistically stimulated 14-fold by the addition of glutathione S-transferase-DnaJ and glutathione S-transferase-GrpE fusion proteins. DnaK hydrolyzed not only ATP but also CTP, UTP, and GTP at about 40% of the efficiency of ATP. The specific activity of DnaK-ATPase was 7.25x10-3 unit/mg protein (the turnover number against ATP was 0.47 min-1) under our assay conditions. The DnaK dimers dissociated into monomers on addition of ATP, GTP, CTP, UTP and ATPgammaS, but not ADP or AMP. DnaK formed a stable complex with permanently unfolded carboxymethylated alpha-lactalbumin but not with native alpha-lactalbumin, and this complex was dissociated by addition of ATP/Mg. Formation of this complex was inhibited in the presence of inorganic phosphate.
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PMID:Molecular cloning of the dnaK locus, and purification and characterization of a DnaK protein from Bacillus brevis HPD31. 974 7

In 32 published reports in surgical patients, the preponderance of evidence from standard clinical measures of renal function (BUN and Cr) indicates the absence of renal toxicity following sevoflurane anesthesia. Studies of surgical patients receiving intermediate-duration sevoflurane with high or low fresh gas flow and long-duration sevoflurane with high fresh gas flow included sensitive measures of renal function and/or injury, which also indicate the absence of renal toxicity following sevoflurane anesthesia. Studies of surgical patients receiving long-duration sevoflurane with low fresh gas flow did not include sensitive measures. Seven studies in volunteers are not directly relevant to clinical practice but do raise the issue of whether it is important to apply sensitive measures of renal function and/or injury such as urine concentrations and/or excretion of NAG, beta 2M, alpha 1M, AAP, alpha GST, pi GST, gamma GTP, albumin, protein, and glucose and Cr clearance. Two studies of volunteers receiving prolonged sevoflurane anesthesia with fresh gas flow no greater than 2 L/min concluded that the potential for adverse renal effects of sevoflurane may exist. The other studies of volunteers did not. In 14 published reports of surgical patients in special conditions, the preponderance of evidence from standard clinical measures of renal function indicates the absence of renal toxicity. Studies with sensitive measures have been reported for some conditions where the kidney may be at increased risk (e.g., sevoflurane-induced hypotension, advanced age, and renal insufficiency and failure), are incomplete in others (e.g., hypertension and ischemic heart disease), and are missing in others (e.g., morbid obesity). Studies with sensitive measures of renal function and/or injury are also missing in an important group where the kidney may not be at increased risk--pediatric patients. Studies of other risk conditions, such as temporary ischemia, hemorrhagic hypotension, nephrotoxic antibiotics, kidney transplantation, and diabetes may provide additional information about the renal effects of sevoflurane.
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PMID:Renal effects of sevoflurane during conditions of possible increased risk. 980 93

From a brain cDNA library of Bombyx mori, we cloned cDNA for BRab, which encoded a 202-amino-acid polypeptide sharing 60-80% similarity with rab1 family members. To characterize its biochemical properties, cDNA for BRab was inserted into an expression vector (pGEX2T) and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The purified GST-BRab bound [35S]-GTP gamma S and [3H]-GDP with association constants of 1.5 x 10(6) M-1 and 0.58 x 10(6) M-1, respectively. The binding of [35S]-GTP gamma S was inhibited with GTP and GDP, but with no other nucleotides. The GTP-hydrolysis activity was evaluated to be 5 m mole/min/mole of BRab. In the presence of 6 mM MgCl2, bound [35S]-GTP gamma S and [3H]-GDP were exchanged with GTP gamma S most efficiently. These results suggest that BRab, having a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolysis activity and returns to the GTP-bound state with the exchange of GDP with GTP.
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PMID:Molecular cloning of cDNA for BRab from the brain of Bombyx mori and biochemical properties of BRab expressed in Escherichia coli. 983 23


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