Lack of inhibitory effects of the Ju-myo protein on development of glutathione S-transferase placental form-positive foci in the male F344 rat liver. (25/8048)

The effects of the 77 kDa Ju-myo protein, isolated from Drosophila melanogaster, on the development of glutathione S-transferase placental form (GST-P) positive foci in the male F344 rat liver were evaluated using a medium-term bioassay system. No modifying potential was evident in terms of the numbers or areas of GST-P positive foci. Ju-myo protein did not exert any influence on cell proliferation, as reflected by ornithine decarboxylase (ODC) or spermidine/spermine N1-acetyltransferase (SAT) activity and BrdU labeling. These results demonstrated that Ju-myo protein is unlikely to have inhibitory or promoting effects on rat liver carcinogenesis.  (+info)

Determinants of specificity for aflatoxin B1-8,9-epoxide in alpha-class glutathione S-transferases. (26/8048)

We have used homology modelling, based on the crystal structure of the human glutathione S-transferase (GST) A1-1, to obtain the three-dimensional structures of rat GSTA3 and rat GSTA5 subunits bound to S-aflatoxinyl-glutathione. The resulting models highlight two residues, at positions 208 and 108, that could be important for determining, either directly or indirectly, substrate specificity for aflatoxin-exo-8,9-epoxide among the Alpha-class GSTs. Residues at these positions were mutated in human GSTA1-1 (Met-208, Leu-108), rat GSTA3-3 (Glu-208, His-108) and rat GSTA5-5 (Asp-208, Tyr-108): in the active rat GSTA5-5 to those in the inactive GSTA1-1; and in the inactive human GSTA1-1 and rat GSTA3-3 to those in the active rat GSTA5-5. These studies show clearly that, in all three GSTs, an aspartate residue at position 208 is a prerequisite for high activity in aflatoxin-exo-8,9-epoxide conjugation, although this alone is not sufficient; other residues in the vicinity, particularly residues 103-112, are important, perhaps for the optimal orientation of the aflatoxin-exo-8,9-epoxide in the active site for catalysis to occur.  (+info)

cAMP-dependent phosphorylation of the tetrodotoxin-resistant voltage-dependent sodium channel SNS. (27/8048)

1. Protein kinase A (PKA) modulation of tetrodotoxin-resistant (TTX-r) voltage-gated sodium channels may underly the hyperalgesic responses of mammalian sensory neurones. We have therefore examined PKA phosphorylation of the cloned alpha-subunit of the rat sensory neurone-specific TTX-r channel SNS. Phosphorylation of SNS was compared with that of a mutant channel, SNS(SA), in which all five PKA consensus sites (RXXS) within the intracellular I-II loop had been eliminated by site-directed mutagenesis (serine to alanine). 2. In vitro PKA phosphorylation and tryptic peptide mapping of SNS and mutant SNS(SA) I-II loops expressed as glutathione-S-transferase (GST) fusion proteins confirmed that the five mutated serines were the major PKA substrates within the SNS I-II loop. 3. SNS and SNS(SA) channels were transiently expressed in COS-7 cells and their electrophysiological properties compared. In wild-type SNS channels, forskolin and 8-bromo cAMP produced effects consistent with PKA phosphorylation. Mutant SNS(SA) currents, however, were not significantly affected by either agent. Thus, elimination of the I-II loop PKA consensus sites caused a marked reduction in PKA modulation of wild-type channels. 4. Under control conditions, the voltage dependence of activation of SNS(SA) current was shifted to depolarized potentials compared with SNS. This was associated with a slowing of SNS(SA) current inactivation at hyperpolarized potentials and suggested a tonic PKA phosphorylation of wild-type channels under basal conditions.5. We conclude that the major substrates involved in functional PKA modulation of the SNS channel are located within the intracellular I-II loop.  (+info)

Developmental aspects of glutathione S-transferase B (ligandin) in rat liver. (28/8048)

The postnatal development in male Sprague-Dawley rats of hepatic glutathione S-transferase B (ligandin) in relation to the other glutathione S-transferases is described. The concentration of glutathione S-transferase B in 1-day-old male rats is about one-fifth of that in adult animals. The enzyme reaches adult concentrations 4-5 weeks later. When assessed by substrate specificity or immunologically, the proportion of transferase B relative to the other glutathione S-transferases is high during the first week after birth. At this age, 67.5% of the transferase activity towards 1-chloro-2,4-dinitrobenzene is immunoprecipitable by anti-(transferase B), compared with about 50% in adults and older pups. Between the second and the fifth postnatal week, the fraction of transferase B increases in parallel fashion with the other transferases in hepatic cytosol. Neither L-thyroxine nor cortisol induce a precocious increase in glutathione S-transferase activity. Phenobarbital did induce transferase activity towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene in both pups and adults. The extent of induction by phenobarbital was a function of basal activity during development such that the percentage stimulation remained constant from 5 days postnatally to adulthood.  (+info)

Functional analysis of the evolutionarily conserved proline 53 residue in Proteus mirabilis glutathione transferase B1-1. (29/8048)

The role of the evolutionarily conserved residue Pro-53 in Proteus mirabilis glutathione transferase B1-1 has been examined by replacing it with a serine residue using site-directed mutagenesis. The effect of the replacement on the activity, thermal stability and antibiotic binding capacity of the enzyme was examined. The results presented support the view that Pro-53 participates in the maintenance of the proper conformation of the enzyme fold rather than playing a direct role in the catalytic reaction. Furthermore, this residue appears to be an important determinant of the antibiotic binding to the enzyme. Experiments with wild type and mutated enzymes provide evidence that glutathione transferases may play an important role in antibiotic resistance exhibited by bacteria.  (+info)

Purification of a glutathione S-transferase and a glutathione conjugate-specific dehydrogenase involved in isoprene metabolism in Rhodococcus sp. strain AD45. (30/8048)

A glutathione S-transferase (GST) with activity toward 1, 2-epoxy-2-methyl-3-butene (isoprene monoxide) and cis-1, 2-dichloroepoxyethane was purified from the isoprene-utilizing bacterium Rhodococcus sp. strain AD45. The homodimeric enzyme (two subunits of 27 kDa each) catalyzed the glutathione (GSH)-dependent ring opening of various epoxides. At 5 mM GSH, the enzyme followed Michaelis-Menten kinetics for isoprene monoxide and cis-1, 2-dichloroepoxyethane, with Vmax values of 66 and 2.4 micromol min-1 mg of protein-1 and Km values of 0.3 and 0.1 mM for isoprene monoxide and cis-1,2-dichloroepoxyethane, respectively. Activities increased linearly with the GSH concentration up to 25 mM. 1H nuclear magnetic resonance spectroscopy showed that the product of GSH conjugation to isoprene monoxide was 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB). Thus, nucleophilic attack of GSH occurred on the tertiary carbon atom of the epoxide ring. HGMB was further converted by an NAD+-dependent dehydrogenase, and this enzyme was also purified from isoprene-grown cells. The homodimeric enzyme (two subunits of 25 kDa each) showed a high activity for HGMB, whereas simple primary and secondary alcohols were not oxidized. The enzyme catalyzed the sequential oxidation of the alcohol function to the corresponding aldehyde and carboxylic acid and followed Michaelis-Menten kinetics with respect to NAD+ and HGMB. The results suggest that the initial steps in isoprene metabolism are a monooxygenase-catalyzed conversion to isoprene monoxide, a GST-catalyzed conjugation to HGMB, and a dehydrogenase-catalyzed two-step oxidation to 2-glutathionyl-2-methyl-3-butenoic acid.  (+info)

Distribution of microsomal glutathione transferase 1 in mammalian tissues. A predominant alternate first exon in human tissues. (31/8048)

An extensive Northern blot analysis of microsomal glutathione transferase 1 in human and rat tissues was performed. When normalized against the glyceraldehyde-3-phosphate dehydrogenase or actin expression it was evident that the predominant expression occurs in liver and pancreas. An ontogenetic, as well as a functional, basis for the high levels in these two organs is possible. The relative expression levels in man ranged from: liver and pancreas (100%), to kidney, prostate, colon (30-40%), heart, brain, lung, testis, ovary, small intestine (10-20%), placenta, skeletal muscle, spleen, thymus and peripheral blood leucocytes (1-10%). Liver-enriched expression was detected in human fetal tissues with lung and kidney displaying lower levels (10-20%). No transcripts could be detected in fetal brain or heart. When comparing the expression levels between rat and man it is apparent that human extrahepatic mRNA levels are much higher relative to liver. Rat microsomal glutathione transferase mRNA expression ranges from 0.2 to 10% that of liver, with adrenal, uterus, ovary and stomach displaying the highest levels of the organs tested. Based on these observations, and the fact that the enzyme is encoded by a highly conserved single-copy gene, it is suggested that microsomal glutathione transferase 1 performs essential functions vital to most mammalian cell types. We suggest that protection against oxidative stress constitutes one such function. Human expressed sequence tag (EST) characterization yielded four alternate mRNA transcripts with different 5'-ends (four alternate noncoding exons 1). The predominant exon (based on the observed EST frequency) revealed a tissue distribution similar to that obtained using the reading frame as probe. Thus, it appears that one exon preferentially gives rise to mature mRNA in the human tissues examined. This exon is different from the one reported in the original cDNA characterized.  (+info)

DNA binding protein dbpA binds Cdk5 and inhibits its activity. (32/8048)

Progress in the cell cycle is governed by the activity of cyclin dependent kinases (Cdks). Unlike other Cdks, the Cdk5 catalytic subunit is found mostly in differentiated neurons. Interestingly, the only known protein that activates Cdk5 (i.e. p35) is expressed solely in the brain. It has been suggested that, besides its requirement in neuronal differentiation, Cdk5 activity is induced during myogenesis. However, it is not clear how this activity is regulated in the pathway that leads proliferative cells to differentiation. In order to find if there exists any Cdk5-interacting protein, the yeast two-hybrid system was used to screen a HeLa cDNA library. We have determined that a C-terminal 172 amino acid domain of the DNA binding protein, dbpA, binds to Cdk5. Biochemical analyses reveal that this fragment (dbpA(Cdelta)) strongly inhibits p35-activated Cdk5 kinase. The protein also interacts with Cdk4 and inhibits the Cdk4/cyclin D1 enzyme. Surprisingly, dbpA(Cdelta) does not bind Cdk2 in the two-hybrid assay nor does it inhibit Cdk2 activated by cyclin A. It could be that dbpA's ability to inhibit Cdk5 and Cdk4 reflects an apparent cross-talk between distinct signal transduction pathways controlled by dbpA on the one hand and Cdk5 or Cdk4 on the other.  (+info)