Beatrice Dragani

DEVELOPMENTAL ASPECTS OF DETOXIFYING ENZYMES IN FISH (SALMO IRIDAEUS)

The activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione transferase and glyoxalase I have been studied during the embryologic development of rainbow trout (Salmo iridaeus) and in several other trout tissues to investigate the protective development metabolism. A gradual increase of superoxide dismutase, catalase, glutathione reductase, glyoxalase I and glutathione transferase activities was noted throughout embryo development. In all trout tissues investigated glutathione peroxidase was found to be extremely low compared to catalase activity. The highest activity of superoxide dismutase, glyoxalase I and glutathione reductase was found in liver followed by kidney. No change in the number of GST subunits was noted with the transition from the embryonic to the adult stages of life according to the SDS/PAGE and HPLC analyses performed on the GSH-affinity purified fractions.

Developmental aspects of Bufo bufo embryo glutathione transferases

The expression of glutathione transferase isoenzymes has been studied during the development of Bufo bufo embryo. By analysing the GSH-affinity purified materials in terms of substrate specificities, SDS-PAGE pattern, HPLC elution profile, we conclude that, up to stage 22, no significant changes in the expression of glutathione transferases isoenzymes occurred during Bufo bufo embryo development. At stage 25 the distribution of glutathione transferases was found to be slightly different from those of all other foregoing stages. A marked decrease of embryonic glutathione transferases subunits with a parallel appearance of new structurally and immunologically different subunits was noted in toad liver and kidney. Toad ovary continued to express embryonic glutathione transferase subunits.

Glutathione transferase isoenzymes in olfactory and respiratory epithelium of cattle

Glutathione transferase (GST) was investigated in the olfactory and respiratory epithelium of cattle. A significantly more abundant GST in terms of either protein amount or activity was found in the olfactory rather than in the respiratory epithelium. No apparent qualitative differences in the isoelectric focusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC profiles were noted in the reduced glutathione (GSH) affinity purified GST pool of olfactory and respiratory epithelium. Both tissues have at least six GST isoenzymes with isoelectric point values of 4.9 (peak I), 5.3 (peak II), 5.95 (peak III), 6.5 (peak IV), 7.1 (peak V) and 9.3 (peak VI). From both tissues at least seven different GST subunits can be resolved by HPLC analysis. The GST isoenzymes having pI at 5.3 and 9.3 were predominantly expressed in the olfactory than in the respiratory epithelium. These latter forms conjugate GSH efficiently with alkenals and hydroperoxides, respectively. Kinetic, immunological and structural properties, including HPLC analysis and N-terminal region amino acid sequence seem to indicate that the bovine nasal mucosa tissue in addition to a GST subunit which is orthologue to rat subunit 8 (alpha class) express tissues specific subunits.

Structural characterization of acid-induced intermediates of human glutathione transferase P1-1.

The acid denaturation of human glutathione transferase P1-1 (hGSTP1-1) has been performed to investigate the unfolding intermediates of the protein and their possible involvement in the refolding mechanism. The acid-induced structures of GSTP1-1 have been characterized by activity, gel filtration, intrinsic fluorescence and far-u.v. circular dichroism (CD) techniques. Because of the non-identity of the different transitions monitored, the acid denaturation of hGSTP1-1 appears to be a multistep process during which several intermediates coexist in equilibrium. The dependence of inactivation on the protein concentration, as well as gel-filtration experiments, indicate that the inactivation transition, centred at about pH 4.0, corresponds to the monomerization of the protein. At pH 2.0, when the enzyme is completely inactive, the protein retains a small, but significant, amount of secondary structure. This means that the dimeric arrangement of the molecule is important for maintaining the native-like secondary structure of the monomer. The results show that, at low pH, the compact state of the GST monomer, even upon the addition of salts, does not possess native-like secondary structure as described for many monomeric proteins (molten globule). In the presence of physiological concentrations of salts, the protein solution at pH 2.0 leads to a dead-end aggregation process, suggesting that this compact state cannot represent a productive intermediate of the refolding pathway.

Analysis by limited proteolysis of domain organization and GSH-site arrangement of bacterial glutathione transferase B1-1

Limited proteolysis method has been used to study the structure-function relationship of bacterial glutathione transferase (GSTB1-1). In absence of three-dimensional structural data of prokaryote GST, the results represent the first information concerning the G-site and domains organization of GSTB1-1. The tryptic cleavages occur mainly at the peptide bonds Lys35-Lys36 and Phe43-Leu44, generating two major molecular species of 20-kDa, 3-kDa and traces of 10-kDa. 1-chloro-2,4-dinitrobenzene favoured the proteolysis of the 20-kDa fragment markedly enhancing the production of the 10-kDa peptide by cleaving the chemical bonds Lys87-Ala88 and Arg91-Tyr92. The tryptic cleavage sites of GSTB1-1 was found to be located close to those previously found for the mammalian GSTP1-1 isozyme. It was concluded that despite their low sequence homology (18%), GSTB1-1 and GSTP1-1 displayed similar structural features in their G-site regions and probably a common organization in structural domains.

A novel glutathione transferase from Haemophilus influenzae which has high affinity towards antibiotics

Cytosolic glutathione transferase (GSTs) are a family of multi-functional proteins which catalyse the conjugation of glutathione (GSH) to a large variety of endogenous and exogenous electrophilic compounds. Much is known about cytosolic mammalian GSTs, however, the presence of GSTs in several aerobic and anaerobic micro-organisms has also been demonstrated. Several findings seem to suggest that bacterial GSTs are involved in processes of biodegradation of xenobiotics, including antibiotics. However, the function played by these enzymes in the bacterial cell still remains to be clarified. At present, it is ill-defined whether bacterial GST can be classified, as in the case of mammalian enzymes, into several distinct classes. Here we report the purification of a GST isoform from Haemophilus influenzae using GSH-affinity chromatography. The purified protein was characterised by immunological and kinetic properties different from other known GSTs. The dissociation constants of chloramphenicol, ampicillin, rifampicin and tetracycline to the purified enzyme were 0.62, 9.06, 4.08 and 1.77 microM, respectively, as determined by following the quenching of the protein intrinsic fluorescence. These values were much lower than those previously determined for the same drugs with other mammalian or bacterial GSTs. The present results indicate that the enzyme purified from H. influenzae is a novel GST isoform well distinguished from other known mammalian or bacterial GSTs.

Dynamic monitoring of restricted eating disorders by indirect calorimetry: a useful cognitive approach

Objective: Outpatient treatment in restricted eating disorder: indirect calorimetry during dynamic monitoring. Design: A retrospective observational study. Subjects: Twenty seven women affected by restricted eating disorder (essentially anorexia nervosa) with a body mass index [weight (kg)/height (m2)] of 17.29±2.47 were studied. The sample was compared as itself control during rehabilitative way. Interventions: Fat mass (FM) and fat free mass (FFM) were determined by anthropometry technique. REE/day and respiratory quotient (RQ,VCO2/VO2) were measured by indirect calorimetry using a Calorimeter Vmax 29n-Sensor Medics-California. Skinfold thickness and circumferences were also measured. Arm muscle area (AMA) and fat area were calculated by formulas reported in Frisancho. Results: The data indicated a positive correlation between AMA, VO2/ml/min and resting energy expenditure (REE)/day values examined during follow-up of patients. The increase of these parameters indicated a good monitoring index correlated to a FFM recovery during psychonutritional rehabilitation. Conclusion: Indirect calorimetry represents a useful approach for determining REE and prescribing diets in these patients. Moreover, the combined use of anthropometric techniques allows to accurately assess and adjust therapy according to the patient’s progress. This study shows that restricted eating disorders are characterized by a recovery of FFM related to improvement of body weight and REE/day. On the contrary, the increase of AFA revealed a recovery of fat-metabolism (corresponding to RQ decrease) and lipid/carbohydrates oxidation improvement, only in the presence, at the same time, of O2 consumption increase.

Purification and characterization of glutathione transferase from psoriatic skin.

The glutathione transferases (GSTs) comprise a family of enzymes that catalyze the conjugation of glutathione with certain hydrophobic compounds, bind various hydrophobic compounds, and have selenium-independent glutathione peroxidase activity. Of the four classes of GST, the pi class is the only one present in keratinocytes, and pi-class GST is elevated in psoriatic epidermis. We have purified and characterized GST from psoriatic scales. Immunoreactivity was observed with pi class antisera, and amino terminal sequencing showed identity with GST from human placenta and cultured human keratinocytes. We conclude that the majority of GST activity in psoriatic skin is due to a pi-class isoenzyme, and pi-class GST may represent an index for hyperproliferation.

Irreversible thermal denaturation of glutathione transferase P1-1. Evidence for varying structural stability of different domains

Abstract Glutathione transferases (GSTs) belong to a dimeric multifunctional enzyme family in which each subunit is organized into two domains. Despite numerous studies, the degree of the structural dependence of GST domains, as well as the functional significance of interdomain interactions, are still unknown. In order to investigate these important aspects of folding we decided to study the thermal denaturation of GSTP1-1. The protein transitions were followed by monitoring the loss of activity, far ultraviolet CD, intrinsic fluorescence and the aggregation state of the protein. The results show that thermal denaturation of the enzyme is a multistep process. This substantially confirms our previous unfolding studies and indicates that the presence of intermediates during unfolding of the protein reflects an inherent property of the native structure. Thermal denaturation of GSTP1-1 was essentially irreversible because of the formation of inactive scrambled structures which were unable to refold spontaneously by lowering the temperature. The fact that the fluorescence changes occur before any other transitions indicates that the GST domain I region containing Trp28 and Tryp38 residues is relatively more flexible than the molecule as a whole. Moreover, the present results, together with our previous unfolding and limited proteolysis studies on GSTP1-1, support the suggestion that the high mobility of the G-site polypeptide portion encompassing the Trp38 residue could have a role during catalysis. The present study also indicates that domain II of GSTP1-1 has a higher intrinsic stability than the other part of the molecule, including some regions of domain I and the active site.

Structural characterization of human glyoxalase II as probed by limited proteolysis

Human glyoxalase II is partially proteolyzed by trypsin, under non denaturing conditions, only at the level of the C‐terminal region. The proteolytic cleavage resulted in an inactivation of the enzyme without loss of the secondary structure. Sodium dodecyl sulphate polyacrylamide gel‐electrophoresis and microsequence analysis showed that the glyoxalase II is proteolyzed at the level of Arg 184 and Lys 230 and undergoes a third cleavage in a region located at the beginning of the supposed C‐terminal domain. The proteolysis occurs either in the presence or in the absence of specific inhibitors. Our limited proteolysis experiments and secondary structure prediction give evidence for the presence of two domains characterized by different pattern of secondary structure.