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.
Inhibition of bovine liver glutamate dehydrogenase by pyridoxal-5'-phosphate was studied by measuring the full time course of the oxidation of NADPH. Progress curves were determined before and after ...incubation of the enzyme with PLP in the presence of saturating concentrations of alpha-ketoglutarate and ammonium ion, at pH 7.4 and 25 degrees C. The data were fitted to the integrated Michaelis-Menten equation and an inhibition model derived. According to the model, PLP inhibits the enzyme non-competitively, by reversible formation of the complexes E--PLP and E--PLP--NADPH; the oxidation of NADPH is also inhibited by NADP+. After incubation with PLP, the dissociation constants of E--NADPH and E--NADP+ (Km and Kp) show a very definite decrease, while the maximum rate of oxidation (Vm) is increased. The inhibition constants for PLP were also computed.
There is much evidence that oxygen free radicals (OFR) may be the final mediators of biochemical and molecular damage in many kidney diseases of different etiology (toxic, ischemic and ...immunologically mediated), involving mainly endothelium, basement membrane and tubular cells, but direct demonstration of a role in inducing mesangiolysis is lacking. An experimental model of renal damage caused by OFR was carried out in 6 rabbits using a mixture of xanthine-oxidase and xanthine, which produces a large amount of the radical superoxide anion. Both enzyme (0.0150 and 0.150 U/ml) and substrate (0.2 and 2 mM) were simultaneously infused in one kidney, while the controlateral kidneys perfused with buffer only were used as controls. Treated kidneys were compared to controls by light and electron microscopy. A further experiment was carried out in 4 other rabbits to evaluate the protection afforded by superoxide dismutase, the specific enzyme-scavenging superoxide anion. Microscopic studies showed dose-related ingravescent damage in the treated kidneys: capillary enlargement, subendothelial swelling, detachment of the endothelium from the basement membrane, mesangiolysis and microaneurysms. Control kidneys appeared to be normal. No significant differences were observed in the kidneys treated with addition of superoxide dismutase. These results are the first direct demonstration of a role of superoxide anion in the induction of mesangiolysis in rabbits. The lack of a protective effect by superoxide dismutase could mean that the superoxide anion triggers a chain of other OFR, further responsible for damage.
In rat adrenal gland and gastric mucosa putrescine is efficiently oxidized to GABA via γ-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 γ-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,
K
m values are equal to 18 and 14 μM, respectively. Affinity for betaine aldehyde is much lower (
K
m=285 μM), but the efficiency is equally good, thanks to a high value of
V. Unaffected by disulfiram and Mg
2+, the enzyme is activated by high NAD
+ concentrations (
V
nn=1.6×
V
n) 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 μM, in the synthesis of GABA.
Rat hepatocytes in culture take up 14C‐agmatine by both a high‐affinity transport system KM = 0.03 mm; Vmax = 30 pmol·min·(mg protein)−1 and a low‐affinity system. The high‐affinity system also ...transports putrescine, but not cationic amino acids such as arginine, and the polyamines spermidine and spermine. The rate of agmatine uptake is increased in cells deprived of polyamines with difluoromethylornithine. Of the agmatine taken up, 10% is transformed into polyamines and 50% is transformed into 4‐guanidinobutyrate, as demonstrated by HPLC and MS. Inhibition by aminoguanidine and pargyline shows that this is due to diamine oxidase and an aldehyde dehydrogenase. 14C‐4‐aminobutyrate is also accumulated in the presence of an inhibitor of 4‐aminobutyrate transaminase.
In the presence of Mg2+ saturation curves of aldehyde dehydrogenase show a sharp maximum at capronaldehyde concentrations lower than 1 microM. Since the native enzyme is a dimer, kinetic data have ...been analyzed with a general rate equation (given as a ratio of two polynomials) that takes into account the presence of two binding sites for both substrates and two for Mg2+. Simulation of the saturation curves was only successful after allowing the formation of the stable complexes ES, ES2, ES2M, ES2M2, EM and EM2. Since ESM and ESM2 are highly reactive but very unstable, activity at low aldehyde concentration can be explained by assuming a direct reaction mediated by Mg2+. At concentrations higher than 1 microM, capronaldehyde effectively binds to the enzyme in a highly cooperative process, but the formation of ES2M and ES2M2 results in slower reaction rates. Since ES2M2 is inactive, increase of the Mg2+ concentration eventually leads to strong inhibition. Experiments at different NAD+ concentrations show that the enzyme binds two NAD+, but reaction takes place at one binding site.
A simple rate equation for alcohol dehydrogenase was obtained by assuming independent binding sites for ethanol and NAD+ and fully competitive inhibition by the products of the reaction, acetaldehyde ...and NADH. A random binding order was also assumed. The rate equation is described by six parameters: four association constants (two for the substrates and two for the products of the reaction), Vf for the forward direction, and the equilibrium constant of the reaction. The six parameters were determined at pH 7.4 by numerical analysis of progress curves of reactions started with different concentrations of ethanol and NAD+. The parameters for alcohol dehydrogenase partially purified from rat liver were: Km for ethanol = 0.746 mM, Km for NAD+ = 0.0563 mM, Km for acetaldehyde = 7.07 microM, Km for NADH = 4.77 microM and Keq = 2.36 X 10(-4). The computed values allowed a very good simulation of the experimental progress curves and little variation was observed in the kinetic parameters when the reactions were started in the presence of either NADH or acetaldehyde.
Intracellular beta-glucosidase is strongly inhibited by its own substrate p-nitrophenyl-beta-glucoside which displays high affinity for two binding sites. A non-productive complex is formed also by ...cellobiose, but its lower affinity results in a much lower inhibition. As shown by inhibition experiments performed with glucono-delta-lactone, the hydrolytic reaction proceeds through the formation of a carbonium ion, very similar in its half-chair conformation to the delta-lactone. Carboxylic groups (pK = 3.19) appear involved in the catalytic process together with a histidine residue (pK = 5.64): while the carboxylate ions stabilize the carbonium ion, the displaced group accepts a proton from the protonated imidazole.
Intracellular beta-glucosidase was extracted from the mycelium of Th. aurantiacus, concentrated by DEAE-cellulose treatment, separated from alpha-glucosidase by hydroxylapatite chromatography and ...purified to electrophoretic homogeneity. Optimally active at 75 degrees C and pH 4.2, beta-glucosidase displayed complex kinetics with p-nitrophenyl-beta-glucoside which inhibited the enzyme at concentrations greater than 0.5 mM. With cellobiose the kinetics were practically hyperbolic at 70 degrees C (Hill coefficient nH = 1.09 and Km = 0.83 mM), but faint inhibition was observed at 50 degrees C. beta-glucosidase shares with alpha-glucosidase a high number of physicochemical properties: with similar aminoacid composition, very close isoelectric point (4.5 and 4.2), high molecular weight in the native state (175,000 and 140,000), the two enzymes showed the same behaviour on DEAE-cellulose, were equally stable at high temperature and were dissociated by 6 M urea to still active proteins. Furthermore, the carbohydrate contents of beta-glucosidase (17.6%) is not far from that previously determined for some forms of alpha-glucosidase (14-16%).
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