[Show abstract][Hide abstract] ABSTRACT: beta H-crystallin was exposed to radiolytically generated hydroxyl radicals at defined radical concentrations, and its capacity to act as an amine-acceptor substrate and as an amine-donor substrate for transglutaminase were investigated. [14C]Methylamine was used as a probe for labelling amine-acceptor sites; a novel biotinylated hexapeptide was used to label amine-donor sites. The results demonstrate that both primary amine incorporation and hexapeptide incorporation by transglutaminase are considerably increased after oxidative attack on the crystallin. The identity of the labelled subunits was established, and it is shown that, in both cases, this increased incorporation is not due to the production of new substrates, but that the existing incorporation sites become more susceptible. Moreover, using the newly developed probe, we could identify, for the first time, the major crystallin subunits active as amine-donor substrates (both before and after treatment) to be beta B1-, beta A3- and beta A4-crystallin. These data support the proposal that oxidative stress and transglutaminase activity may be jointly involved in the changes found in lens crystallins with age and in the development of cataract.
[Show abstract][Hide abstract] ABSTRACT: 1. The effects of free radicals on the capacity of beta L-crystallin to act as a substrate for the enzyme transglutaminase were investigated. 2. beta L-Crystallin was exposed to defined radical species that were generated radiolytically, and transglutaminase activity, using the modified protein as substrate, was subsequently measured by monitoring the incorporation of [14C]putrescine. 3. Exposure of beta L-crystallin to hydroxyl radicals, thymine peroxyl radicals and acetone peroxyl radicals at concentrations of up to 135 microM increased the capacity of the protein to incorporate putrescine. With higher concentrations of these radicals this capacity of beta L-crystallin to act as a transglutaminase substrate declined to control levels or lower. 4. Superoxide radicals were inactive in this regard; hydroperoxyl radicals were active only at high concentrations. 5. It has previously been suggested that changes in the crystallins that occur during aging and with cataract may be due to oxidative reactions and to transglutaminase activity. This study suggests that these phenomena may be considered together rather than separately.
[Show abstract][Hide abstract] ABSTRACT: 4-hydroxynonenal (HNE) is one of the major breakdown products of cellular lipid peroxidation. Its effects on proliferation, ornithine decarboxylase (ODC) activity and DNA synthesis have been investigated in leukemic cell lines. The cells were incubated for 1 hour with different aldehyde concentrations, then washed and resuspended in medium with fresh foetal calf serum. HNE concentrations ranging from 10(-5) to 10(-6) M significantly inhibited ODC activity when induced by addition of fresh foetal calf serum both in K562 and HL-60 cells. 3H-Thymidine incorporation in K562 cells was also inhibited from 6 to 12 hours after the treatment. The same HNE concentrations did not inhibit ODC activity when added to cytosol, thus a direct action on the enzyme can be excluded. Moreover, HNE did not affect the half-life of ODC, so that a specific effect on ODC synthesis may be supposed. These data indicate a reduction of proliferative capacity of the cells and are consistent with the possibility that HNE, at concentrations close to those found in normal cells, plays a role in the control of cell proliferation.
Free radical research communications 02/1991; 14(2):81-9. DOI:10.3109/10715769109094120
[Show abstract][Hide abstract] ABSTRACT: Calvatic acid (p-carboxyphenylazoxycyanide) is an antibiotic containing an azoxycyano group that displays carcinostatic activity. In the present work it has been shown that in AH-130 hepatoma and K562 leukemia cells the antibiotic, at low concentration, decreases ornithine decarboxylase (ODC) levels. The change depends on two summative effects of the drug, impairment of overall protein synthesis and inhibition of enzyme activity. Some analogs of calvatic acid have been tested in order to gain more insight into the structure-activity relationship. The decarboxylated derivative phenylazoxycyanide proved to be more effective in reducing protein synthesis and ODC activity in the whole tumor cells. The rapidly growing K562 cells displayed high sensitivity to this compound. Calvatic acid analogs devoid of the cyano group were less effective on the same parameters.
Research communications in chemical pathology and pharmacology 12/1990; 70(2):143-53.
[Show abstract][Hide abstract] ABSTRACT: In order to test whether a mild oxidative stress could promote the transglutaminase damaging effect on eye lens proteins, total lens soluble proteins and purified beta L-crystallin have been exposed to H2O2 slowly produced by the glucose-glucose oxidase reaction. Soon after the pretreatment, the substrate capacity of the lens proteins for an exogenous transglutaminase has been evaluated. Exposure to the oxidative stress increased the susceptibility of the lens proteins to transglutaminase. When ferrous ions were added to the preincubation medium, in order to convert the H2O2 into the hydroxyl radical, the increase was more evident.
Free radical research communications 02/1990; 11(4-5):223-9. DOI:10.3109/10715769009088919
[Show abstract][Hide abstract] ABSTRACT: Liver microsomes have a strong ornithine decarboxylase (ODC) inactivating capacity in vitro. The present results suggest that this may be involved in regulation of ODC activity in vivo: (1) the ODC inactivating capacity of microsomes appears susceptible to in vivo modulation: a single administration of thioacetamide, which induces ODC. also causes a significant increase in the inactivating capacity of the microsomes; (2) under conditions leading to increased microsome-bound ODC-inactivating capacity (e.g. liver from thioacetamide-treated rates versus regenerating liver) ODC displays a greater thermal lability and inactivability in vitro.
A possible involvement of this microsomal activity in an autoregulatory pathway of ODC is suggested by the fact that it is induced by the administration of polyamines. However, inhibition of ODC activity by α-difluoromethylornithine does not prevent the increase of the microsomal activity caused by thioacetamide. Thus, polyamine biosynthesis does not appear to be an absolute requirement for induction of the microsomal ODC-inactivating capacity.
The apparent half-life of ODC in vivo, as evaluated after cycloheximide administration, does not appear to correlate with the microsomal ODC-inactivating capacity content and the stability properties of ODC in vitro.
Cell Biochemistry and Function 04/1988; 6(2):107-14. DOI:10.1002/cbf.290060205 · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Cell Biology International Reports 09/1987; 11(8):573-82. DOI:10.1016/0309-1651(87)90137-8
[Show abstract][Hide abstract] ABSTRACT: 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.
Cancer Letters 08/1987; 36(1):35-43. DOI:10.1016/0304-3835(87)90100-5 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Phosphonic (4a,b) and phosphinic (5a-d) analogues of ornithine were synthesized and evaluated for their inhibitory activity against ornithine decarboxylase and against the lymphocytic leukemia P388. The title compounds possess a low degree of inhibition against rat liver ornithine decarboxylase as compared to alpha-(difluoromethyl)ornithine. Thus, compounds 4a and 5a inhibit by 40% the ornithine decarboxylase activity at a 5 mM concentration. The other derivatives are less potent. Compounds 4a, 4b, 5b, and 5d are inactive against P388 tumor in CD2F1 mice at doses of 50 and 150 mg/kg.
Journal of Medicinal Chemistry 10/1985; 28(9):1350-4. · 5.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rat liver (hydrocortisone-induced) ornithine decarboxylase has been shown to be stable when the cytosolic fraction is incubated alone at 37 degrees C, although there is a very rapid and drastic loss of activity after addition of microsomes to the incubation medium. The present paper is concerned with the behaviour of ornithine decarboxylase induced in rat liver by a growth stimulus (partial hepatectomy); comparative studies have been carried out on the enzyme induced by sham operation, or by hydrocortisone. Results show that ornithine decarboxylase from regenerating liver is more stable when incubated with microsomes (from the same source); this higher stability depends both on a lower microsome-bound inactivating capacity and a limited susceptibility of the enzyme to the inactivation. A critical role in modulating the microsome-dependent inactivation appears to be played by low molecular weight cytosolic factors, whose greater content in regenerating liver is likely to be included with the factors above in determining the relative stability of ornithine decarboxylase.
Cell Biochemistry and Function 04/1985; 3(2):139-45. DOI:10.1002/cbf.290030210 · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Isolated rat hepatocytes have been treated with cumene hydroperoxide at concentrations not inducing irreversible cell damage. Under these experimental conditions cells show an enhanced lipid peroxidation, a decrease of glucose-6-phosphatase activity and of cytochrome P-450 content, and a stimulation of aminopyrene demethylation. Furthermore, the hepatocyte incorporation of amino acids is slightly but significantly reduced by the tested compound. Finally, because of the inhibitory effect of cumene hydroperoxide on cell lipoprotein but not on protein secretion, a mechanism of damage acting at the level of the assembly and maturation of lipoprotein micelles is postulated.
[Show abstract][Hide abstract] ABSTRACT: A very rapid and drastic microsome-dependent in vitro inactivation of the hydrocortisone-induced ornithine decarboxylase in rat liver was reported recently (M. F. Zuretti and E. Gravela, Biochim. Biophys. Acta. 742: 269-277, 1983). Present results show that ornithine decarboxylase from preneoplastic nodules and hepatomas, which have been induced in rats by N-2-fluorenylacetamide, is much more stable, its greater stability not being accounted for by a lower microsome-bound inactivating capacity. The possibility of a relationship between the in vitro enzyme stability and the increase of enzyme activity in neoplastic tissues is suggested.
Cancer Research 06/1983; 43(5):2298-300. · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Isolated rat hepatocytes, treated with CCl4 or ADP-Fe3+ complex show an enhanced lipid peroxidation and a decreased glucose 6-phosphatase activity. Lipid peroxidation is much more stimulated by ADP-Fe3+ or Fe3+ than by CCl4, when the metal and the haloalkane are used at a similar concentration. Increasing rates of lipid peroxidation in the different experimental conditions do not correlate with the degree of glucose 6-phosphatase inactivation, which is produced by CCl4 and not by a similar amount of ferric iron. In the case of iron, its intracellular concentration must be higher to give the enzyme inactivation exerted by CCl4. Higher intracellular levels of iron are reached when the metal is added to the cell suspension together with ADP. Under these conditions there is inactivation of glucose 6-phosphatase. Possible mechanisms accounting for a different enzyme sensitivity to iron and CCl4 are discussed.
[Show abstract][Hide abstract] ABSTRACT: 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).
[Show abstract][Hide abstract] ABSTRACT: Isolated rat liver cells have been exposed to 3 different lipid peroxidation-inducing agents, CCl4, FeCl3 and cumene hydroperoxide, and the rates of malonaldehyde production and of lipoprotein secretion have been compared. Results indicate that it is possible to induce a high degree of lipid peroxidation without inducing strong changes in lipoprotein secretion. Only in CCl4-poisoned hepatocytes is lipoprotein secretion strongly impaired. In this experimental condition, the effect of free radical scavengers, or inhibitors of lipid peroxidation, as well as the behavior of both lipid peroxidation and lipoprotein secretion, have been evaluated. Promethazine and propyl gallate prevented malonaldehyde production, but neither agent reduced covalent binding nor improved secretion. Menadione, on the contrary, besides inhibiting malonaldehyde production, decreased covalent binding and protected against the impairment of secretion. These data lead to the conclusion that covalent binding of CCl4 metabolites, rather than lipid peroxidation products, accounts for the derangement of lipoprotein secretion in CCl4-poisoned liver cells.
[Show abstract][Hide abstract] ABSTRACT: In this paper the Authors describe a new method they have adopted (in studies reported in accompanying papers II and III) for measuring S-adenosylmethionine decarboxylase activity in entire cells and cell fractions. The new method is a general one for recovering and measuring labelled carbon dioxide formed "in vitro" during enzymatic reactions. It presents several advantages when compared to traditional methods.
Bollettino della Società italiana di biologia sperimentale 07/1981; 57(11):1195-201.