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)

To further investigate the role of p53 gene inactivation in gastric tumorigenesis, the mutational status of the p53 gene in primary human gastric cancer samples was examined. Reverse transcriptase polymerase chain reaction and subsequent direct sequencing of the p53 gene from gastric cancer samples revealed frequent point mutations of the p53 gene: some of these coincided with those previously identified in gastric cancer cell lines. In addition, both allelic deletion analysis using pYNZ 22 and polymerase chain reaction-restriction fragment length polymorphism analysis demonstrated an allelic deletion of the p53 gene in cancer tissue which contained a point mutation of the p53 gene in the remaining allele. Transfection of the wild-type or mutant p53 genes into gastric cancer cells showed that the wild-type but none of the mutated p53 genes suppressed the colony formation of gastric cancer cells. Furthermore, the incorporation of thymidine into DNA was reduced in cancer cells expressing the wild-type p53 gene. The glutathione S-transferase-wild type p53 fusion protein bound to simian virus 40 large T antigen in COS-1 cell lysate. None of the p53 fusion proteins containing mutations at codons 143, 175, 248, or 273 bound to simian virus 40 large T antigen. By contrast, two different mutant p53 fusion proteins containing mutations specifically observed in gastric cancer bound to simian virus 40 large T antigen. These results indicate that inactivation of the p53 gene through mutations and the allelic deletion may play an important role in gastric tumorigenesis. These mutations may cause a conformational change in the p53 protein resulting in the loss of the suppression by p53 of the growth of gastric cells, partly through disruption of the association of p53 protein with a cellular component.
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PMID:p53 gene mutations in human gastric cancer: wild-type p53 but not mutant p53 suppresses growth of human gastric cancer cells. 132 85

The aim of this work was to define a transfection procedure that is compatible with the sorting and propagation of cells that transiently express a heterologous gene. Three requirements were established for the procedure and were met with COS monkey kidney cells that express a recombinant glutathione S-transferase (GST) gene. The transfection procedure used had to generate (i) populations in which at least 10% of the cells expressed recombinant GST, (ii) cellular morphological homogeneity throughout the population, and (iii) viable cells with at least a 5% colony-forming ability. Of the transfection techniques tested, only electroporation satisfied all three requirements. Usually 20-22% of the cells that survived electroporation expressed recombinant GST 3 days after electroporation as measured by flow cytometry, and 25% of the cells that survived electroporation formed colonies in cloning assays. Transfection with DEAE-dextran and chloroquine did enable 40% of the surviving cells to express GST, but only 0.01% of the cells that survived transfection formed colonies in cloning assays. Finally, with lipofection, only 1% of the surviving cells expressed recombinant GST, although 25-40% of the cells that survived transfection formed colonies. These studies define the merits and limitations of transfection techniques relative to the analysis and sorting of transfected cells by flow cytometry.
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PMID:Gene transfer by electroporation, lipofection, and DEAE-dextran transfection: compatibility with cell-sorting by flow cytometry. 154 55

COS cells transiently expressing glutathione S-transferase (GST) pi, Ya, or Yb1 (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony-forming assays to show that GST confers cellular resistance to the carcinogen benzo[a]pyrene (+/-)-anti-diol epoxide (anti-BPDE). We developed a sorting technique to viably separate recombinant GST+ cells (20%) from the nonexpressing electroporated population (80%) on the basis of a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane (mClB). The concentration of mClB, length of time cells are exposed to mClB, and activity of the expressed GST isozyme determined the degree to which recombinant GST+ cells fluoresced more intensely than controls. On-line reagent addition ensured that all cells were exposed to 25 microM mClB for 30-35 s during transit before being analyzed for fluorescence intensity and sorted. The apparent Km for mClB of the endogenous COS cell GST-catalyzed intracellular reaction was 88 microM. Stained GST Ya+ or Yb1+ cells catalyzed the conjugation 2 or 5 times more effectively than GST pi+ cells. Enzyme activity in cytosolic fractions prepared from sorted recombinant GST+ cells was 1.8 +/- 0.3-fold greater than that of the control (80 +/- 4 nmol/min/mg protein). Upon a 5-fold purification of GST pi+ cells in the electroporated population, resistance to anti-BPDE in colony-forming assays increased 5 times, from 1.1-fold (unsorted) to 1.5-fold (sorted) (P less than 0.001).
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PMID:Recombinant glutathione S-transferase (GST) expressing cells purified by flow cytometry on the basis of a GST-catalyzed intracellular conjugation of glutathione to monochlorobimane. 166 13

Monoclonal antibodies were raised against the recently discovered subtilisin-like proprotein processing enzyme furin. As immunogen, a bacterially expressed hybrid protein was used which consisted of glutathione S-transferase fused to almost the entire human furin protein. Ten monoclonal antibodies were obtained and these could be divided into four categories on the basis of their reactivity towards a number of bacterially expressed hybrid proteins, each of which contained a different portion of human furin. Four of the monoclonal antibodies did not recognize mouse furin. All monoclonal antibodies were tested for their applicability in Western blot and immunofluorescence analysis. Western blot analysis was performed with COS-1 cells in which biologically active forms of human and mouse furin were expressed transiently under control of the SV40 late promoter. This approach was necessary, since physiological levels of fur gene encoded proteins appeared to be very low. In cells transfected with human or mouse fur cDNA, a protein of about 100 kDa and a doublet of about 90 kDa could be detected with most of the monoclonal antibodies. Some of these antibodies appeared to be also reactive in immunofluorescence analysis of transfected COS-1 cells.
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PMID:Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin. 173 42

Expression vectors were designed and constructed to achieve optimum production of two different isozymes of rat glutathione S-transferase (GST) (EC 2.5.1.18) in COS cells, for studies of drug resistance. Promoter-enhancer elements from the simian virus 40 (SV40) early-region or the mouse alpha 2(I)-collagen gene, GST cDNAs encoding the rat Ya or Yb1 isozymes, and an SV40 replicative origin (ori) were positioned in the vector to express two GSTs at high levels in the same cell. The optimized construct yielded levels of both GST proteins (1% of postmitochondrial protein fraction) that were up to 1.3-fold greater than the sum of those produced individually by two single-unit expression constructs. The best production of the tandem recombinant gene products was observed when the genes were placed in a head to head orientation in close proximity (1 kilobase). With the recombinant genes configured in this way, the plasmid DNA was also amplified in COS cells to higher levels (30% increase over single-unit expression constructs), as ori elements were placed on both DNA strands. Cells expressing the recombinant GSTs were viably sorted by flow cytometry on the basis of a GST-catalyzed conjugation of glutathione to monochlorobimane. Sorted COS cells that expressed both GST Ya and Yb1 from recombinant genes in a tandem, head to head configuration were 25 or 70% more resistant to the alkylating agent chlorambucil than cells that expressed GST Ya or Yb1 alone.
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PMID:Expression of tandem glutathione S-transferase recombinant genes in COS cells for analysis of efficiency of protein expression and associated drug resistance. 185 90

Fatty acid ethyl ester synthase-III (FAEES-III), previously purified to homogeneity from human heart, metabolizes ethanol nonoxidatively. Using a derived partial amino acid sequence and corresponding oligonucleotide probes, the cDNA for this enzyme has been cloned from a human heart lambda gtll library. Of the five positive clones obtained, one contained a complete coding region (630 base pairs) and the entire 3'-noncoding region (41 base pairs). From this nucleotide sequence the complete 210 amino acid sequence of FAEES-III (Mr 23,307) is reported. Comparison of its amino acid sequence with that of glutathione S-transferase pi-1 suggests that they belong to the same gene family since they differ in only six nucleotides and four amino acids. The sequence of FAEES-III was also compared with those of placental glutathione S-transferase and the basic glutathione S-transferase. FAEES-III was 84% homologous with placental glutathione S-transferase but only less than 10% homologous with the basic glutathione S-transferase. Northern blots demonstrate expression of FAEES-III mRNA in normal human liver, placenta, and heart. In all cases, the mRNA for the enzyme is 0.7 kilobase in size. MCF-7 cells transfected with FAEES-III cDNA have a 14-fold increase in synthase activity and a 12-fold increase in glutathione S-transferase (GST) activity compared with control cells. MCF-7 cells transfected with GST pi-1 cDNA have a 13-fold increase in GST activity compared with control cells but no increase in synthase activity. When the supernatant of COS-7 cells transfected with FAEES-III cDNA were immunoblotted with rabbit FAEES-III antibody, a band at 24 kilodaltons was demonstrated. Thus, we have obtained the first cDNA and amino acid sequence for a human FAEES-III which also has significant GST activity, and we have identified 4 residues potentially responsible for conferring ethanol recognition to GSTs.
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PMID:Molecular cloning, sequencing, and expression of human myocardial fatty acid ethyl ester synthase-III cDNA. 188 4

Increased levels of glutathione S-transferase (GST; RX:glutathione R-transferase; EC 2.5.1.18) mRNA, protein, and activity in tumor biopsy samples and in drug-resistant cultured cells are associated with resistance to anticancer drugs. We report that each of three full-length cloned GST cDNAs, that for pi (acidic), Ya (basic), and Yb1 (neutral), can confer drug resistance when expressed in cultured mammalian cells. In one approach, stably transfected mouse C3H/10T1/2 cells that express GST pi, Ya, or Yb1 were cloned and analyzed for drug resistance in colony-forming assays. Transiently transfected COS cells that were sorted on a fluorescence-activated cell sorter were used in the second approach to avoid interclonal variation in factors other than the recombinant GST and to show that reversion of transient GST expression correlated with loss of drug resistance. A sorting technique, developed to separate the 20% of the electroporated COS cell population that transiently expressed GST pi, Ya, or Yb1 from the nonexpressing population, was based on a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane. GST Ya conferred the greatest increase in resistance to chlorambucil and melphalan (1.3- to 2.9-fold), Yb1 conferred the greatest increase in resistance to cisplatin (1.5-fold), and pi conferred the greatest increase in resistance to a racemic mixture of 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene and 7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene and doxorubicin (1.5- and 1.3-fold) relative to controls. These resistance values to alkylating agents are commensurate with values observed clinically. Cytotoxicity curves representing recombinant GST+ populations were significantly different from their controls with P values ranging from 0.005 to 0.0001. No resistance to vinblastine was detected. Conferred drug resistance was proportional to the magnitude of GST Ya expression, and reversion of transient expression in GST Ya+ COS cell clones to a GST Ya- phenotype was associated with total loss of drug resistance.
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PMID:Expression of recombinant glutathione S-transferase pi, Ya, or Yb1 confers resistance to alkylating agents. 232 May 66

cDNA clones encoding the third member of the RAC protein kinase family, termed RAC-PK gamma, were isolated from a rat brain cDNA library. The deduced amino acid sequence of RAC-PK gamma was highly related to those of previously identified family members, RAC-PK alpha and beta, that have a pleckstrin homology domain and a protein-serine/threonine kinase catalytic domain at the amino- and carboxyl-terminal regions, respectively. Northern blot analysis indicated that RAC-PK gamma was expressed abundantly in brain and testis. Specific activities of RAC-PK alpha, beta, and gamma purified from transfected COS-7 cells were similar when measured by using myelin basic protein as a phosphate acceptor. Analysis using fusion proteins of glutathione S-transferase revealed that the pleckstrin homology domain of the three subtypes of RAC-PK associate with both protein kinase C subspecies and beta gamma subunits of G proteins. These results suggest that the pleckstrin homology domains of RAC protein kinase family could associate more than one protein to regulate the activity and/or intracellular distribution of this enzyme family by different ways.
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PMID:Molecular cloning and characterization of a new member of the RAC protein kinase family: association of the pleckstrin homology domain of three types of RAC protein kinase with protein kinase C subspecies and beta gamma subunits of G proteins. 748 43

Growth hormone (GH) has been shown to stimulate the mitogen-activated protein (MAP) kinases designated ERKs (extracellular signal regulated kinases) 1 and 2. One pathway by which ERKs 1 and 2 are activated by tyrosine kinases involves the Src homology (SH)-2 containing proteins SHC and Grb2. To gain insight into pathways coupling GH receptor (GHR) to MAP kinase activation and signaling molecules that might interact with GHR and its associated tyrosine kinase JAK2, we examined whether SHC and Grb2 proteins serve as signaling molecules for GH. Human GH was shown to promote the rapid tyrosyl phosphorylation of 66-, 52-, and 46-kDa SHC proteins in 3T3-F442A fibroblasts. GH also promoted binding of GHR and JAK2 to the SH2 domain of 46/52-kDa SHC protein fused to glutathione S-transferase (GST). Constitutively phosphorylated JAK2, from COS-7 cells transiently transfected with murine JAK2 cDNA, bound to SHC SH2-GST fusion protein, demonstrating that the SHC SH2 domain can bind tyrosyl-phosphorylated JAK2 in the absence of GHR. Regions of GHR required for GH-dependent tyrosyl phosphorylation of SHC were examined using Chinese hamster ovary cells expressing mutated rat GHR. In cells expressing GHR1-638 and GHR1-638(Y333,338F), GH stimulated phosphorylation of all 3 SHC proteins whereas GH stimulated phosphorylation of only the 66- and 52-kDa SHC proteins in cells expressing GHR1-454. GH had no effect on SHC phosphorylation in cells expressing GHR1-294 or GHR delta P, the latter lacking amino acids 297-311 containing the proline-rich motif required for JAK2 activation by GH. In contrast to SHC, Grb2 appeared not to interact directly with GHR or JAK2. However, Grb2 was shown to associate rapidly with SHC proteins in a GH-dependent manner. These findings suggest that GH stimulates: 1) the association of SHC proteins with JAK2.GHR complexes via the SHC-SH2 domain, 2) tyrosyl phosphorylation of SHC proteins, and 3) subsequent Grb2 association with SHC proteins. These events are likely to be early events in GH activation of MAP kinases and possibly of other responses to GH.
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PMID:Growth hormone-promoted tyrosyl phosphorylation of SHC proteins and SHC association with Grb2. 753 73

A protein called Tip (tyrosine kinase interacting protein) of herpesvirus saimiri associates with Lck in virus-transformed human T cells and is an in vitro substrate for Lck kinase. Mutational analyses of a GST-Tip fusion protein revealed that binding to Lck requires putative SH3 binding sequences and a sequence homologous to the carboxyl terminus of Src-related kinases. These sequences are referred to as SH3-Binding (SH3B) and C-terminal Src-related Kinase Homology (CSKH) elements. Peptide fragments as short as 37 amino acids containing both SH3B and CSKH elements were sufficient to form a stable complex with Lck in vitro. Furthermore, these same sequences of Tip were necessary for in vivo association with Lck when Tip and Lck were expressed transiently in COS-1 cells or stably in Rat-1 cell lines. These results demonstrate that the CSKH element of Tip participates in the binding of sequences within Lck. Tip of herpesvirus saimiri has apparently acquired such CSKH and SH3B elements for the purpose of targeting cellular protein kinases. The interaction of Tip with Lck may influence Lck kinase activity or its binding to other cellular proteins and thereby alter Lck function in T cells infected by h. saimiri.
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PMID:Identification of Lck-binding elements in tip of herpesvirus saimiri. 754 93


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