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.
Addition of glucose-6-phosphate (0.03-0.06 mM) to a suspension of rat lung mitochondria promotes solubilization of 30 to 85% of the bound form of hexokinase. This effect is significantly enhanced by ...the simultaneous addition of glucose (10 mM), fructose (10 mM) or mannose (10 mM). Conversely addition of N-acetylglucosamine (10 mM), mannose-6-phosphate (10 mM) or inorganic phosphate (1 mM) reduces hexokinase solubilization. Insulin is involved in the regulation of the interaction between mitochondrial membrane and hexokinase. Treatment with alloxan causes an increase of the free form of the enzyme in the lung and a correspondent reduction of the bound form. Insulin administration restores the normal intracellular distribution of the enzyme.
c-erbB-2 and ras expression was measured on tumor extracts from 132 human primary breast carcinomas, by immunoblotting analysis. Expression of the c-erbB-2-encoded p185 protein was observed in 39% of ...the samples and found to correlate with c-erbB-2 gene amplification, detected by Southern analysis in 19 of the 77 available tumor DNAs. p185 expression was linked to the absence of progesterone receptors, but it was not related to lymph-node status or to other clinico-pathological parameters. Levels of the ras-encoded p21 proteins higher than in normal breast tissues were found in 71% of the samples. No significant correlation was seen between p21 level and the available clinical parameters. Conversely, there was a strong positive correlation between p21 and p185 levels. Analysis of follow-up data revealed that p185 expression was associated with a shorter time to relapse and death. Most notably, the contemporaneous expression of p185 and of high p21 levels was more effective than p185 expression alone in identifying cases with poor prognosis. The prognostic value of p185/p21 co-expression was particularly significant in progesterone-receptor-positive tumors. Our data suggest that c-erbB-2 and ras may act synergistically to endow breast-tumor cells with a highly aggressive phenotype.
1. In chicken embryo cartilage putrescine levels, maximal at day 8, fall by day 16 to a four-fold lower value, which remains unchanged through hatching and in the 12-day-old chick. 2. Spermine and ...spermidine, initially higher than putrescine, are almost halved between days 8 and 11, and remain constant afterwards. 3. Ornithine decarboxylase is down to 20% of the day 8 value by day 16, and is further reduced in the newly hatched chick. 4. S-Adenosyl-methionine decarboxylase activity shows a 50% reduction between days 8 and 11, and no further changes. 5. Spermidine acetyltransferase activity at day 11 is 30% lower than at day 8, goes back up to the initial level by day 16, and progressively decreases through hatching and the first 12 days of life.
Two proteins were isolated, in a stable form, from bovine brain by ion exchange chromatography, gel filtration and ultracentrifugation on glycerol gradient. They were identified as 20S and 26S ...proteasomes on the basis of molecular mass, migration velocity on non-denaturing gels, immunoreactivity, multipeptidase activity and the 26S proteasome also for dependence on ATP for the degradation of short peptides and ubiquitinylated proteins. However, the 26S proteasome has some properties not yet described for its counterpart of other tissues and from brain of this and other species. In particular, the ATP concentration required by the 26S proteasome to reach maximal peptidase activity was approximately 40-fold lower than the one required for maximal proteolytic activity on polyubiquitinylated substrates. Moreover, plots of substrate concentration vs. velocity gave a saturation curve for the 26S proteasome only, which, for the trypsin-like and post-glutamyl peptide hydrolase activities fitted the Michaelis–Menten equation, whereas for the chymotrypsin-like activity indicated multibinding site kinetics with positive cooperativity (
n=2.32±0.38). As concerns the 20S proteasome, its electrophoretic pattern on native gel revealed a single protein band, a feature, to our knowledge, not yet described for the brain particle of any species.
Hexosokinase of the rat lung Tazartes Pesando, O
Bollettino della Societa italiana di biologia sperimentale,
1978-Apr-15, Letnik:
54, Številka:
7
Journal Article
Oxalic, succinic, glutaric and pimelic acid (5 mM) had no effect on lactate formation from glucose if added to a crude extract of chicken embryo at the same time as substrate and cofactors; ...conversely malonic, adipic, suberic, azelaic and sebacic acid had an inhibitory effect ranging from 20% to 35%. When the enzyme preparation was pre-incubated with the dicarboxylic acids for one hour before beginning the experiments, all compounds tested, with the exception of succinate, inhibited anaerobic glycolysis. Hexokinase activity was significantly reduced by saturated dicarboxylic acids from C3 to C10, but not by oxalic acid. Phosphofructokinase was inhibited only by oxalic, pimelic and suberic acid. Pyruvate kinase appeared sensitive only to oxalic acid (64% inhibition).
Platelets from patients with known red blood cell G-6-PD deficiency were investigated to find out whether this genetic defect is associated with changes in platelet aggregation. The enzyme defect in ...platelets could be verified by a decreased G-6-PD activity which was as low as 15% compared to control subjects. The decreased enzyme activity was reflected by lowered levels of NADPH and GSH and by a diminished maximum capacity of the hexose monophosphate shunt. Aggregation measurements in platelet-rich plasma from deficient patients revealed an enhanced dose response to ADP which was higher by about one order of magnitude compared to controls. The same effect was observed in a smaller degree in arachidonic acid induced aggregation.
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