Amphibian embryo glutathione transferase: amino acid sequence and structural properties.
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Amino Acid Sequence↗
Biomedical subjects
R Petruzzelli
Publications and source records attributed to R Petruzzelli.
Identification of an N-capping box that affects the alpha 6-helix propensity in glutathione S-transferase superfamily proteins: a role for an invariant aspartic residue.
We have identified an N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) that is strictly conserved, at the beginning of alpha 6 helix, in all glutathione S-transferases (GSTs) and most of the related superfamily proteins. By using CD and peptide modelling we have demonstrated that the capping box residues have an important role in determining the helical conformation adopted by this fragment in the hydrophobic environment of the protein. This is an example in which a local motif, contributing to nucleation of a structural element essential to the global folding of the protein, is strictly conserved in a superfamily of homologous proteins.
Amino Acid Sequence↗
Characterization of Cu,Zn superoxide dismutase from the bathophile fish, Lampanyctus crocodilus.
Cu,Zn SOD from the bathophile teleost Lampanyctus crocodilus (LSOD) shows a high degree of homology with the sequence of the enzymes from other teleostean fish species. The catalytic properties of LSOD are very similar to those of the bovine enzyme, albeit with higher sensitivity to thermal denaturation. The apparent molecular mass of LSOD (37.6 KDa) is higher than the other Cu,Zn SOD variants studied. The aminoacid sequence of LSOD reveals interesting substitutions compared to the bovine enzyme. These are discussed in view of the particular environmental conditions to which L. crocodilus is adapted.
Amino Acid Sequence↗
Effect of anaerobic environment on the glutathione transferase isoenzymatic pattern in Proteus mirabilis.
When Proteus mirabilis was cultured anaerobically in the presence of nitrate as terminal electron acceptor, a dramatic reduction of glutathione transferase production occurred. The analysis of the glutathione affinity purified materials in terms of substrate specificity, SDS-PAGE pattern, IEF pattern and immunoblotting revealed that a significantly different glutathione transferase pattern also occurred: two new glutathione transferase forms with an isoelectric point at pH 4.8 and 5.0 appeared. Their N-terminal amino acid sequence analysis as well as the ability to bind to a glutathione affinity column indicate that major differences between anaerobic and aerobic glutathione transferase forms are mainly located in the C-terminal region of the primary structure. In contrast, no significant changes occurred in the production of glutathione transferase isoenzymes when P. mirabilis was grown anaerobically in the absence of a terminal electron acceptor. These results support the idea that bacterial glutathione transferase expression is not strictly related to the absence of oxygen stress.
Anaerobiosis↗
Diving behaviour and haemoglobin function: the primary structure of the alpha- and beta-chains of the sea turtle (Caretta caretta) and its functional implications.
The amino acid sequence of the alpha- and beta-chains of haemoglobin (Hb) from the loggerhead sea turtle (Caretta caretta) has been determined. Comparison with that of human Hb shows differences in several residues involved in both alpha 1 beta 1 and alpha 1 beta 2 packing contacts. On the whole, in spite of the mutations, the essential characteristics of both interfaces seem to be maintained. The functional properties of the sea turtle Hb have been investigated at different temperatures and as a function of proton, chloride and organic phosphate concentrations. In addition to overall similarities shared with most of the vertebrate Hbs previously described, this molecule shows significant differences which could be related to the life behaviour of the turtle. In fact, while the shape of the Bohr-effect curve is well adapted for gas exchange during prolonged dives, the very small enthalpy change for O2 binding ensures that O2 delivery becomes essentially insensitive to the temperature changes of the environment. Moreover, and similarly to the case of emperor penguin Hb, the small alkaline Bohr effect appears to be only choride-linked, since the pH dependence of the O2 affinity is abolished in the absence of this ion. These functional characteristics are discussed on the basis of the primary structure of alpha- and beta-chains.
Adaptation, Physiological↗
Alteration of glutathione transferase subunits composition in the liver of young and aged rats submitted to hypoxic and hyperoxic conditions.
In the present work, we have studied glutathione transferase (GST) activity and GST subunits distribution in the liver of young and aged rats kept under hypoxic or hyperoxic normobaric conditions as model of oxidative stress. A significant decrease of GST activity was detected in young hypoxic rat liver, whereas a significant increase occurred in aged hypoxic liver. No significant alteration of activity was obtained in both young and aged rat livers subjected to hyperoxic treatment. Substrate specificity measurements, SDS/PAGE analysis and reverse-phase HPLC, of GSH-affinity purified fractions were used to study the changes in the GST subunits pattern occurring in the liver of rat as a consequence of hypoxic and hyperoxic treatment. The results demonstrate that young and aged rat liver has a different constitutive GST subunit pattern which are markedly and differentially altered in hypoxia or hyperoxia. The hyperoxic treatment caused an increase of GST subunit 3 in aged, but not in young liver. In aged liver, both the hypoxic and hyperoxic treatment produced a decrease of GST subunit 4. After hypoxic treatment GST subunit 3 significantly increased in both young and aged liver. GST subunit 1a increased in both young and adult liver after hyperoxia. Following hypoxia a decrease of subunit 1a was seen in both young and aged liver. After hypoxic treatment, subunit 6 doubled in young, but not in aged, livers. It was concluded that the alterations in GST subunit expression occurring in the liver as a consequence of hypoxic or hyperoxic treatment respond to the necessity of a better protection of liver against the products of oxidative metabolism.
Aging↗
Purification and characterization of a heterodimeric 23/20-kDa proteolytic fragment of bacterial glutathione transferase B1-1.
The proteolytic attack of bacterial glutathione S-transferase (GSTB1-1) by trypsin cleaves and inactivates the enzyme. The polypeptide portion of GSTB1-1 encompassing the cleavage site (Lys35-Lys36) constitutes an exposed and flexible region of the GSTB1-1 G-site. By sequentially using a benzamidine-affinity chromatography and GSH-affinity column, a proteolyzed form of GSTB1-1 (23/20 kDa), in which only one subunit has been cleaved has been purified and characterized. Gel filtration, sequence analysis of subunits separated by HPLC, and CD experiments indicate that the 23/20-kDa GSTB1-1 form is a dimer and maintains its secondary structure. In addition, kinetic determinations reveal that the proteolytic cleavage of one polypeptide chain inactivates one active site but does not influence the catalytic efficiency of the second one. Previous refolding studies on GSTB1-1 have shown that the formation of a correct dimer precedes the recovery of the full activity of the enzyme, indicating that the dimeric structure is essential for catalytic activity of GSTB1-1. Thus, although GSTB1-1 active sites are catalytically independent and, probably, mainly located on each monomer, interactions deriving from the dimeric arrangement of the molecule appear essential for maintaining each active site in a fully active conformation. The catalytic independence of the two active sites, as well as the importance of dimeric structure for catalytic activity, has already been established for other GSTs. Thus, despite the very low sequence identity and kinetic differences between bacterial and other distant members of the GST superfamily, the results reported here indicate that important properties of the GST active site are conserved.
Amino Acid Sequence↗
Bovine hemoglobin cross-linked through the beta chains: functional and structural aspects.
2-Nor-2-formylpyridoxal (NFPLP) has been synthesized and coupled to bovine Hb according to the procedure developed by Benesch and Benesch. The reaction of bovine Hb with NFPLP leads to a cross-linkage between the beta subunits, which greatly stabilizes the low affinity T state of the molecule and simultaneously abolishes the tendency of the tetramer to dissociate into alpha beta dimers. The functional properties, examined from both the equilibrium and kinetic points of view, indicate that the chemical modification affects the O2 affinity, abolishes cooperativity, and induces a slight decrease of the Bohr effect. From modeling studies we are confronted with two different structural alternatives; the cross-link of beta chains may be formed between lysine 82 of beta2 and the N terminus of methionine 2 of beta1 or between the two lysine 82 residues of both beta2 chains. Digestion of modified beta globin chains and isolation of the cross-linked peptide have showed that NFPLP cross-links Met-beta2 and Lys-beta82. This allowed discussion in some detail of the molecular basis of the Bohr effect of the modified bovine hemoglobin. On the whole, NFPLP-modified bovine Hb could be considered as a first step toward the synthesis of a potential blood substitute.
Animals↗
Divalent cation binding to ceruloplasmin.
Binding of calcium to human and sheep ceruloplasmin was investigated by metal substitution with manganese and competitive displacement of bound manganese by calcium monitored by electron paramagnetic resonance spectroscopy. The Kd for calcium was found to be 1.4 mM. Magnesium also bound to ceruloplasmin, with Kd = 0.3 and 0.7 mM for the human and sheep protein, respectively. The thermal stability of ceruloplasmin, as studied by differential scanning calorimetry, was affected by calcium but not by magnesium. A considerable increase of the Tm value, from 73.8 to 83.1 degrees C, was observed for sheep ceruloplasmin in the presence of calcium. The Tm value of the human protein was only slightly altered by calcium (from 85.1 to 87 degrees C). The interaction of ceruloplasmin with the chromatographic material used for its isolation, Sepharose 4B derivatized with chloroethylamine, was weakened by calcium. This allowed us to set up a novel purification scheme that made it possible to efficiently isolate ceruloplasmin and prothrombin from plasma with the same single-step chromatography.
Animals↗
Purification and characterization of three Pi class glutathione transferase from monkey (Macaca fascicularis) placenta.
Three forms of glutathione transferase (GST) with an apparent isoelectric point of pH 4.65 (GST I), 4.75 (GST II) and 4.9 (GST III) were resolved from the monkey (Macaca fascicularis) placenta after GSH-affinity chromatography followed by chromatofocusing. Substrate specificity, immunological reactivity, as well as N-terminal aminoacid sequences indicate that the three enzymes belongs to the pi class of GST. Reverse phase HPLC analysis indicates that the three GST arise from the combination of two different subunits eluting respectively at 29.60 +/- 0.10 min and 32.43 +/- 0.13 min. GST I is an homodimer of the 29.60 +/- 0.10 min subunit, GST III is an homodimer of the 32.43 +/- 0.13 min subunit, whereas the GST II is an heterodimer of the 29.60 +/- 0.10 min and 32.43 +/- 0.13 min subunits. Our results strongly suggest that unlike human, multiple forms of pi class GST exist in monkey placenta.
Animals↗
Modifying filamentous phage capsid: limits in the size of the major capsid protein.
Ff filamentous phages are long thin cylindrical structures that infect bacteria displaying the F pilus and replicate without lysing the host. These structures are exploited to display peptides by fusing them to the amino terminus of either the bacterial receptor protein (pIII) or the major coat protein (pVIII). We have analysed a vast collection of phage mutants containing substitutions and insertions in the amino terminus of pVIII to ask whether any chemical group of this solvent exposed region of the phage capsid has any key function in the phage life cycle. Any of the five amino-terminal residues can be substituted by most amino acids without affecting phage assembly suggesting that this region does not play any essential role in morphogenesis. However, a deletion of three residues delta (Gly3Asp4Asp5) results in a phage clone with an decreased ability to produce infective particles. By engineering phages designed to display peptides by fusion to the amino terminus of the major coat protein we have found that phage viability is affected by peptide length while peptide sequence plays a minor "tuning" role. Most peptides of six residues are tolerated irrespective of their sequence while only 40% of the phages carrying an amino-terminal extension of eight residues can form infective particles. This fraction drops to 20% and 1% when we attempt to insert peptides 10 and 16 amino acids long. We have used this information to build phage libraries where each phage displays approximately 2700 copies of a different octapeptide all over the phage surface.
Amino Acid Sequence↗
Reptile heme protein structure: X-ray crystallographic study of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin at 2.0 A resolution.
The X-ray crystal structures of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin have been determined at 2.0 A resolution (R = 0.182, and 0.178, respectively). The results here reported, representing the first reptile globin solved by X-ray crystallography, have been analyzed in parallel with data for related monomeric hemoproteins, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian myoglobins, reflected by the 63% amino acid identity of their primary structures. The root-mean-square deviation between the entire polypeptide backbones of loggerhead sea turtle and sperm whale myoglobins, after structure superposition, is 0.83 A. Upon cyanide binding to the protein distal site, the iron-bound water molecule present in the aquo-met form is displaced by the incoming ligand. Cyanide is oriented towards the inner part of the heme distal site forming a Fe-C-N angle of 133 degrees.
Animals↗
Cytoplasmic and periplasmic production of human placental glutathione transferase in Escherichia coli.
An expression vector yielding large amounts of GST P1-1 in the cytoplasm of Escherichia coli was constructed. The recombinant enzyme, obtained after purification, was characterized in its physicochemical and kinetic properties and appeared to be indistinguishable from that purified from human placenta. However, N-terminal amino acid sequencing revealed that about 50% of the recombinant GST still contained methionine as the N-terminal amino acid. Such an incomplete processing was not simply due to overproduction of GST. In fact, under growth conditions that lead to a sharp decrease in the production of the protein the N-terminal methionine was not removed. GST was unable to translocate across the bacterial membrane when it was fused to the leader peptide of the pelB gene from Erwinia carotovora and accumulated in the cytoplasm in a soluble and active conformation. However, when this fusion protein was produced in a bacterial strain overexpressing the bacterial chaperonins GroEL and GroES, a fraction of GST was exported into the periplasmic space with the correct N-terminal sequence. The yield of correctly processed GST accounted for 12% of total GST present in the E. coli cells. Our results suggest that chaperonins are able to interact with nascent GST, thus maintaining the protein in an export-competent form and that E. coli strains with enhanced secretory characteristics may be obtained by genetic engineering technology.
Amino Acid Sequence↗
Pathogenicity of cat-scratch disease bacilli.
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Animals↗
Downregulation of human polymorphonuclear cell activities exerted by microorganisms belonging to the alpha-2 subgroup of Proteobacteria (Afipia felis and Rochalimaea henselae).
Intracellular pathogens have evolved effective mechanisms in order to survive in an intracellular environment, thus avoiding destruction by phagocytic cells. In this regard, a correlation between resistance to phagocytic killing and expression of pathogenic potency has been established. In this report, we have studied the interaction between human polymorphonuclear cells (PMN) and two gram-negative microorganisms, Afipia felis and Rochalimaea henselae, which belong to the alpha-2 subgroup of the class Proteobacteria. A. falis has been previously proposed as the causative agent of Cat Scratch Disease (CSD), but several recent lines of evidence attribute a major role to R. henselae. Of note, CSD is a syndrome characterized by a chronic lymphoadenopathy, involving macrophages and endothelial cells with a progression towards a granulomatous process and/or angiogenesis. Since members of the alpha-2 subgroup of Proteobacteria have the property to survive intracellularly, we have evaluated the effects exerted by A. felis and R. henselae on human PMN in terms of chemotaxis locomotion, degranulation and oxidative metabolism. Results will show an impairment of PMN activities as a consequence of the challenge with both microrganisms. In particular, inhibition of PMN oxidative function occurred either as result of a direct exposure to both A. felis and R. henselae or when PMN were primed by bacteria for the N-formyl-methionyl-leucyl-phenylalanine enhancement of the oxidative burst. These findings may account for the ability of A. felis and R. henselae to survive within PMN as expression of a further mechanism of pathogenic potency, influencing also the nature and the evolution of inflammatory response in the lesion sites.
Bartonella henselae↗
Cat-scratch disease in Italy: a serological approach.
The results of studies on the serologic responses to Afipia felis and Rochalimaea henselae in suspected patients for Cat Scratch Disease (CSD) are illustrated. This preliminary study performed using Indirect Immunofluorescence Assay, proved negative for A. felis and R. henselae in some patients and positive in others; in a few instances the test was positive for both organisms. Additional microbiological and serological studies are needed to clarify the exact role of these microorganisms in causing CSD.
Adolescent↗
Rochalimaea henselae organisms possess an elevated capacity of binding to peripheral blood lymphocytes from patients with cat scratch disease.
Cat scratch disease (CSD) is a clinical condition whose aetiological agent, according to recent findings, is of bacterial origin. Two Gram-negative bacteria are invoked as causative agents of CSD, namely Afipia felis and Rochalimaea henselae. In this paper, five patients with suspected CSD were studied in terms of binding capacity of A. felis and R. henselae to their own peripheral blood lymphocytes (PBL). This parameter was correlated with serum antibody titres to both A. felis and R. henselae, as determined by an indirect fluorescence assay (IFA). Results demonstrate that in four out of five cases binding of R. henselae to PBL was higher than that observed with A. felis. In two cases serum antibody titres to both bacteria were lower or absent, while in the other two patients serum antibody titres to R. henselae were significantly high. In one case only, characterized by elevated titres of serum antibodies to A. felis, values of cytoadherence exhibited by this bacterium were similar to those observed with R. henselae. The results suggest that bacterial binding to lymphocytes may represent an additional parameter to support diagnosis of CSD.
Adolescent↗
Functional, spectroscopic and structural properties of haemoglobin from chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex).
The functional and spectroscopic properties of chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex) haemoglobin (Hb) have been studied with special reference to the action of allosteric effectors and temperature. Moreover, the amino acid sequences of the N-terminal segments of the alpha- and beta-chains have been determined. The present results indicate that chamois and steinbock Hbs display a low affinity for O2, which appears to be modulated in vivo by Cl- ions rather than 2,3-bisphosphoglycerate. The Bohr effect for O2 binding to chamois and steinbock Hb is higher than for reindeer and bovine Hbs, being similar to that of human Hb. Moreover, the temperature-dependence of oxygenation appears intermediate between that of human and reindeer Hbs. E.p.r. and absorption spectroscopic properties of the ferrous nitrosylated derivative of chamois and steinbock Hbs suggest that both haemoproteins are in a low-affinity conformation even in the absence of InsP6. The reduced effect of polyphosphates on the functional and spectroscopic properties of chamois and steinbock Hb agree with amino acid differences in the N-terminal segment of the beta-chains (i.e. the deletion of Val(NA1) and the replacement of His(NA2), present in human Hb, and Gln(NA2), present in horse Hb, by Met). The molecular mechanism modulating the basic reaction of O2 with chamois and steinbock Hb may be linked to specific physiological needs related to the high-altitude habitats of these two animals.
Amino Acid Sequence↗