AS-30D hepatoma cells, a highly oxidative and fastgrowing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mm glucose or 10 mm fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular P i (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca 21 and Mg 21 were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low P i (0.6 mm) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.Keywords: Crabtree effect; fast-growth tumor cells; multisite control; oxidative phosphorylation.Fast-growing tumor cells are characterized by a high glycolytic activity, even in the presence of saturating oxygen. In these cells it is also known that cellular oxidative phosphorylation is strongly inhibited by exogenous glucose [1]. A diminished or absent Pasteur effect would explain the former phenomenon. However, for the glucose-induced inhibition of oxidative phosphorylation, known as the Crabtree effect [2], there is no satisfactory explanation. The Crabtree effect is not restricted to malignant cells as it is also observed in normal proliferative cells [3]. In fact, in several nontumoral tissues (pig platelets, hamster embryos, proliferating thymocytes, retina, intestinal mucosa) [4±7] and in bacteria and yeast [8,9], it has been reported that oxidative phosphorylation is inhibited by exogenous hexoses.Eigenbrodt et al. proposed that this process could be a selective advantage for tumor cells, as the consequence of a glucose-dependent accumulation of essential metabolic intermediaries, such as serine, phosphoribosyl-pyrophosphate, fructose 1,6-bisphosphate, and glycerol 3-phosphate, which can trigger the mitogenic events In this work, we have evaluated some of these hypotheses, using the oxidative ascites tumor AS-30D cell line as the experimental model. We show that several factors are involved in the onset of the Crabtree effect. M A T E R I A L S A N D M E T H O D SCell preparation AS-30D ascites tumor cells were propagated by inoculation of 2 Â 10 8 cells into the peritoneal cavity of female Wistar rats of 200±250 g weight. The cells were harvested, and washed as described previously [17]. They were stored in ice at a density of 2±3 Â 10 8 cells´mL 21 until use. Isolation of mitochondriaAS-30D mitochondria were isolated by the digitonin permeabilization procedure...

The F(1)F(O) and F(1)-ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome-reconstituted holoenzyme preserves its tight coupling between F(1) and F(O) sectors, as evidenced by its high sensitivity to the F(O) inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N-terminal sequencing of the band profile in SDS-PAGE of the F(1) and F(1)F(O) preparations showed a novel 11-kDa protein in addition to the 5 canonical alpha, beta, gamma, delta, and epsilon subunits present in all known F(1)-ATPase complexes. BN-PAGE followed by 2D-SDS-PAGE confirmed the presence of this 11-kDa protein bound to the native F(1)F(O)-ATP synthase of P. denitrificans, as it was observed after being isolated. The recombinant 11 kDa and epsilon subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F(1)F(O) and soluble F(1)-ATPase complexes. The 11-kDa protein, but not the epsilon subunit, inhibited the F(1)F(O) and F(1)-ATPase activities of P. denitrificans. The 11-kDa protein was also found in Rhodobacter sphaeroides associated to its native F(1)F(O)-ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11-kDa protein on the F(1)F(O)-ATPase nanomotor of P. denitrificans and closely related alpha-proteobacteria.

Aldehyde dehydrogenases (ALDHs) are involved in the detoxification of aldehydes generated as byproducts of lipid peroxidation. In this work, it was determined that, among the three most studied human ALDH isoforms, ALDH2 showed the highest catalytic efficiency for oxidation of acrolein, 4-hydroxy-2-nonenal (4-HNE), and malondialdehyde. ALDH1A1 also exhibited significant activity with these substrates, whereas ALDH3A1 only showed activity with 4-HNE. ALDH2 was also the most sensitive isoform to irreversible inactivation by these compounds. Remarkably, ALDH3A1 was insensitive to these aldehydes even at concentrations as high as 20 mM. Formation of adducts of ALDH1A1 and ALDH2 with acrolein increased their K(d) values for NAD(+) by 2- and 3-fold, respectively. NADH exerted a higher protection than propionaldehyde to the inactivation by acrolein, and this protection was additive. These results suggested that both binding sites, those for aldehyde and NAD(+) in ALDH2, are targets for the inactivation by lipid peroxidation products. Thus, with the advantage of being relatively inactivation-insensitive, ALDH1A1 and ALDH3A1 may be actively participating in the detoxification of these aldehydes in the cells.

The effect of increased expression or reconstitution of the mitochondrial inhibitor protein (IF1) on the dimer/monomer ratio (D/M) of the rat liver and bovine heart F1F0-ATP synthase was studied. The 2-fold increased expression of IF1 in AS-30D hepatoma mitochondria correlated with a 1.4-fold increase in the D/M ratio of the ATP synthase extracted with digitonin as determined by blue native electrophoresis and averaged densitometry analyses. Removal of IF1 from rat liver or bovine heart submitochondrial particles increased the F1F0-ATPase activity and decreased the D/M ratio of the ATP synthase. Reconstitution of recombinant IF1 into submitochondrial particles devoid of IF1 inhibited the F1F0-ATPase activity by 90% and restored partially the D/M ratio of the whole F1F0 complex as revealed by blue native electrophoresis and subsequent SDS-PAGE or glycerol density gradient centrifugation. Thus, the inhibitor protein promotes or stabilizes the dimeric form of the intact F1F0-ATP synthase. A possible location of the IF1 protein in the dimeric structure of the rat liver F1F0 complex is proposed. According to crystallographic and electron microscopy analyses, dimeric IF1 could bridge the F1-F1 part of the dimeric F1F0-ATP synthase in the inner mitochondrial membrane.