Changes in the structure and function of plant cell protoplast due to energy deficit

The influence of glycolysis inhibitors and of the respiratory chain inhibitors on the structure and function of protoplast were investigated in <i>Allium cepa</i> and <i>Tradescantia bracteata</i> meristematic cells. Hypertrophy of rough ER and modification of active transport were found, which was expressed by changes in the permeability of the plasmalemma and tonoplast and by depressed level of active ATP-ase. It is supposed that the formation of the characteristic rough endoplasmic reticulum (ER) configurations plays a significant role in bioenergetic processes by increasing the surface area active in glycolysis and in the respiratory chain connected with ER membranes.

Oligomycin inhibits HIF-1α expression in hypoxic tumor cells

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to reduced oxygen availability. The contribution of mitochondria in regulation of HIF-1α in hypoxic cells has received recent attention. We demonstrate that inhibition of electron transport complexes I, III, and IV diminished hypoxic HIF-1α accumulation in different tumor cell lines. Hypoxia-induced HIF-1α accumulation was not prevented by the antioxidants Trolox and N-acetyl-cysteine. Oligomycin, inhibitor of F0F1-ATPase, prevented hypoxia-induced HIF-1α protein accumulation and had no effect on HIF-1α induction by hypoxia-mimicking agents desferrioxamine or dimethyloxalylglycine. The inhibitory effect of mitochondrial respiratory chain inhibitors and oligomycin on hypoxic HIF-1α content was pronounced in cells exposed to hypoxia (1.5% O2) but decreased markedly when cells were exposed to severe oxygen deprivation (anoxia). Taken together, these results do not support the role for mitochondrial reactive oxygen species in HIF-1α regulation, but rather suggest that inhibition of electron transport chain and impaired oxygen consumption affect HIF-1α accumulation in hypoxic cells indirectly via effects on prolyl hydroxylase function.

Effects of glucose starvation on the oxidation of fatty acids by maize root tip mitochondria and peroxisomes: evidence for mitochondrial fatty acid β-oxidation and acyl-CoA dehydrogenase activity in a higher plant

Fatty acid beta-oxidation was studied in organellar fractions from maize root tips by h.p.l.c. and radiometric analysis of the products of incubations with [1-14C]octanoate and [1-14C]palmitate. In crude organellar fractions containing both mitochondria and peroxisomes, octanoate and palmitate beta-oxidation, as determined by the production of acetyl-CoA, was functional and, for palmitate, was activated 4-12-fold after subjecting the root tips to 48 h of glucose starvation. The sensitivity to a ‘cocktail’ of respiratory-chain inhibitors containing cyanide, azide and salicylhydroxamate depended on the conditions of incubation, with no inhibition in a medium facilitating peroxisomal beta-oxidation and a significant inhibition in a medium potentially facilitating mitochondrial beta-oxidation. Indeed, preparations of highly purified mitochondria from glucose-starved root tips were able to oxidize octanoate and palmitate to give organic acids of the tricarboxylic acid cycle. This activity was inhibited 5-10-fold by the above cocktail of respiratory-chain inhibitors, with no parallel accumulation of acetyl-CoA, thus showing that the inhibition affected beta-oxidation rather than the pathway from acetyl-CoA to the organic acids. This provides the first evidence that the complete beta-oxidation pathway from fatty acids to citrate was functional in mitochondria from a higher plant. Moreover, an acyl-CoA dehydrogenase activity was shown to be present in the purified mitochondria. In contrast with the peroxisomal activity, mitochondrial beta-oxidation showed the same efficiency with octanoate and palmitate and was strictly dependent on glucose starvation.

Evidence that the inhibition sites of the neurotoxic amine 1-methyl-4-phenylpyridinium (MPP+) and of the respiratory chain inhibitor piericidin A are the same

1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), interrupts mitochondrial electron transfer at the NADH dehydrogenase-ubiquinone junction, as do the respiratory chain inhibitors rotenone, piericidin A and barbiturates. Proof that these classical respiratory chain inhibitors and MPP+ react at the same site in the complex NADH dehydrogenase molecule has been difficult to obtain because none of these compounds bind covalently to the target. The 4′-alkyl derivatives of MPP+ inhibit NADH oxidation in submitochondrial particles at much lower concentrations than does MPP+ itself, but still dissociate on washing the membrane preparations, with consequent re-activation of the enzyme. The MPP+ analogues with short alkyl chains prevent the binding of 14C-labelled piericidin A to the membrane and thus must act at the same site, but analogues with alkyl chains longer than heptyl do not prevent binding of [14C]piericidin.

Energy coupling mechanisms in host-grown Mycobacterium lepraemurium

Energy coupling mechanisms of Mycobacterium lepraemurium isolated from Sprague-Dawley rats lepromata were investigated. Cell-free extracts catalyzed phosphorylation coupled to the oxidation of generated NADH, added NADH, and succinate yielding P/O ratios of approximately 0.8, 0.6, and 0.4, respectively. Ascorbate oxidation alone or in the presence of cytochrome c or N,N,N′,N′-tetramethyl-p-phenylenediamine was not coupled to ATP synthesis.The oxidative phosphorylation was completely uncoupled by 2,4-dinitrophenol, 2,6-dibromophenol, pentachlorophenol, m-chlorocarbonylcyanide phenylhydrazone, dicumarol, and gramicidin at concentrations which did not cause any inhibition of oxygen uptake. While the NADH oxidation and associated phosphate esterification was markedly sensitive to rotenone and other flavoprotein inhibitors, these inhibitors had no effect, however, on the phosphorylation coupled to succinate oxidation. The respiratory chain inhibitors such as antimycin A or 2-n-heptyl-4-hydroxyquinoline-N-oxide, and cyanide were the potent inhibitors of the phosphorylation associated with the oxidation of NADH and succinate. The ATP formation coupled to the oxidation of NADH and succinate was also inhibited by oligomycin as well as by the thiol-binding agents, p-hydroxymercuribenzoate and N-ethylmaleimide. The results indicated that NADH and succinate oxidation by in vivo grown M. lepraemurium was mediated by oxidative enzymes involving first and second energy coupling sites.