RESUMO
Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.
Assuntos
Glutationa Transferase/química , Glutationa Transferase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cisteína/química , Primers do DNA/genética , DNA Complementar/genética , Dimerização , Dissulfetos/metabolismo , Escherichia coli/genética , Glutationa/metabolismo , Glutationa Transferase/genética , Técnicas In Vitro , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Oxirredução , Conformação Proteica , Subunidades Proteicas , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Levels of the hGSTM3 glutathione S-transferase (GST) subunit in testis of the human fetus and infant were found to be only a small fraction of those in adults. To understand these observations and to determine whether hGSTM3 subunit expression is developmentally and/or hormonally regulated, an experimental model based on the rat testis homologue (subunit rGSTM5) was used. For prepubertal rats, testicular rGSTM5 subunit levels were very low, but a sharp increase was observed between weeks 6 and 7 of development, when testicular growth includes increased numbers of germ cells associated with spermatogenesis. In adult hypophysectomized rats, the rGSTM5 subunit content of testis decreased progressively over 5 weeks, at which time the subunit was barely detectable. In contrast, the other GST subunit types did not vary significantly during development or after hypophysectomy. These results suggest that rGSTM5 subunits in rat testis could originate from spermatogenic cells. Accordingly, GSTs were purified from human sperm, and it was shown that the hGSTM3 subunit was, by a large measure, the predominant form. These data are consistent with the notion that the differential expression of hGSTM3 during human testicular development can also be explained on the basis of its preferential location in germs cells.
Assuntos
Glutationa Transferase/biossíntese , Espermatogênese , Espermatozoides/enzimologia , Adulto , Animais , Encéfalo/enzimologia , Humanos , Hipofisectomia , Lactente , Masculino , Ratos , Ratos Sprague-Dawley , Testículo/citologia , Testículo/embriologia , Testículo/crescimento & desenvolvimentoRESUMO
Cytosolic glutathione S-transferase (GST) subunits from human testis were resolved by HPLC and unambiguously identified by combined use of peptide sequence-specific antisera and electrospray ionization mass spectrometry (ESI MS). Allelic variants of hGSTP1, hGSTM1 and hGSTA2 were distinguished on the basis of observed differences in their molecular masses. Relative amounts of the multiple different subunit types in various human tissues were determined from HPLC profiles. From this type of analysis, tissues from hGSTM1 null allele individuals were readily discerned at the protein level; liver was the only tissue in which the hGSTM1 subunit was the major mu-class GST. hGSTM4 and hGSTM5 subunits were found at very low levels in all tissues examined. By far the tissue richest in the unique hGSTM3 subunit was testis, although brain also has significant levels.
Assuntos
Glutationa Transferase/química , Glutationa Transferase/genética , Testículo/enzimologia , Idoso , Alelos , Animais , Encéfalo/enzimologia , Cromatografia Líquida de Alta Pressão , Citosol/enzimologia , Expressão Gênica , Variação Genética , Glutationa Transferase/classificação , Humanos , Masculino , Espectrometria de Massas , Camundongos , Peso Molecular , Conformação Proteica , Ratos , Especificidade da Espécie , Distribuição TecidualRESUMO
The aim of this investigation was to determine the frequency of adverse pregnancy outcomes in Kiev during the period surrounding the Chernobyl accident on April 26, 1986. Additional effective equivalent doses resulting from the catastrophic irradiation in 1986-1991 was 8.04 mSv for Kiev inhabitants. We retrospectively analyzed the archives of the two largest obstetric hospitals between 1969 and 1990. Spontaneous miscarriages, congenital anomalies, and perinatal mortality varied during the two decades without any pronounced changes in any direction. Additional long-term follow-up is needed to determine mutagenic or carcinogenic effects.