Calcineurin activity is regulated both by redox compounds and by mutant familial amyotrophic lateral sclerosis-superoxide dismutase.

Calcineurin (CN) is a protein phosphatase involved in a wide range of cellular responses to calcium-mobilizing signals, and a role for this enzyme in neuropathology has been postulated. We have investigated the possibility that redox modulation of CN activity is relevant to neuropathological conditions where an imbalance in reactive oxygen species has been described. We have monitored CN activity in cultured human neuroblastoma SH-SY5Y cells and obtained evidence that CN activity is promoted by treatment with ascorbate or dithiothreitol and impaired by oxidative stress. Evidence for the existence of a redox regulation of this enzyme has been also obtained by overexpression of wild-type antioxidant Cu,Zn superoxide dismutase (SOD1) that promotes CN activity and protects it from oxidative inactivation. On the contrary, overexpression of mutant SOD1s associated with familial amyotrophic lateral sclerosis (FALS) impairs CN activity both in transfected human neuroblastoma cell lines and in the motor cortex of brain from FALS-transgenic mice. These data suggest that CN might be a target in the pathogenesis of SOD1-linked FALS.

Imbalance in corneal redox state during herpes simplex virus 1-induced keratitis in rabbits. Effectiveness of exogenous glutathione supply.

A significant decrease in the antioxidant glutathione (GSH) was found in the corneal tissue of rabbits with Herpes Simplex 1 (HSV-1)-induced keratitis. Such a decrease was due to a loss of the reduced species, since no increase in its oxidized form was observed. Topical administration of purified GSH was able to reduce the virus titre in corneal tissue and, at the same time, was effective in reducing the severity and progression of keratitis and conjunctivitis. This effect was paralleled by a partial recovery in the corneal GSH content. In vitro experiments performed on HSV-1 infected corneal-derived rabbit cells showed that exogenous GSH reduced virus titre in the supernatant of infected cells. These results are in agreement with our previous findings that an oxidative environment, due to GSH depletion, is necessary for virus replication and suggest that topical GSH treatment could be considered as complementary therapy in HSV-1-induced keratitis.

The conformation of peptide thymosin alpha 1 in solution and in a membrane-like environment by circular dichroism and NMR spectroscopy. A possible model for its interaction with the lymphocyte membrane.

The 28-residue peptide thymosin alpha1 was studied by circular dichroism and two-dimensional NMR. Circular dichroism indicates that thymosin alpha1 in water solution does not assume a preferred conformation, while in the presence of small unilamellar vesicles of dimiristoylphosphatidylcholine and dimiristoylphosphatidic acid (10:1) and in sodium dodecyl sulphate, it assumes a partly structured conformation. Presence of zinc ions produces similar effects. In a more hydrophobic environment like a solution of a mixed solvent water-2,2,2 trifluoroethanol, it adopts a structured conformation. NMR spectra indicated that in this mixture as solvent, thymosin alpha1 has a structure characterized by two regions. A beta-turn is present between residue 5 and residue 8, while the region between residues 17 and 24 shows an alpha helix conformation. These changes of conformation in different environments may be considered structural requirements in the steps of its interaction with the lymphocyte membrane. In fact, these conformational changes may correspond to the first event of the mechanism of lymphocyte activation in the immune response modulation by thymosin alpha1.

Decrease of immunoreactive catalase protein in specific areas of ageing rat brain.

The activity of catalase, the main enzyme responsible for detoxification against hydrogen peroxide, decreases in specific brain areas of aged rats. The reduction of enzyme activity appears to be the consequence of a decreased protein expression rather than an impaired function of the native enzyme. In fact, diminution of the immunoreactive protein parallels enzyme activity decrease. Since the extent of decrease of both activity and protein content was observed to be area dependent, we hypothesise that this phenomenon may underlie, at least in part, the increased susceptibility of specific brain regions to oxidative insults observed in pathological situations related to ageing.

Inhibition of copper amine oxidase by haloamines: a killer product mechanism.

The observation that the alkylamines 2-Br-ethylamine and 2-C1-ethylamine and 1,2-diaminoethane, the shortest diamine, are irreversible inhibitors of several copper amine oxidases led to the investigation of the mechanism by which these compounds react with the highly active amine oxidase from lentil seedlings. 1,2-Diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine were found to be both poor substrates and irreversible inhibitors of lentil amine oxidase; inactivation took place in both the presence and absence of oxygen. All three compounds strongly affected the spectrum of the enzyme, leading to the formation of a stable band at 336 nm both in anaerobiosis and in aerobiosis, consistent with an interaction with the enzyme cofactor 6-hydroxydopa. On the contrary, the corresponding propylamine compounds 1,3-diaminopropane, 3-Br-propylamine, and 3-C1-propylamine were reversible inhibitors of lentil amine oxidase. Inhibition was shown to be due to the aldehyde oxidation products rather than the short chain amines themselves; a reaction mechanism is presented which involves attack of the aldehyde on the 6-hydroxydopa-derived free radical catalytic intermediate. With 1,2-diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine, the complex produced will form a stable 6-membered ring, causing irreversible inhibition of the enzyme.

The reaction mechanism of copper amine oxidase: detection of intermediates by the use of substrates and inhibitors.

Intermediate states in the catalytic mechanism of lentil copper amine oxidase have been investigated by ESR and optical spectroscopy. Using highly purified apo- and holoenzyme in combination with a poor substrate and a range of inhibitors, under both aerobic and anaerobic conditions, the single steps of the reaction mechanism can be slowed down or 'frozen' completely. In this way, a sequence of six intermediate species in the catalytic cycle has been established. Oxidative deamination of p-(dimethylamino)benzylamine is 5 x 10(5) times slower than for putrescine; the rate-limiting step is shown to be release of the aldehyde product. This process is not affected in the apoenzyme, but subsequent intramolecular electron transfer to form the characteristic free radical intermediate is completely blocked, and the apoenzyme is trapped as an aminoresorcinol species. Classic hydrazine and hydrazide inhibitors bind to the 6-hydroxydopa cofactor in the same way as active substrates, but rearrangements lead to formation of stable intermediate adducts at the step preceding release of aldehyde. The semicarbazide-6-hydroxydopa adduct is shown to bind simultaneously to Cu(II), providing the first direct evidence for localization of 6-hydroxydopa close to the copper site.

Metal uptake of recombinant cambialistic superoxide dismutase from Propionibacterium shermanii is affected by growth conditions of host Escherichia coli cells.

We constructed the complete nucleotide sequence coding for the cambialistic superoxide dismutase from Propionibacterium shermanii by ligation of a synthetic linker to a polymerase chain reaction amplification product obtained using degenerate primers. We set up an expression system yielding large amounts of recombinant superoxide dismutase in the cytoplasm of Escherichia coli and purified the enzyme from cells grown in a complex medium. The physicochemical properties of the recombinant enzyme were identical to those of the natural protein. Under anaerobic conditions the enzyme produced in an iron-supplemented medium incorporated iron as metal cofactor, while the enzyme purified from cells grown under aerobic conditions contained a variable amount of iron and manganese depending on metal availability. Functional equivalence of the two metals in this superoxide dismutase variant was indicated by independence of enzyme activity from Fe/Mn ratio.

Copper-dependent metabolism of Cu,Zn-superoxide dismutase in human K562 cells. Lack of specific transcriptional activation and accumulation of a partially inactivated enzyme.

The regulation of Cu,Zn-superoxide dismutase by copper was investigated in human K562 cells. Copper ions caused a dose- and time-dependent increase, up to 3-fold, of the steady-state level of Cu,Zu-superoxide dismutase mRNA. A comparable increase was also observed for actin and ribosomal protein L32 mRNAs, but not for metallothionein mRNA which was augmented more than 50-fold and showed a different induction pattern. The copper-induced mRNAs were actively translated as judged from their enhanced loading on polysomes, the concomitantly increased cellular protein levels and an augmented incorporation of [3H]lysine into acid-precipitable material. Cu,Zn-superoxide dismutase protein followed this general trend, as demonstrated by dose- and time-dependent increases in immunoreactive and enzymically active protein. However, a specific accumulation of Cu,Zn-superoxide dismutase was noticed in cells grown in the presence of copper, that was not detectable for other proteins. Purification of the enzyme demonstrated that Cu,Zn-superoxide dismutase was present as a reconstitutable, copper-deficient protein with high specific activity (kcat./Cu = 0.89 x 10(9) M-1.s-1) in untreated K562 cells and as a fully metallated protein with low specific activity (kcat./Cu = 0.54 x 10(9) M-1.s-1) in copper-treated cells. Pulse-chase experiments using [3H]lysine indicated that turnover rates of Cu,Zn-superoxide dismutase in K562 cells were not affected by growth in copper-enriched medium, whereas turnover of total protein was significantly enhanced as a function of metal supplementation. From these results we conclude that: (i) unlike in yeast [Carrì, Galiazzo, Ciriolo and Rotilio (1991) FEBS Lett. 278, 263-266] Cu,Zn-superoxide dismutase is not specifically regulated by copper at the transcriptional level in human K562 cells, suggesting that this type of regulation has not been conserved during the evolution of higher eukaryotes; (ii) copper ions cause an inactivation of the enzyme in intact K562 cells; and (iii) the metabolic stability of Cu,Zn-superoxide dismutase results in its relative accumulation under conditions that lead to increased protein turnover.

Differential decrease of copper content and of copper binding to superoxide dismutase in liver, heart and brain of copper-deficient rats.

Dietary copper-deficiency in rats produced a organ-specific decrease of copper content. This was paralleled by a decrease of the activity of the copper-enzyme superoxide dismutase. In liver such a decrease is partially due to the existence of an apo-form of superoxide dismutase, which can be reactivated by addition of exogenous copper to tissue extracts. These results demonstrate in vivo that superoxide dismutase is post-translationally modulated by copper in higher vertebrates as previously found for yeast and mammalian cells in culture.

In vivo incorporation of cobalt into Propionibacterium shermanii superoxide dismutase.

Propionibacterium shermanii, an aerotolerant anaerobic bacterium, has already been shown to incorporate, depending on the metal supplementation to the medium, either iron or manganese or copper into the same superoxide dismutase protein. The in vivo incorporation of cobalt in the same superoxide dismutase was obtained in an iron-, manganese- and copper-depleted medium. The protein was isolated and characterized by NMR which offers the possibility to identify the amino acid residues at the active site exploiting isotropically shifted proton resonance.

A substrate-cofactor free radical intermediate in the reaction mechanism of copper amine oxidase.

Reduction of copper amine oxidase with substrate led to the appearance of a free radical which can be detected in anaerobiosis by ESR and optical spectroscopy. The origin of this radical was examined through studies of the semiquinones of 6-hydroxydopamine, an analogue of the recently identified cofactor 6-hydroxydopa. The ESR spectrum of the 6-hydroxydopamine radical was too narrow to account for the enzyme radical signal; however, after spontaneous reaction with primary amines the hyperfine splittings and spectral width obtained by modulation broadening became very similar to those observed for the oxidase radical species. This effect was ascribed to covalent binding of a nitrogen atom directly to the aromatic ring structure, suggesting that the amine oxidase radical is an amino-6-hydroxydopa semiquinone. Identical ESR spectra were obtained using the amines putrescine, cadaverine, p-[(dimethylamino)methyl]benzylamine, and ethylenediamine; these oxidase substrates gave identical enzyme radical spectra as well. The interaction between cofactor and substrate was proved unambiguously by the technique of isotopic labeling: addition of [15N2]ethylenediamine instead of the normal 14N-labeled compound changed the ESR spectra of both the enzyme radical and its 6-hydroxydopamine counterpart. The results were confirmed by optical spectroscopy measurements; 6-hydroxydopamine and oxidized 6-hydroxydopamine gave spectra identical to those of reduced and oxidized amine oxidase, respectively. The 6-hydroxydopamine radical showed a sharp peak at 440 nm; upon addition of amines the maximum shifted to 460 nm, as found for the enzyme. It is proposed that copper amine oxidase represents the first example of a mixed substrate-cofactor radical within the family of tyrosine radical enzymes.

NGF restores decrease in catalase activity and increases superoxide dismutase and glutathione peroxidase activity in the brain of aged rats.

The effects of ageing on the activity of copper-zinc superoxide dismutase (SOD), selenium-dependent and independent glutathione peroxidase (GSH-Px) and catalase in several areas of the brain in 3-, 12-, and 24-month-old rats were studied. In addition, the effects of a subacute intracerebroventricular treatment of NGF (1 microgram daily for 28 consecutive days) on SOD, GSH-Px, and catalase activity in the same areas of the brain were assessed. The effects of ageing on the activities of antioxidant enzymes varied considerably in the different brain areas studied. Copper-zinc SOD was alone in being unaffected by ageing. Intraventricular infusion of NGF significantly increased SOD activity in the prefrontal cortex, hypothalamus, caudate nucleus, and mesencephalon of 24-month-old rats. Selenium-dependent GSH-Px activity did not significantly change in 12-month-old rats but it increased in the lower brain stem of 24-month-old animals. In comparison to vehicle-treated rats, NGF significantly increased selenium-dependent GSH-Px activity in all brain areas studied in 12- and 24-month-old rats. Catalase activity decreased significantly in the majority of the brain areas studied in 12- and 24-month-old rats. NGF completely restored the fall in catalase activity in 12- and 24-month-old animals to levels similar to those occurring in young rats. In conclusion, the present experiments show, for the first time, that long-term intraventricular administration of NGF significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of superoxide radicals and hydrogen peroxide.

Age-related changes in Cu,Zn superoxide dismutase, Se-dependent and -independent glutathione peroxidase and catalase activities in specific areas of rat brain.

Oxidative injury of tissues involves both accumulation of damage due to persistent oxidative stress and loss of the proper balance of antioxidative enzymes. These events may produce a faster rate of tissue senescence. In this regard, we have assayed the antioxidative enzyme activities (Cu,Zn superoxide dismutase, glutathione peroxidase and catalase), in various areas of rat brain (prefrontal cortex, parietal cortex, hippocampus, hypothalamus, caudate nucleus, mesencephalon and lower brain stem) for the age groups of 3, 6, 12, 24 months. The results obtained show that the levels of antioxidant enzyme activities differed considerably in the various brain parts studied. Furthermore, changes in the specific activities of superoxide dismutase, catalase, and glutathione peroxidase did not follow the same pattern as a function of aging. In particular, in prefrontal cortex and caudate nucleus, superoxide dismutase and glutathione peroxidase activities did not change, while catalase activity decreased. In parietal cortex and mesencephalon, superoxide dismutase and glutathione peroxidase activities increased, but the catalase activity decreased in parietal cortex and did not change in mesencephalon. In lower brain stem, the activities of glutathione peroxidase and catalase decreased in 3-12-month-old rats. The activity of glutathione peroxidase was increased in the hippocampus and was decreased in hypothalamus during aging. In this area the catalase activity was also significantly diminished.

NGF restores decrease in catalase and increases glutathione peroxidase activity in the brain of aged rats.

The effects of subchronic administration of nerve growth factor (NGF) into the lateral ventricle on catalase and selenium-dependent glutathione-peroxidase (GSH-Px) activity in several areas of the brain in 3-, 12- and 24-month-old rats were studied. NGF given daily (1 microgram for 28 consecutive days) produced in all brain areas studied a significant increase in catalase activity in 12- and 24-month-old rats. The most important finding was a complete restoration in 12- and 24-month-old rats of catalase activity to levels similar to those occurring in young (3-month-old) rats. In addition, NGF produced in comparison to 3-month-old rats and to same age vehicle-treated rats a significant increase in selenium-dependent GSH-Px in all the brain areas studied in 12- and 24-month-old animals, whereas selenium-independent GSH-Px was unaffected. In conclusion, the present results show that long-term administration of NGF into the lateral ventricle significantly increases in old animals the activity of key enzymes involved in the metabolic degradation of hydrogen peroxide.

1H and 31P NMR studies of the binding of low-affinity anions to Cu,Zn superoxide dismutase.

Anions that do not coordinate to the catalytically active copper ion of Cu,Zn superoxide dismutase, but still affect the activity of the enzyme by weaker interactions with the protein moiety surrounding the active site (low affinity anions), uniformly perturbed the 1H NMR line of the NH group of the copper ligand His 46. This effect was detected on the enzyme having Co(II) substituted for the native Zn(II), in which the resonances of residues bound to the copper are detected because of the antiferromagnetic coupling between Cu(II) and Co(II). The interaction with the enzyme of phosphate, a good representative of low-affinity anions, was also studied by 31P NMR of the native enzyme and of enzyme samples covalently modified at all lysines or at the Arg 141, which is 5 A away from the copper. The results obtained indicate that Arg 141 is a likely candidate for binding of low-affinity anions in the vicinity of the copper and that the 1H NMR line of His 46 NH is diagnostic for such an interaction.

Oxygen-independent induction of enzyme activities related to oxygen metabolism in yeast by copper.

Aerobic growth of Saccharomyces cerevisiae in the presence of CuSO4 (between 0.1 and 1 mM) caused a generalized induction of major enzyme activities involved in 'housekeeping' routes of oxygen metabolism (cytochrome oxidase, glutathione peroxidases and catalase) which were comparable to or higher than that observed with Cu,Zn-superoxide dismutase. Fumarase and glutathione transferase, tested as controls for oxygen-unrelated activities, were found to decrease under the same conditions. In the absence of oxygen, copper addition to yeast resulted in significant increases of Cu,Zn-superoxide dismutase and glutathione peroxidases and a slight increase of cytochrome oxidase, with catalase remaining undetectable irrespective of whether or not copper was present. Other metal ions tested (Mn2+, Co2+) were unable to produce such effects. It is concluded that copper has a general inducing effect on enzymes related to metabolism of oxygen and oxygen derivatives, which is mediated neither by formation of O2-. and H2O2 nor by interaction with copper-specific apoproteins. These results point to a general role of copper as regulator of the expression of major enzyme activities involved in biological oxygen activation.

Reaction of beef plasma and lentil seedlings Cu-amine oxidases with phenylhydrazine.

The reaction of copper amine oxidases from beef plasma and lentil seedlings with hydrazine derivatives has been studied. A 1:1 stoichiometry was always found for the irreversible binding to the dimeric proteins. The formation of the adduct does not require the presence of oxygen or copper. Substrates compete with hydrazine derivatives for the binding to the enzymes. The binding of hydrazines and of substrate has different effects on the EPR spectra of enzymic copper.