Filippo Martini

Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis

The cytosolic and mitochondrial alcohol dehydrogenases from Kluyveromyces lactis (KlADHs) were purified and characterised. Both the N-terminally blocked cytosolic isozymes, KlADH I and KlADH II, were strictly NAD-dependent and exhibited catalytic properties similar to those previously reported for other yeast ADHs. Conversely, the mitochondrial isozymes, KlADH III and KlADH IV, displayed Ala and Asn, respectively, as N-termini and were able to oxidise at an increased rate primary alcohols with aliphatic chains longer than ethanol, such as propanol, butanol, pentanol and hexanol. Interestingly, the mitochondrial KlADHs, at variance with cytosolic isozymes and the majority of ADHs from other sources, were capable of accepting as a cofactor, and in some case almost equally well, either NAD or NADP. Since Asp-223 of horse liver ADH, thought to be responsible for the selection of NAD as coenzyme, is strictly conserved in all the KlADH isozymes, this amino-acid residue should not be considered critical for the coenzyme discrimination with respect to the other residues lining the coenzyme binding pocket of the mitochondrial isozymes. The relatively low specificity of the mitochondrial KlADHs both toward the alcohols and the cofactor could be explained on the basis of an enhanced flexibility of the corresponding catalytic pockets. An involvement of the mitochondrial KlADH isozymes in the physiological reoxidation of the cytosolic NADPH was also hypothesized. Moreover, both cytosolic and KlADH IV isozymes have an additional cysteine, not involved in zinc binding, that could be responsible for the increased activity in the presence of 2-mercaptoethanol.

Primary structure of hemoglobins from trout (Salmo irideus). Partial determination of amino acid sequence of Hb trout IV

Recent work on the properties of the hemoglobin components from the trout Salmo irideus has brought to focus a number of interesting features bearing on the problem of molecular adaptation to physiological requirements [ 1,2] . Hb trout IV is characterized by a strong dependence of both ligand affinity and cooperativity on pH and organic phosphates; this is the so-called Root effect, which is related with delivery of O2 to the swim bladder in teleost fishes. Hb trout I lacks completely proton and phosphate linked heterotropic effects, although ligand binding is strongly cooperative, it is supposed to be an important O2 delivery-system present in hyperactive fish, which may undergo serious O2 debts under conditions of acidosis. Thus the presence of multiple hemoglobin components with different functional properties can be given a rational interpretation on the basis of multiple physiological demands. Any attempt to interpret the functional properties of these hemoglobins in terms of structural features demands, as a necessary prerequisite, the knowledge of their primary structure. Therefore a major goal of the work on the structure-function relationships in the hemoglobin components from trout is the determination of their amino acid sequence. In a previous communication [3] we reported information on the Cand N-terminal regions of both Hb trout I and Hb trout IV, and commented on the implications of

The structure of mitochrondrial aspartate aminotransferase from pig heart and comparison with that of the cytoplasmic isozyme

Donatella BARRA+, Francesco BOSSA*, Shawn DOONAN **, Hisham M. A. FAHMY**, Graham J. HUGHES**, Khalida Y. KAKOZ**, Filippo MARTINI+ and Raffaele PETRUZZELLI+ *Istituto di Chimica Biologica, Universitci di Roma, *Laboratorio di Biochimica Applicata, Universitci di Camerino e Centro di Biologia Molecolare de1 CNR, Cittci Universitaria, 00185 Roma, Italy and **Department of Chemistry, University College London, 20 Gordon Street, London WCIH OAJ, England

The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart, and inferences on evolution of the isoenzymes

SummaryWe report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme fromEscherichia coli. The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the geneduplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 109 years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme fromE. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3×109 years ago.

The primary structure of mitochondrial aspartate aminotransferase from human heart

The complete amino acid sequence of the mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from human heart has been determined based mainly on analysis of peptides obtained by digestion with trypsin and by chemical cleavage with cyanogen bromide. Comparison of the sequence with those of the isotopic isoenzymes from pig, rat and chicken showed 27, 29 and 55 differences, respectively, out of a total of 401 amino acid residues. Evidence for structural microheterogeneity at position 317 has also been obtained.

Purification and basic properties of the aspartate aminotransferases from a variety of sources

1. 1. The following aspartate aminotransferases have been isolated: cytosolic and mitochondrial isozymes from horse heart, rat liver and mouse liver, the mitochondrial isozyme from turkey liver, and the single isozymes from yeast and E. coli. 2. 2. Comparisons of the electrophoretic characteristics of the various isozymes are reported. In the case of the mitochondrial isozyme from turkey liver, studies have been made of the subforms of the protein and of its behaviour on hydrophobic chromatography. 3. 3. Studies of the distribution and stabilities of aspartate aminotransferase isozymes in homogenates of trout liver are reported.

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.

Interspecies comparisons of aspartate aminotransferases based on terminal and active site sequences

Abstract 1. 1. N-terminal amino acid sequences of mitochondrial aspartate aminotransferases from horse heart and rat liver, and of the cytosolic isozyme from rat liver have been determined by solid phase sequencing of intact proteins. 2. 2. Evidence is presented to show that the N-terminal residue of the cytosolic isozyme from horse heart is blocked. 3. 3. C-terminal amino acid sequences of the mitochondrial isozymes from horse heart and turkey liver, and of the cytosolic isozyme from horse heart have been determined by digestion with carboxypeptidases. 4. 4. Pyridoxyl-lysine peptides have been isolated from the mitochondrial isozymes of horse heart, turkey liver and rat liver and from the cytosolic isozyme of horse heart. Sequences of these peptides have been determined. 5. 5. The results obtained are compared with those for aspartate aminotransferases from other species.

An IL-15 dependent CD8 T cell response to selected HIV epitopes is related to viral control in early-treated HIV-infected subjects

In some early-treated HIV+ patients, Structured Treatment Interruption (STI) is associated to spontaneous control of viral rebound. Thus, in this clinical setting, we analyzed the immunological parameters associated to viral control. Two groups of early treated patients who underwent STI were retrospectively defined, according to the ability to spontaneously control HIV replication (Controller and Non-controller). Plasma cytokine levels were analyzed by multiplex analysis. CD8 T cell differentiation was determined by polychromatic flow cytometry. Antigen-specific IFN-Γ production was analyzed by ELISpot and intracellular staining after stimulation with HIV-peptides. Long-term Elispot assays were performed in the presence or absence of IL-15. Plasma IL-15 was found decreased over a period of time in Non-Controller patients, whereas a restricted response to Gag (aa.167–202 and 265–279) and Nef (aa.86–100 and 111–138) immunodominant epitopes was more frequently observed in Controller patients. Interestingly, in two Non-Controller patients the CD8-mediated T cells response to immunodominant epitopes could be restored in vitro by IL-15, suggesting a major role of cytokine homeostasis on the generation of protective immunity. In early-treated HIV+ patients undergoing STI, HIV replication control was associated to CD8 T cell maturation and sustained IL-15 levels, leading to HIV-specific CD8 T cell responses against selected Gag and Nef epitopes.

Primary structure of mitochondrial aspartate aminotransferase from turkey liver: Cysteine-containing peptides

The determination of the complete primary structures of both cytoplasmic [ 1,2] and mitochondrial [3,4] isoenzymes of aspartate aminotransferase (EC 2.6.1 .l) from pig heart coincides with a renewed interest in this system, as well documented by recent papers aimed at unraveling the three dimensional structures of these enzymes [5-71, their evolutionary history [89] and the structural basis and molecular mechanism of their intracellular compartmentation [ 10-121. In this context we have studied some structural features of aspartate aminotransferase isoenzymes purified from various sources. Here we report results obtained with the mitochondrial isoenzyme from the liver of turkey, an animal evolutionarily rather distant from pig. This is of interest since the homotopic isoenzyme from another avian source, chicken, is being successfully subjected to crystallographic studies [7]. In particular, we have investigated the reactivity of sulphydryl groups in the turkey liver enzyme and have carried out primary structure studies of the enzyme labeled with radioactive iodoacetate. The latter studies allowed determination of 75% of the sequence of the polypeptide chain. The results are compared with those obtained from the homotopic isoenzyme from pig heart.