Stefano Bedino

Proteasomes are a target of the anti-tumour drug vinblastine

Proteasomes, the proteolytic machinery of the ubiquitin/ATP-dependent pathway, have a relevant role in many processes crucial for cell physiology and cell cycle progression. Proteasome inhibitors are used to block cell cycle progression and to induce apoptosis in certain cell lines. Here we examine whether proteasomal function is affected by the anti-tumour drug vinblastine, whose cytostatic action relies mainly on the disruption of mitotic spindle dynamics. The effects of vinblastine on the peptidase activities of human 20 S and 26 S proteasomes and on the proteolytic activity of 26 S proteasome were assessed in the presence of specific fluorogenic peptides and (125)I-lysozyme-ubiquitin conjugates respectively. The assays of ubiquitin-protein conjugates and of inhibitory kappa B alpha (I kappa B alpha), which are characteristic intracellular proteasome substrates, by Western blotting on lysates from HL60 cells incubated with or without vinblastine, illustrated the effects of vinblastine on proteasomes in vivo. We also evaluated the effects of vinblastine on the signal-induced degradation of I kappa B alpha. Vinblastine at 3--110 microM reversibly inhibited the chymotrypsin-like activity of the 20 S proteasome and the trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of both proteasomes, but only at 110 microM vinblastine was the chymotrypsin-like activity of the 26 S proteasome inhibited; furthermore, at 25--200 microM the drug inhibited the degradation of ubiquitinated lysozyme. In HL60 cells exposed for 6 h to 0.5--10 microM vinblastine, the drug-dose-related accumulation of polyubiquitinated proteins, as well as that of a high-molecular-mass form of I kappa B alpha, occurred. Moreover, vinblastine impaired the signal-induced degradation of I kappa B alpha. Cell viability throughout the test was approx. 95%. Proteasomes can be considered to be a new and additional vinblastine target.

Regulation of polyamine synthesis in chicken liver

Abstract 1. 1. Insulin injected into fasting chickens promotes a marked increase of liver S -adenosylmethionine decarboxylase, with maximum activity 6 hr after administration. 2. 2. The apparent half-life of the enzyme is 22 min. 3. 3. Ornithine and putrescine in vivo give rise only to small modifications in enzyme activity; spermidine and spermine induce the enzyme. 4. 4. Putrescine activates the decarboxylase in vitro . Spermidine and spermine, however, appear to be inhibitors. 5. 5. Insulin promotes a decrease of arginine, ornithine and all other amino acids, except for proline which increases. Putrescine is enhanced, spermidine slightly decreased and spermine unchanged. 6. 6. Levels of polyamines are significantly altered by administration of methionine which promotes synthesis of spermidine, and by spermine which blocks transformation of putrescine into spermidine.

Stimulation by insulin of chicken liver ornithine decarboxylase

Abstract 1. 1. Insulin injected in fasting chickens promotes a marked increase of liver ornithine decarboxylase activity. Maximum activity, 90-fold the normal value, is observed 6 hr after administration. 2. 2. Apparent half-life of the enzyme, measured after induction with insulin, is 8 min. 3. 3. Ornithine promotes a reduction of arginase and an induction of ornithine decarboxylase. When given in addition to insulin, enhancement is even higher. 4. 4. Putrescine and spermidine given with insulin have no effect on arginase, but partially prevent ornithine decarboxylase induction. 5. 5. Putrescine, spermidine and spermine all inhibit ornithine decarboxylase activity in vitro .

Induction of an ornithine decarboxylase-antizyme in chicken liver

Abstract 1. 1. The insulin-induced increase in ornithine decarboxylase and in S-adenosylmethionine decarboxylase in chicken liver is considerably inhibited by diaminopropane. This effect does not last long: after 5 hr, ornithine decarboxylase has returned to normal, though S-adenosylmethionine decarboxylase is more active. 2. 2. There is a good correlation between diaminopropane and ornithine decarboxylase activity for 1 hr. after which ornithine decarboxylase remains low, even when diaminopropane disappears from the liver. 3. 3. Liver diaminobutane concentration is fairly constant throughout the treatment, while spermidine and spermine concentrations rise following the increase in S-adenosylmethionine decarboxylase. 4. 4. Diaminopropane induces the synthesis of an ornithine decarboxylase-antizyme (mol. wt 19,500 and t 1 2 12.8 min). A similar antizyme is also induced by diaminobutane, spermidine and spermine. 5. 5. The effect of the antizyme on ornithine decarboxylase is lower in the presence of high concentrations of pyridoxal phosphate. The antizyme loses its activity when treated with pyridoxal phosphate and reduced with NaBH4.

Regulation of polyamine synthesis in the chicken.

Abstract 1. 1. Ornithine decarboxylase is active in kidney and heart muscle, less so in brain, pancreas and intestinal mucosa extracts, but in every case more than in liver. 2. 2. S-adenosylmethionine decarboxylase is also active in the same tissues, with maximal activity in heart, brain and pancreas. 3. 3. Insulin induces both enzymes in all tissues, with the exception of brain, where activity is somewhat decreased. 4. 4. Spermidine partially prevents induction by insulin in pancreas. 5. 5. In the pancreas putrescine, spermidine and spermine levels are very high, with a spermidine/spermine ratio 1.80. Insulin promotes an increase of putrescine and a decrease of spermidine. Administration of spermine promotes modifications in polyamine levels lower than in liver, but of the same kind.

Purification and kinetic characterization of gamma-aminobutyraldehyde dehydrogenase from rat liver.

Abstract Oxidative deamination of putrescine, the precursor of polyamines, gives rise to γ-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD + -dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3–8.4. Temperatures higher than 28°C promote slow activation and the process is favoured by the presence of at least one substrate. K m for aliphatic aldehydes decreases from 110 μM for ABAL and acetaldehyde to 2–3 μM for capronaldehyde. The highest relative V -values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD + is affected by the aldehyde present at the active site: K m for NAD + is ∼70 μM with ABAL, ∼200 μM with isobutyraldehyde and capronaldehyde, and >800 μM with acetaldehyde. The kinetic behaviour at 37°C is quite complex; according to enzymatic models, NAD + activates the enzyme ( K act ∼ 500 μ M) while NADH competes for the regulatory site ( K in ∼ 70 μ M). In the presence of high NAD + concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site ( K act ∼ 10 mM). The data show that the enzyme is probably an E 3 aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.

Kinetic behaviour and properties of aldehyde dehydrogenase from rat testis mitochondria. Effect of Mg2+ ions

1. Mitochondrial aldehyde dehydrogenase is purified to near homogeneity by hydroxylapatite-, affinity- and hydrophobic interaction-chromatography. 2. The enzyme is an oligomeric protein and its molecular weight, as determined by gel-filtration, is 117,000 +/- 5000. 3. Active only in the presence of exogenous sulfhydryl compounds and NAD(+)-dependent, aldehyde dehydrogenase works optimally with linear-chain aliphatic aldehydes and is practically inactive with benzaldehyde. The pH-optimum is at about pH 8.5. 4. Km-Values for aliphatic aldehydes (C2-C6) range between 0.17 and 0.32 microM. The Km for NAD+ increases from 16 microM with acetaldehyde to 71 microM with capronaldehyde. 5. Millimolar concentrations of Mg2+ promote high increases of both V and Km for NAD+. At the same time, saturation curves with C4-C6 aldehydes can be simulated with a substrate inhibition model. 6. Inhibition by NADH is competitive: with capronaldehyde, the inhibition constant for NADH is 52 microM in the absence of Mg2+ and 14 microM in the presence of 4 mM Mg2+; with acetaldehyde, the inhibition constant is about three times higher (36 and 159 microM, respectively).

Allosteric regulation of rat testis mitochondrial aldehyde dehydrogenase by capronaldehyde and magnesium ion

1. The influence of Mg2+ on the kinetic behaviour of mitochondrial aldehyde dehydrogenase from rat testis has been investigated using capronaldehyde as substrate. 2. The kinetic data, obtained by numerical analysis of the progress curves of aldehyde oxidation, were fitted to a modified version of the Monod-Wyman-Changeux model and the fitting procedure resulted in a good correspondence between theoretical and experimental reaction rates over a wide range of capronaldehyde and Mg2+ concentrations. 3. According to the model, the tetrameric enzyme is in equilibrium between two conformational states R and T which display comparable affinities for capronaldehyde (the dissociation constants are 0.17 and 0.3 microM, respectively), but different catalytic power (VT = 2VR). The T state can bind with lower affinity a second molecule of aldehyde (K = 2.5 microM). 4. Mg2+ stabilizes the T state (the dissociation constants for the R and T states are 2.2 and 0.12 mM, respectively) and acts as a strong activator of the R state, but as a weak inhibitor of the T state. In the absence of substrates and Mg2+, the R<-->T equilibrium favors the R state ([T]/[R] = 0.16). 5. The model is able to predict the kinetic behaviour also when the NAD+ concentrations are not saturating and when inhibitory effects by NADH are taken into account.

Aldehyde dehydrogenase from rat intestinal mucosa: purification and characterization of an isozyme with high affinity for γ-aminobutyraldehyde

In rat adrenal gland and gastric mucosa putrescine is efficiently oxidized to GABA via gamma-aminobutyraldehyde (ABAL) by action of diamine oxidase and aldehyde dehydrogenase. Having turned our attention on the rat intestinal mucosa, where putrescine uptake and diamine oxidase are active, we have purified and characterized an aldehyde dehydrogenase optimally active on gamma-aminobutyraldehyde. A dimer with a subunit molecular weight of 52,000, the native enzyme binds ABAL and NAD+ with high affinity: at pH 7.4, Km values are equal to 18 and 14 microM, respectively. Affinity for betaine aldehyde is much lower (Km = 285 microM), but the efficiency is equally good, thanks to a high value of V. Unaffected by disulfiram and Mg2+, the enzyme is activated by high NAD+ concentrations (Vnn = 1.6 x Vn) and is competitively inhibited by NADH. According to the best fitting model, the dimeric enzyme only binds one NADH and the mixed complex enzyme-NAD(+)-NADH is inactive. The increase of activity promoted by NAD+ can therefore be ascribed to an allosteric effect, rather than to the activation of a second reaction center. Highly stable at pH 6.8 in the presence of dithiothreitol and high phosphate concentrations, ABALDH is inactivated by ion-exchange resins and by cationic buffers. Our results show that the enzyme can be effectively involved in the metabolism of biogenic amines and, with a K(m) for ABAL lower than 20 microM, in the synthesis of GABA.

Regulation by progesterone and pregnenolone of dimeric aldehyde dehydrogenase from rat testis cytoplasm.

1. Aldehyde dehydrogenase from rat testis cytosol has been purified to electrophoretic homogeneity. With an isoelectric point of 9.5, the enzyme appears a dimer with a subunit molecular weight of 52,500. 2. The influence of pregnenolone and progesterone on the kinetic behaviour has been investigated using valeraldehyde as substrate. 3. The kinetic data were fitted to a modified version of the Monod-Wyman-Changeux model and the fitting procedure resulted in a good correspondence between theoretical and experimental reaction rates over a wide range of valeraldehyde concentrations. 4. According to the model, the dimeric enzyme is in equilibrium between two conformational states R and T. The R state displays higher affinity for valeraldehyde, but lower catalytic power. In the absence of substrates and effectors the [T]/[R] ratio is near to 1. 5. Pregnenolone and progesterone activate the enzyme by stabilizing the more active state T and by increasing the catalytic power of the R state. The increase of activity is counteracted by the inhibition exerted by both steroids on the T state.