A fructan: fructan fructosyltransferase activity from Lolium rigidum

Fructan:fructan fructosyltransferase (FFT) activity was purified about 300-fold from leaves of Lolium rigidura Gaudin by a combination of affinity chromatography, gel filtration, anion exchange and isoelectric focusing. The FFT activity was free of sucrose:sucrose fructosyltransferase and invertase activities. It had an apparent pI of 4.7 as determined by isoelectric focusing, and a molecular mass of about 50000 (gel filtration). The FFT activity utilized the trisaccharides 1-kestose and 6(G)-kestose as sole substrates, but was not able to use 6-kestose as sole substrate. The FFT activity was not saturated when assayed at concentrations of 1-kestose, 6(G)-kestose or (1,1)-kestotetraose of up to 400 mM The rate of reaction of the FFT activity was most rapid when assayed with 1-kestose and was less rapid when assayed with 6(G)-kestose, (1,1)-kestotetraose or (1,1,1)-kestopentaose. The FFT activity when assayed at a relatively high concentration of enzyme activity (approximately equivalent to about half the activity in crude extracts per gram fresh mass) did not synthesize fructan of degree of polymerization > 6, even during extended assays of up to 10 h. When assayed with a combination of 1-kestose and uniformly labelled [C-14]sucrose as substrates, the major reaction was the transfer of a fructosyl residue from 1-kestose to sucrose resulting in the re-synthesis of 1-kestose. Tetrasaccharide and 6(G)-kestose were also synthesized. When assayed with 6(G)-kestose and [C-14]sucrose as substrates, the major reaction of the FFT activity was the synthesis of tetrasaccharide. However, some synthesis of 1-kestose and re-synthesis of 6(G)-kestose also occurred. When 6, kestose was the sole substrate for the FFT activity, synthesis of tetrasaccharide was 2.7 to 3.4-fold slower than when 1-kestose was used as the sole substrate. Owing to differences in the fructan:sucrose fructosyltransferase activity of the FFT with each of the trisaccharides, net synthesis of tetrasaccharide by the FFT was altered significantly in the presence of sucrose. The magnitude of this effect depended on the concentration of the trisaccharides. In the presence of sucrose, 6(G)-kestose could be a substrate of equivalent importance to 1-kestose for synthesis of tetrasaccharide.

Identification of products formed by a fructan:fructan fructosyltransferase activity from Lolium rigidum

The products formed by a fructan:fructan fructosyltransferase (FFT) activity purified from Lolium rigidum Gaudin were identified after gas chromatography-mass spectrometry of partially methylated alditol acetates, electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography. The FFT activity synthesized oligofructans up to degree of polymerization (DP) 6, but did not synthesize fructans of DP > 6 even when assayed with (1,1,1)-kestopentaose for up to 10 h. The FFT activity when assayed with 1-kestose or 6(G)-kestose synthesized fructan with fructosyl residues almost exclusively linked by beta-2,1-glycosidic linkages. When assayed with 1-kestose, the FFT activity synthesized tetrasaccharides and pentasaccharides with an internal glucosyl residue. The predominant tetrasaccharide was (1&6(G))-kestotetraose and the predominant pentasaccharide was (1&6(G),1)-kestopentaose. By comparison, tetrasaccharides and pentasaccharides extracted from L. rigidum also contained predominantly beta-2,1-glycosidic linked fructans with an internal glucosyl residue. The only exception was that one of the pentasaccharides contained beta-2,1- and beta-2,6-glycosidic linked fructosyl residues. This pentasaccharide was not synthesized by the FFT activity. The role of this FFT activity in formation of oligofructans in L. rigidum is discussed.

Synthesis of fructans by fructosyltransferase from the tuberous roots of Viguiera discolor (Asteraceae)

Sucrose:sucrose fructosyltransferase (SST) and fructan:fructan fructosyl-transferase (FFT) activities from crude extracts of tuberous roots of Viguiera discolor growing in a preserved area of cerrado were analyzed in 1995-1996. SST activity was characterized by the synthesis of 1-kestose from sucrose and FFT activity by the production of nystose from 1-kestose. The highest fructan-synthesizing activity was observed during early dormancy (autumn), when both (SST and FFT) activities were high. The increase in synthetic activity seemed to start during the fruiting phase in the summer, when SST activity was higher than in spring. During winter and at the beginning of sprouting, both activities declined. The in vitro synthesis of high molecular mass fructans from sucrose by enzymatic preparations from tuberous roots collected in summer showed that long incubations of up to 288 h produced consistently longer polymers which resembled those found in vivo with respect to chromatographic profiles.

Influência do treinamento físico aeróbio no transporte mitocondrial de ácidos graxos de cadeia longa no músculo esquelético: papel do complexo carnitina palmitoil transferase

O ácido graxo (AG) é uma importante fonte de energia para o músculo esquelético. Durante o exercício sua mobilização é aumentada para suprir as necessidades da musculatura ativa. Acredita-se que diversos pontos de regulação atuem no controle da oxidação dos AG, sendo o principal a atividade do complexo carnitina palmitoil transferase (CPT), entre os quais três componentes estão envolvidos: a CPT I, a CPT II e carnitina acilcarnitina translocase. A função da CPT I durante o exercício físico é controlar a entrada de AG para o interior da mitocôndria, para posterior oxidação do AG e produção de energia. Em resposta ao treinamento físico há um aumento na atividade e expressão da CPT I no músculo esquelético. Devido sua grande importância no metabolismo de lipídios, os mecanismos que controlam sua atividade e sua expressão gênica são revisados no presente estudo. Reguladores da expressão gênica de proteínas envolvidas no metabolismo de lipídios no músculo esquelético, os receptores ativados por proliferadores de peroxissomas (PPAR) alfa e beta, são discutidos com um enfoque na resposta ao treinamento físico.

Predictive values of aspartate aminotransferase and gamma-glutamyl transferase for the hepatic accumulation of copper in cattle and buffalo

Ten cattle and 10 buffalo were divided into 2 groups (control [n = 8] and experimental [n = 12]) that received daily administration of copper. Three hepatic biopsies and blood samples were performed on days 0, 45, and 105. The concentration of hepatic copper was determined by spectrophotometric atomic absorption, and the activities of aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) were analyzed. Regression analyses were done to verify the possible existing relationship between enzymatic activity and concentration of hepatic copper. Sensitivity, specificity, accuracy, and positive and negative predictive values were determined. The serum activities of AST and GGT had coefficients of determination that were excellent predictive indicators of hepatic copper accumulation in cattle, while only GGT serum activity was predictive of hepatic copper accumulation in buffalo. Elevated serum GGT activity may be indicative of increased concentrations of hepatic copper even in cattle and buffalo that appear to be clinically healthy. Thus, prophylactic measures can be implemented to prevent the onset of a hemolytic crisis that is characteristic of copper intoxication.

Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa

Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 angstrom, alpha = 63.45, beta = 80.66, gamma = 94.55 degrees. xfGST crystals diffracted to 2.23 angstrom resolution on a rotating-anode X-ray source.

Cloning, expression, molecular modelling and docking analysis of glutathione transferase from Saccharum officinarum

Sugarcane yield and quality are affected by a number of biotic and abiotic stresses. In response to such stresses, plants may increase the activities of some enzymes such as glutathione transferase (GST), which are involved in the detoxification of xenobiotics. Thus, a sugarcane GST was modelled and molecular docked using the program LIGIN to investigate the contributions of the active site residues towards the binding of reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). As a result, W13 and I119 were identified as key residues for the specificity of sugarcane GSTF1 (SoGSTF1) towards CDNB. To obtain a better understanding of the catalytic specificity of sugarcane GST (SoGSTF1), two mutants were designed, W13L and I119F. Tertiary structure models and the same docking procedure were performed to explain the interactions between sugarcane GSTs with GSH and CDNB. An electron-sharing network for GSH interaction was also proposed. The SoGSTF1 and the mutated gene constructions were cloned and expressed in Escherichia coli and the expressed protein purified. Kinetic analyses revealed different Km values not only for CDNB, but also for GSH. The Km values were 0.2, 1.3 and 0.3 mM for GSH, and 0.9, 1.2 and 0.5 mM for CDNB, for the wild type, W13L mutant and I119F mutant, respectively. The V(max) values were 297.6, 224.5 and 171.8 mu mol min(-1) mg(-1) protein for GSH, and 372.3, 170.6 and 160.4 mu mol min(-1) mg(-1) protein for CDNB.

Regulation of the gene encoding glutathione S-transferase M1 (GSTM1) by the Myb oncoprotein

The identification of Myb 'target' genes will not only aid in the understanding of how overexpression of Myb, or expression of activated forms of Myb, leads to cellular transformation but will also shed light on its role in normal cells. Using a combination of an estrogen-regulated Myb-transformed cell line (ERMYB) and PCR-based subtractive hybridization, we have identified the gene (GSTM1) encoding the detoxification enzyme glutathione S-transferase M1 as being transcriptionally upregulated by Myb. Functional analysis of the GSTM1 promoter using reporter assays indicated that both the DNA binding and transactivation domains of Myb were required for transcriptional activation. Mutational analysis of consensus Myb-binding sites (MBS) in the promoter and electrophoretic mobility gel shift analysis indicated that one of the three potential MBS can bind Myb protein, and is the primary site involved in the regulation of this promoter by Myb.

Association between glutathione S-transferase polymorphisms and triglycerides and HDL-cholesterol

Objective: Null genotypes of glutathione S-transferase (GSTs) exhibit absence of enzymatic activity and are hypothesized to modulate an increased risk of developing cardiovascular disease. The aim of this study was to identify the potential association between GSTM1 and GSTT1 deleted polymorphisms with cardiovascular risk factors and coronary atherosclerosis in two independent urban populations. Methods and results: Genotype distribution of GSTM1 and GSTT1 deleted polymorphism were examined in a sample of 1577 individuals from the general population and a replication sample of 871 individuals submitted to coronary angiography. Triglycerides, HDL-cholesterol and the triglycerides/HDL ratio were significantly associated with a double-deleted genotype in individuals from the general population. These findings were replicated in a second, independent, population of individuals submitted to coronary angiography. In addition, coronary artery disease severity was also associated with GSTs genotypes and the risk conferred from GSTs genotype was mainly due to triglycerides/HDL ratio information. Conclusions: The data suggest that the presence of a double deletion genotypes of the GSTM1 and GSTT1 genes is associated with hypertriglyceridemia and low HDL-cholesterol levels in humans. These novel findings may provide a new unexplored link between lipid metabolism and GST homeostasis. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Farnesoic acid O-methyl transferase (FAMeT) isoforms: Conserved traits and gene expression patterns related to caste differentiation in the stingless bee, Melipona scutellaris

Farnesoic acid O-methyl transferase (FAMeT) is the enzyme that catalyzes the formation of methyl farnesoate (MF) from farnesoic acid (FA) in the biosynthetic pathway of juvenile hormone (JH). This work reports the cloning, sequencing, and expression of FAMeT gene from the stingless bee Melipona scutellaris (MsFAMeT). The MsFAMeT in silica analysis showed that greatest sequence similarity is found in Apis mellifera and other insects, while relatively less similarity is shown in crustaceans. Evidence of alternative splicing of a 27 nucleotide (nt) microexon explains the presence of the detected isoforms, 1 and 2. The expression analysis of the two isoforms showed a marked difference when castes were compared, suggesting that they could be involved differently in the JH metabolism in M. scutellaris, providing new insights for the comprehension of female plasticity.

Expression of placental glutathione S-transferase in rat tongue mucosa exposed to cigarette smoke

Glutatione S-transferases (GSTs) are a family of enzymes involved in detoxification of xenobiotics. Placental GST, known as GST-P, has been detected in tissues following exposure to carcinogenic agents being regarded a reliable biomarker of exposure and susceptibility in early phases of carcinogenesis. The aim of this study was to investigate the expressivity of GST-P positive foci in the rat tongue mucosa exposed to cigarette smoke by means of immunohistochemistry. A total of twelve male Wistar rats were distributed into two groups: negative control and experimental group exposed to cigarette smoke during 75 days. After experimental period, no histopathological changes in the tongue mucosa were evidenced in the negative control and the experimental group. However, a total of five GST-P positive foci were detected in two out of six animals exposed to cigarrette smoke. None control animals were noticed GST-P positive foci. These data indicate that expression of GST-P may reflect the carcinogenic effect of cigarette smoke as well as the genetic susceptibility of animals in relation to continuous carcinogens exposure.

Polymorphisms at the glutathione S-transferase GSTM1, GSTT1 and GSTP1 loci: risk of ovarian cancer by histological subtype

The phase II glutathione S-transferases (GSTs) GSTT1, GSTM1 and GSTP1 catalyse glutathione-mediated reduction of exogenous and endogenous electrophiles. These GSTs have broad and overlapping substrate specificities and it has been hypothesized that allelic variants associated with less effective detoxification of potential carcinogens may confer an increased susceptibility to cancer. To assess the role of GST gene variants in ovarian cancer development, we screened 285 epithelial ovarian cancer cases and 299 unaffected controls for the GSTT1 deletion (null) variant, the GSTM1 deletion (null) variant and the GSTP1 codon 104 A-->G Ile-->Val amino acid substitution variant, The frequencies of the GSTT1, GSTM1 and GSTP1 polymorphic variants did not vary with tumour behaviour (low malignant potential or invasive) or p53 immunohistochemical status. There was a suggestion that ovarian cancers of the endometrioid or clear cell histological subtype had a higher frequency of the GSTT1 and GSTM1 deletion genotype than other histological subgroups. The GSTT1, GSTM1 and GSTP1 genotype distributions did not differ significantly between unaffected controls and ovarian cancer cases (overall or invasive cancers only). However, the GSTM1 null genotype was associated with increased risk of endometrioid/clear cell invasive cancer [age-adjusted OR (95% CI) = 2.04 (1.01-4.09), P = 0.05], suggesting that deletion of GSTM1 may increase the risk of ovarian cancer of these histological subtypes specifically. This marginally significant finding will require verification by independent studies.