Maria Franca Zuretti

Studies on the mechanisms of ornithine decarboxylase in vitro inactivation.

Hydrocortisone-induced rat liver ornithine decarboxylase appears quite stable in the soluble fraction of the homogenate incubated at 37 degrees C. In contrast, the incubation of the whole homogenate causes a rapid loss of activity. The ornithine decarboxylase-inactivating capacity appears mainly bound to microsomes. Lysosomes seem to play a role only after the microsome-induced inactivation. Different reducing agents (dithiothreitol, NADPH, NADH, GSH) are effective both in preventing and in reversing ornithine decarboxylase inactivation. NADPH is peculiar in that it can reactivate the enzyme at very low concentrations. Oxidized glutathione potentiates the inactivating effect of microsomes. On the basis of present results it is suggested that ornithine decarboxylase may be reversibly inactivated through microsome-catalyzed formation of mixed or enzyme-enzyme disulfides and that NADPH plays a crucial role in ornithine decarboxylase reactivation, probably by cytosolic reductase(s).

Studies on the hepatotoxicity of Amanita phalloides in the rat: II. Biochemical analysis of the lysosomal changes☆

Abstract Marked changes in the integrity and fragility of liver lysosomes have been detected in rats poisoned with a lethal dose of a crude aqueous extract of Amanita phalloides . In the course of the poisoning, four lysosomal hydrolases (acid phosphatase, β- N -acetylglucosaminidase, β-galactosidase and β-glucuronidase) were found to undergo a progressive reduction in both their structure-linked latency and sedimentability. Concurrently, there occurred in the blood plasma a conspicuous increase of acid hydrolase activities, which distinctly preceded the subsequent increase of both aspartate aminotransferase and isocitrate dehydrogenase. Observations on the fractional free activity of the acid hydrolases at different substrate concentrations seem to rule out the possibility that a graded change in the permeability of the lysosomal membrane underlies the activation of lysosomal enzymes. Rather, activation seems to be an all-or-none kind of phenomenon. When submitted to various labilizing procedures in vitro , the liver lysosomal population from poisoned rats exhibited a modified fragility in comparison with control lysosomes. Factors such as the increased size of hydrolase-bearing particles, changes in the properties of the limiting membrane, or modifications in the intra as well as extralysosomal environment are likely to account for these results. The reliability of the observed lysosomal changes as indexes of the modifications that take place in vivo and the relevance of the latter to the production of the liver injury by Amanita phalloides are discussed.

Studies on the hepatotoxicity of Amanita phalloides in the rat: I. Liver cell vacuolation

Abstract Rats poisoned with a lethal dose of a crude Amanita phalloides extract develop a severe liver derangement culminating in haemorrhagic liver necrosis. Light, fluorescence, and electron microscopy as well as enzyme histochemical techniques have been employed to monitor the development of the liver injury, particular attention being paid to lysosomes. The biochemical correlates of the lysosomal changes have been examined in the accompanying paper ( Zuretti and Baccino, 1976 ). The first changes were detected at the level of the hepatocytic plasmalemma, both histochemically (ATPase and 5′-nucleotidase) and ultrastructurally. At the sinusoidal surface, the early development of multiple invaginations, giving rise to cytoplasmic vacuoles, is later followed by the complete disappearance of microvilli. Moreover, microfilaments formed prominent clusters juxtaposed to the inner surface of the plasma membrane at both the sinusoidal and biliary poles. Cytoplasmic vacuoles developed in hepatocytes early after poisoning due to the progressive overloading of cells with blood-derived material: plasma proteins at first, as shown by the stainability of vacuoles with fluoresceinated anti-rat serum antibodies or by the ultrastructural observation of fibrin strands segregated within, then also striking amount of blood cells. Both constituents finally flooded hepatocytes. Not only heterophagic, but also autophagic processes contributed material (cytoplasmic organelles, glycogen) to be segregated into the vacuoles, which thereby acquired an ambiphagosomal character. Autophagosomes of the usual type were increased too. A large proportion of vacuoles, except for the largest ones in the late stages of the poisoning, acquired acid phosphatase activity histochemically detectable; this presumably derived from pre-existing lysosomes, which strongly decreased in number. Many vacuoles also contained diastase-resistant, PAS-positive material.

Biochemical and structural changes of rat liver lysosomes by ethionine

Abstract 1. (1) The administration of ethionine to rats (1 g/kg body wt) was shown to induce marked yet reversible changes in liver lysosomes, detectable both biochemically and histochemically. 2. (2) In liver homogenates the free activity of the lysosomal marker acid phosphatase, still unchanged at three hours, was consistently increased at six hours, and by 13 hours returned to normal levels. The free activity of β- N -acetylglucosaminidase, another lysosomal hydrolase, was similarly increased at six hours. 3. (3) Six hours after ethionine, the proportion of acid phosphatase activity recovered in the soluble phase of the homogenate was in most cases significantly increased. The levels of soluble activity, however, were distincly lower than those for free activity. 4. (4) The ratio of free to total activity for acid phosphatase and β- N -acetyl-glucosaminidase is not modified by varying the substrate concentration in the medium; thereby an acquired permeability of the lysosomal membrane to the substrates is likely ruled out as a mechanism for the increased value of free activity. 5. (5) The fragility of liver lysosomes from rats six hours after ethionine was investigated by exposing the whole homogenate to mechanical or hypoosmotic shock as well as a lysosome-rich fraction (M + L) to hypoosmotic shock or preincubation at pH 5, 37° C. In all cases both hydrolases were shown to be activated to a higher degree than control preparations. Moreover, an enhanced osmotic fragility of lysosomes in liver homogenates was also observed at three and 13 hours after ethionine. These findings suggest the presence in the liver of ethionine-treated animals of an increased proportion of particles particularly fragile. 6. (6) Possible mechanism(s) by which ethionine administration causes the enhanced lysosomal fragility are briefly considered. The relationship between the augmented fragility of lysosomes and the increased value of free and soluble activities of their hydrolases is discussed.

RESIDUAL LATENT ACTIVITY OF ACID-PHOSPHATASE - AUTOPHAGY-RELATED VARIATIONS AND EFFECTS OF CYCLOHEXIMIDE

Abstract The patterns of activation of two lysosomal hydrolases, acid phosphatase and N -acetyl β- d -glucosaminidase, by hypotonic shock have been measured in heavy and light mitochondrial, and in microsomal fractions from rat liver. The osmotic sensitivity of the particles, as inferred from enzyme activation, was highest in the heavy mitochondrial and lowest in the microsomal fraction. An aliquot of activity (referred to as residual latent activity, RLA) remained latent even after incubation in distilled water for 30 min at 0°C. The proportion of RLA has been shown to vary in relation to changes in the dynamic state of the vacuolar apparatus. A significant decrease of RLA occurred in rats fasted for 24 hr as well as after glucagon administration, two situations known to be correlated with increased autophagy. A significant increase of RLA has been found in regenerating liver, where autophagy is reduced. The effect of glucagon was prevented by previous injection of cycloheximide, which has been reported to inhibit the stimulation of autophagy by the hormone. In addition we have found that cycloheximide alone causes a very conspicuous increase of RLA, already noticeable 30 min after the treatment.

Studies on ornithine decarboxylase from liver, kidney and tumoral tissues during activity decay following cycloheximide administration

We have studied the activity, thiol-dependency and Km of ornithine decarboxylase (ODC) from the following sources: liver of rats subjected to partial hepatectomy or administered thioacetamide, the rat 3924A Morris hepatoma, the rat AH130 Yoshida ascites hepatoma, a mouse transplantable mammary carcinoma and kidney of rats administered testosterone propionate. In order to detect possible changes occurring during in vivo ageing of this enzyme we inhibited protein synthesis with cycloheximide. A gradual decrease of Km during ageing was observed in ODC from liver.

Acid phosphatase residual latent activity versus lysosome osmotic fragility in rat liver autophagy

Abstract The fraction of acid phosphatase which remains latent after extensive hypoosmotic shock and requires the action of Triton X-100 to be unmasked, has been defined as residual latent activity (RLA); its level was tentatively related to the activity of the autophagic processes ( M. F. Zuretti, J. W. M. Bouma, M. Messina, G. Barrera, and F. M. Baccino, 1980 , Exp. Mol. Pathol.33, 316–322). Whether the modifications of the RLA under different experimental conditions correlate with changes in the osmostic fragility of lysosomes was investigated. The latter is an index for changes in the physical properties of lysosomes, mainly size, which are known to vary depending on the rate of the autophagic process. In the course of glucagon-induced autophagy, a decrease in RLA and an increase in lysosome osmotic fragility developed concurrently. Not differently from its effect on RLA, cycloheximide prevented the osmotic fragility changes induced by glucagon when given before the hormone, or caused their reversal when given after it. The prompt increase in RLA caused by cycloheximide in normally fed animals is not always accompanied by a discernible reduction of the lysosome osmotic fragility.

Ornithine decarboxylase lability in 2 transplantable highly deviated rat hepatomas

A strong ornithine decarboxylase (ODC)-inactivating capacity has been previously shown (M.F. Zuretti and E. Gravela (1983) Biochim. Biophys. Acta, 742, 269-277) to be bound to rat liver microsomes. Present results show that in 2 fast-growing transplantable tumors, the 3924A Morris hepatoma and the AH 130 Yoshida ascites hepatoma, microsomes are endowed with a greatly enhanced ODC-inactivating capacity, and, concurrently, ODC displays an extreme in vitro liability and an unusual thiol-dependency (most of the activity requires dithiothreitol supply to be determined). These data are at variance with those previously obtained in hepatomas induced by N-2-fluorenylacetamide (E. Gravela et al., (1983) Cancer Res., 42, 2298-2300). The possibility that ODC liability in the 2 hepatomas here studied may result from in vivo exposure to a strong microsomal activity is considered.

Different Factors Possibly Involved in Post-translational Regulation of Ornithine Decarboxylase Activity

Present results concern a microsome-bound enzymatic system which has been recognized as responsible for the rapid inactivation in vitro of ornithine decarboxylase (ODC). Two different models have been investigated: a) rat liver after a single thioacetamide administration, and b) the 3924 A Morris hepatoma. In both these models we observed variations in the microsome-bound ODC-inactivating capacity. In parallel, changes in ODC properties were observed. The possibility of a causal relationship between the two events is discussed. The actual role of the microsome-bound ODC-inactivating system, in ODC activity regulation in vivo cannot be established, but it remains as a fairly plausible working hypothesis.