Bruno Mondovi

Catalase Takes Part in Rat Liver Mitochondria Oxidative Stress Defense

Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalatic mechanism with a rate constant of 0.0346 s-1. The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 ± 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.

Enzyme defense against reactive oxygen derivatives. II. Erythrocytes and tumor cells

SummaryThe enzymatic destruction of oxidizing products produced during metabolic reduction of oxygen in the cell (such as singlet oxygen, H2O2 and OH radical) involves the concerted action of superoxide dismutase-which removes O2- and yields H2O2-and H2O2 removing enzymes such as catalase and glutathione peroxidase. A difference in distribution or ratio of these enzymes in various tissues may result in a different reactivity of oxygen radicals.It was found that in red blood cells superoxide dismutase and catalase are extracted in the same fraction as hemoglobin, while glutathione peroxidase appears to be “loosely” bound to the cellular structure. This suggests that in red blood cells catalase acts in series with superoxide dismutase against bursts of oxygen radicals formed from oxyhemoglobin, while glutathione & peroxidase may protect the cell membrane against low concentrations of H2O2. On the other hand, catalase activity is absent in various types of ascites tumor cells, while glutathione peroxidase and superoxide dismutase are found in the cytoplasm. However, the peroxidase/dismutase ratio is lower than in liver cells, and this may provide an explanation for the higher susceptibility of tumor cells to treatments likely to involve oxygen radicals.

Increased immunogenicity of ehrlich ascites cells after heat treatment

Immunization of Swiss mice with Ehrlich ascites cells which had been exposed in vitro to 42.C for 3 hours is more effective, so far as subsequent tumor transplantation is concerned, than immunization with radiation‐inactivated cells or with cells exposed to 42.5C for 6 hours. These results, which indicate that upon moderate heat treatment there is a definite increase of the immunogencity of these cells, may have some relevance as to the mechanism of the delayed tumor regression which is often clinically observed after hyperthermic perfusion.

Selective removal of type 2 copper from Rhus vernicifera laccase

Rhus vernicifera lactase contains, like fungal lactase, one Type 1, one Type 2 and two Type 3 copper ions [I] , which can all be removed by cyanide at pH 8.0 in a reversible process [2]. In the case of fungal lactase a method is described [3] which allows reversible removal of only the Type 2 copper. In view of the many analogies presented by these two proteins we have investigated the possibility of obtaining, also in the case of Rhus vemicifera lactase, selective removal of Type 2 copper. A method for the preparation of a protein depleted of Type 2 copper is reported in this communication.

Interaction of Biologically Active Amines with Mitochondria and Their Role in the Mitochondrial-Mediated Pathway of Apoptosis

The natural polyamines spermine, spermidine and putrescine, polycationic molecules at physiological pH, interact with mitochondrial membranes at two specific binding sites exhibiting low affinity and high binding capacity. This binding represents the first step in the electrophoretic mechanism of polyamine transport into mitochondria. Spermine accumulated into the mitochondrial matrix is able to flow out by an electroneutral mechanism. This process promotes bi-directional transport of polyamines in and out of mitochondria, driven by electrical potential and pH gradient, respectively. Polyamines and biogenic amines are oxidized by cytosolic and mitochondrial amine oxidases with the production of hydrogen peroxide and aldehydes, both of which are involved in the induction and/or amplification of the mitochondrial permeability transition (MPT). This phenomenon, which provokes a bioenergetic collapse and redox catastrophe, is strongly inhibited by polyamines in isolated mitochondria. Monoamines also exhibit an inhibitory effect at higher concentrations, but at low concentrations behave as inducer agents. MPT is characterized by the opening of a channel, the transition pore, which permits non-specific bi-directional traffic of solutes across the inner membrane, leading to swelling of the organelle and release of cytochrome c and apoptosis-inducing factors. These proteins in turn activate the caspase-cascade, which triggers the apoptotic pathway. Depending on their cytosolic concentration, metabolic conditions and cell type, polyamines act as promoting, modulating or protective agents in mitochondrial-mediated apoptosis. While their protective effect could reflect inhibition of MPT and retention of cytochrome c, the promoting effect can be explained by the generation of reactive oxygen species that induce the opposite effect on MPT and cytochrome c release. Polyamines and other active amines can also participate in the regulation of apoptotic pathways by interacting with the mitochondrial tyrosine phosphorylation/dephosphorylation system. Future studies of the multifaceted interactions of polyamines with mitochondria will thus have a substantial impact on our understanding of the physiology of cell proliferation death at several mechanistic levels.

Erythrocuprein and singlet oxygen

McCord and Fridovich [ 1,2] have shown that erythrocuprein, the copper and zinc containing protein extracted from red blood cells, can enzymatically disproportionate superoxide radicals (Oh), an intermediate of the reduction of oxygen, the generation of which in some Ha 0s -producing biological oxidations has been demonstrated [3]. Khan has recently pointed out [4] that dismutation of Og gives rise to the production of the highly reactive singlet state of oxygen, which can be revealed by its own luminescence; indeed Arneson [5] observed that oxidation of xan’thine by xanthine oxidase produced luminescence, which was abolished by the presence of erythrocuprein. On the basis of these data, the main purpose of the following paper is to show that erythrocuprein has a quenching effect on luminescence phenomena which are reasonably ascribed to singlet oxygen produced even in the absence of superoxide intermediates. Thus the physiological role of erythrocuprein could be to catalyze the singlet-triplet conversion of the oxygen states: in other words to afford an enzyme-regulated dismutation liberating in solution as a product the inert ground state triplet oxygen, rather than simply accelerating the dismutation reaction.

Protection against apoptosis by monoamine oxidase A inhibitors

Several lines of evidence have been accumulating indicating that an important role may be played by mitochondrial homeostasis in the initiation phase, the first stage of apoptosis. This work describes the results obtained by using different inhibitors of monoamine oxidases (MAO), i.e. pargyline, clorgyline and deprenyl, on mitochondrial integrity and apoptosis. Both pargyline and clorgyline are capable of protecting cells from apoptosis induced by serum starvation while deprenyl is ineffective. These data represent the first demonstration that MAO‐A inhibitors may protect cells from apoptosis through a mechanism involving the maintenance of mitochondrial homeostasis.

Purification of bovine plasma amine oxidase

Abstract A new method for the purification of bovine plasma amine oxidase is described. The enzyme is purified by ammonium sulfate precipitation and by affinity chromatography performed with AH-Sepharose 4B and concanavalin A-Sepharose. Three activity peaks were separated, all showing similar properties. Specific activity is the highest described for this enzyme. The enzyme appears to contain 2 copper atoms and 1 carbonyl group/molecule.

Structure and Function of Amine Oxidases

Amine oxidases are enzymes widely distributed among living organisms. They catalyze the oxidative deamination of many biologically important amines with the formation of the corresponding aldehyde, hydrogen peroxide and ammonia according to the equation RCH2NH2 + O2 + H2O → RCHO + H2O2 + NH3

The biochemical mechanism of selective heat sensitivity of cancer cells—IV. Inhibition of RNA synthesis

Abstract The mechanism by which exposure of ascites tumour cells to supranormal temperatures causes an irreversible inhibition of uridine incorporation into RNA was investigated. Heat-treated cells were still able to incorporate labeled nucleotides, and even nucleosides, into RNA, if these precursors were added at sufficiently high concentrations. The passive permeability of the cell membrane increased exponentially with temperature, but this increase was fully reversible. At variance with the results obtained with agents which increase cell permeability, or which inhibit nucleoside permeation, heat treatment of Ehrlich ascites cells did not modify the ability of labeled uridine to be metabolized by these cells. The inhibition of uridine incorporation following heat treatment cannot therefore be attributed to a damage at the cell membrane level; the experimental data can be tentatively accounted for by the hypothesis of a block in the maturation of pre-rRNA into rRNA, with reutilization of the pre-rRNA degradation products.