Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A phage display library was constructed in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different phage, was screened with a glutathione S-transferase (GST) fusion protein containing the Src homology 3 (Src SH3) domain and a protein kinase A phosphorylation site (GST/PKA/Src SH3). A family of proline-rich sequences was isolated following four cycles of enrichment and amplification. Phage containing these sequences were shown to specifically bind to the GST/PKA/Src SH3 protein but not to GST/PKA only. A comparison of the inferred amino acid sequence of the different phage clones revealed a consensus sequence, RPLPXXP, which conforms to a Src SH3 domain binding motif identified independently during an affinity screen of a lambda-lox mouse embryo cDNA library using a 32P-labeled Src SH3 protein fragment as the probe (Y. Ivashchenko, manuscript in preparation). Peptides based upon the 7-amino acid SH3 binding domain core motif displayed strong binding to both the Src and to the Fyn SH3 domains, but failed to bind to the SH3 domain of p21 Ras-GTPase-activating protein (Ras-GAP) and other proteins. We anticipate that further screening of the phage display library will be a useful tool for the rapid identification of additional SH3 domain binding sequences and will also help to establish the essential core motifs that define the specificity of interactions among the diverse proteins containing SH3 domains and those containing SH3 binding motifs.
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PMID:Identification of a Src SH3 domain binding motif by screening a random phage display library. 792 55

Mu A protein, the 75-kDa phage transposase, consists of three domains: a 30-kDa NH2 terminus, a 35-kDa central domain, and a 10-kDa COOH terminus (Nakayama, C., Teplow, D. B., and Harshey, R. M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1809-1813). Genetic and biochemical experiments have demonstrated that the COOH-terminal domain must be present for functional interaction with Mu B protein. To further investigate the COOH-terminal domain of Mu A, we fused this 89-amino acid region to the glutathione S-transferase gene to facilitate subsequent expression and purification. We show that either the glutathione S-transferase-peptide fusion protein or the COOH-terminal peptide severed from glutathione S-transferase is active in Mu B interaction. Addition of the COOH-terminal domain to the in vitro strand transfer reaction inhibits intermolecular strand transfer by a mechanism previously characterized for intact Mu A protein (Baker, T. A., Mizuuchi, M., and Mizuuchi, K. (1991) Cell 65, 1003-1013), although the COOH-terminal domain is 70 times less effective than intact Mu A. The transient interaction between the COOH-terminal domain and Mu B does not inhibit Mu B stimulation of the strand cleavage and intramolecular strand transfer activity of Mu A. Deletion analysis has shown that the last 36 amino acids are sufficient for interaction with Mu B, but that removal of as few as 4 amino acids from the COOH terminus renders the peptide inactive. The recovery of an active COOH-terminal domain of Mu A will facilitate future structure/function studies of the Mu transposase.
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PMID:Characterization of a region in phage Mu transposase that is involved in interaction with the Mu B protein. 796 40

Central to spectrin's function is its association with the plasma membrane. The linking proteins ankyrin and protein 4.1 partly mediate this association, and their interactions with spectrin are well understood. Both beta I (erythrocyte) and beta II (fodrin, beta G) spectrin also associate with unknown protein receptors in crude membrane preparations by ankyrin and protein 4.1 independent mechanisms. As a first step to understanding this interaction, kinetic and equilibrium assays have been used to monitor which regions of beta I and beta II spectrin inhibit the binding of purified 125I-labeled bovine brain spectrin to demyelinated and NaOH-stripped bovine brain membranes. A series of 19 recombinant proteins spanning the entire sequence of beta II spectrin, including an alternatively spliced NH2-terminal isoform (beta II epsilon 2 spectrin), were prepared as glutathione S-transferase fusion proteins. Also prepared were peptides representing the alternatively spliced COOH-terminal domain found in beta I epsilon 2 spectrin ("muscle spectrin"). Two distinct sequence motifs inhibited the binding of native brain spectrin. Membrane association domain 1 (MAD1) was represented in all fusion peptides that included spectrin repeat 1. These peptides slowed the kinetics of brain spectrin binding and inhibited up to 46% of the maximal binding under the conditions of these assays (apparent Ki < or = 0.2 microM). Peptides representative of repeats 2-17 of beta II spectrin were devoid of inhibitory activity. The second membrane association domain (MAD2) was identified in penultimate COOH-terminal sequences (domain III) of both beta II and beta I epsilon 2 spectrin. These sequences were absent in beta I epsilon 1 (erythrocyte) spectrin. MAD2 competitively inhibited over 80% of brain spectrin binding in these assays, with an apparent Ki < or = 0.1 microM. Direct binding studies confirmed that both MAD1 and MAD2 peptides associated with membranes with affinities comparable to their inhibition constants. Sequence comparisons suggest that MAD1 is created by the insertion of two non-homologous sequence motifs into repeat 1, extending it from 106 to 122 amino acids. Similarly, MAD2 encompasses a putative site of beta gamma-heterotrimeric G-protein binding called the pleckstrin homology domain, and MAD2 may in fact be the pleckstrin homology domain although this has not been rigorously proven. Collectively these studies identify two novel functional motifs in spectrin that mediate ankyrin independent association with membranes. We hypothesize that these motifs and their still to be discovered ligands play a primary role in the nascent assembly and stabilization of an ordered and polarized spectrin skeleton.
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PMID:Beta II-spectrin (fodrin) and beta I epsilon 2-spectrin (muscle) contain NH2- and COOH-terminal membrane association domains (MAD1 and MAD2). 796 88

Ankyrin has a spectrin-binding region within a central 62-kDa chymotryptic peptide. We examined the spectrin binding ability of a series of smaller ankyrin fragments and recombinant peptides within the 62-kDa domain using a ligand blot assay. The smallest proteolytic fragment that bound was a 12-kDa tryptic peptide starting at amino acid 1068. Peptides containing this region expressed as glutathione S-transferase fusion products also bound spectrin and suggested that residues 1101-1192 were important. In contrast, a fusion protein containing residues 826-898 did not bind spectrin, a surprising finding since this region is known to influence binding affinity. Proteins that bound spectrin on ligand blots also competed for binding in solution, but did so with one-tenth the affinity of the native peptide. Comparing the 62-kDa domains of erythrocyte and brain ankyrins (species that bind spectrin but with 10-fold differences in affinity), the NH2-terminal regions are 0-40% identical, while the regions (1136-1160) common to all binding peptides are 80-90% identical. We hypothesize that the highly conserved region contains an important spectrin-binding site, while the poorly conserved region controls the binding affinity. We speculate that this unique NH2-terminal region is what gives different members of the ankyrin family their signature set of affinities, and accordingly their distinctive cellular localization.
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PMID:A highly conserved region of human erythrocyte ankyrin contains the capacity to bind spectrin. 822 93

Using a subtractive hybridization method, we have cloned cDNAs corresponding to 10 different mRNAs which share the property of being expressed in the intestine of adult but not baby rabbits. Four could be identified as coding for previously known gene products (sucrase-isomaltase, a glutathione S-transferase, a cytochrome P450, and a long form of ferritin mRNA), while six code for previously unknown proteins. One clone, AdRab-B, codes for a protein of 1458 amino acids, including (i) a putative signal sequence at the NH2 terminus, (ii) four internal repeats, 308-346 amino acids in length, (iii) a hydrophobic stretch near the COOH terminus, which represents a potential membrane anchor, and (iv) a short hydrophilic stretch at the very COOH terminus. The corresponding protein was studied with the aid of antibodies prepared against polypeptides expressed from segments of the cDNA in Escherichia coli. The protein was shown to be proteolytically processed in the intestine (but not when expressed in COS cells) and to be targeted to the brush border membrane of the enterocytes. Finally, the protein was found to have esterase and phospholipase A/lysophospholipase activity.
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PMID:Messenger RNAs expressed in intestine of adult but not baby rabbits. Isolation of cognate cDNAs and characterization of a novel brush border protein with esterase and phospholipase activity. 850 24

The herpes simplex virus type 1 protease is expressed as an 80,000-dalton polypeptide, encoded within the 635-amino acid open reading frame of the UL26 gene. The two known protein substrates for this enzyme are the protease itself and the capsid assembly protein ICP35 (Liu, F., and Roizman, B. (1991) J. Virol. 65, 5149-5156). In this report we describe the use of a rapid and quantitative assay for characterizing the protease. The assay uses a glutathione S-transferase fusion protein containing the COOH-terminal cleavage site of ICP35 as the substrate (GST-56). The protease consists of N0, the NH2-terminal 247 amino acid catalytic domain of the UL26 gene product, also expressed as a GST fusion protein. Upon cleavage with N0, a single 25-mer peptide is released from GST-56, which is soluble in trichloroacetic acid. Using this assay, the protease displayed a pH optimum between 7 and 9 but most importantly had an absolute requirement for high concentrations of an antichaeotrophic agent. Strong salting out salts such as Na2SO4 and KPO4 (> or = 1 M) stimulated activity, whereas NaCl and KCl had no effect. The degree of stimulation by 1.25 M Na2SO4 and KPO4 were 100-150- and 200-300-fold, respectively. Using the fluorescent probe 1-anilino-8-naphthalene sulfonate, the protease was shown to bind the dye in the presence of 1.25 M Na2SO4 or KPO4, but not at low ionic strength or in the presence of 1.25 or 2.2 M NaCl. This binding was most likely at the protease active site because a high affinity cleavage site peptide, but not a control peptide, could displace the dye. In addition to cleaving GST-56, the herpes simplex virus type I protease also cleaved the purified 56-mer peptide. Circular dichroism and NMR spectroscopy showed the peptide to be primarily random coil under physiological conditions, suggesting that antichaeotrophic agents affect the conformation of the substrate as well as the protease.
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PMID:Stimulation of the herpes simplex virus type I protease by antichaeotrophic salts. 853 Apr 25

We report here that calreticulin interacts with protein disulfide isomerase (PDI). The PDI-calreticulin complex can be dissociated by Zn(2+)-iminodiacetate-substituted Sepharose-agarose chromatography, suggesting that these interactions may be Zn2+-dependent. Direct interaction between calreticulin and PDI is also documented by calreticulin affinity chromatography. PDI was the only pancreatic microsomal protein retained on the calreticulum affinity column. Calreticulin and PDI were identified by their NH2-terminal amino acid sequence analysis, mobilities in SDS-polyacrylamide gel electrophoresis, binding of 45Ca2+, and their reactivity with specific antibodies. Using glutathione S-transferase-calreticulin fusion proteins, we show that PDI interacts strongly with the P-domain and only weakly with the N-domain of calreticulin. Expression of calreticulin domains and PDI as fusion proteins with GAL4 in the yeast two-hybrid system revealed that calreticulin interacted with PDI also under normal cellular conditions. Interaction with PDI required only the NH2-terminal region of the N-domain (amino acid residues 1-83) and the P-domain (amino acid residues 150-240) of calreticulin. Importantly, interaction between calreticulin and PDI led to the modulation of their activities. In the presence of PDI, calreticulin does not bind Ca2+ with high affinity. Calreticulin or the N-domain of calreticulin inhibited PDI ability to refold scrambled RNase A.
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PMID:Interaction of calreticulin with protein disulfide isomerase. 853 5

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.
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PMID:Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level. 863 13

Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1; previously named HCP, PTP1C, SH-PTP1, and SHP) is a cytosolic protein tyrosine phosphatase that contains two SH2 domains. Recent data have demonstrated that the gene encoding SHP-1 is mutated in motheaten (mc) and viable motheaten (mc') mice resulting in autoimmune disease. More recently, SHP-1 has been shown to negatively regulate B cell antigen receptor (BCR)-initiated signaling. To elucidate potential mechanisms of SHP-1 action in BCR signal transduction, we studied proteins that interact with SHP-1 in B cells. Both anti-SHP-1 antibody and the two SH2 domains of SHP-1 expressed as glutathione S-transferase fusion proteins precipitated at least three phosphoproteins of approximately 75, 110, and 150 kD upon anti-immunoglobulin M stimulation of the WEHI-231 immature B cell line. Binding of SHP-1 to the 75- and 110-kD proteins appeared to be mediated mainly by the NH2-terminal SH2 domain of SHP-1, whereas both the NH2- and COOH-terminal SH2 domains are required for maximal binding to the 150-kD protein. Immunoprecipitation and Western blot analysis revealed that the SHP-1-associated 75-kD protein is the hematopoietic cell-specific, SH2-containing protein SLP-76. Further, this protein-protein association was constitutively observed and stable during the early phase of BCR signaling. However, significant tyrosine phosphorylation of SLP-76 as well as of SHP-1 was observed after BCR ligation. Constitutive association of SHP-1 with SLP-76 could also be detected in normal splenic B cells. Collectively, these results suggest possible mechanisms by which SHP-1 may modulate signals delivered by BCR engagement.
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PMID:Hematopoietic cell phosphatase, SHP-1, is constitutively associated with the SH2 domain-containing leukocyte protein, SLP-76, in B cells. 876 Jul 99

A protein serine/threonine kinase, p160(ROCK), has been identified as a putative Rho target protein that is activated when bound to the GTP-bound form of the small GTPase Rho (Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatu, A., Fujita, A., Watanabe, N. Saito, Y., Kakizuka, A., Morii, N., and Narumiya, S. (1996) EMBO J. 15, 1885-1893). p160(ROCK) has a serine/threonine kinase domain in its NH2-terminal region, followed by an approximately 600-amino acid-long alpha-helix, a cysteine-rich zinc finger-like motif, and a pleckstrin homology region in the COOH terminus. To identify the Rho binding domain of this protein, we divided p160 into five fragments, expressed each as a His-tagged recombinant protein, and performed a ligand overlay assay using [35S]guanosine-5'-3-O-(thio)triphosphate (GTPgammaS)-bound glutathione S-transferase-RhoA. Specific GTPgammaS-Rho binding was observed only in the fragment M2, which covered most of the carboxyl half of the alpha-helix between amino acids 727 and 1021. This fragment was further subdivided into several fragments, and the ligand overlay assay as well as the yeast two hybrid system was carried out to identify the Rho-binding region. These studies localized the minimum Rho binding region to amino acids 934-1015. To identify critical amino acids for Rho binding, we analyzed the Rho binding activity of the subfragment with various point mutations. This analysis revealed that K934M, L941A, and E1008A mutations significantly weakened Rho binding and an I1009A mutation abolished Rho binding. The amino acid sequence in this region had no significant homology with Rho effector motif class 1, which is shared by putative Rho targets, PKN, rhophilin, and rhotekin, (Reid, T., Furuyashiki, T., Ishizaki, T., Watanabe, G., Watanabe, N., Fujisawa, K., Morii, N., Madaule, P., and Narumiya, S. (1996) J. Biol. Chem. 271, 13556-13560) and may define a distinct class of Rho effector motif.
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PMID:Identification of the Rho-binding domain of p160ROCK, a Rho-associated coiled-coil containing protein kinase. 879 90


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