ATP Synthesis In Cells Research Articles

Structure and in Situ Organization of ATP Sythase and Respiratory Chain Complexes

Mitochondria are the powerhouses of eukaryotic cells and the main site of ATP synthesis in cells performing aerobic respiration. Located on the cristae of the inner mitochondrial membrane, the ATP synthase uses the energy stored in a transmembrane gradient of protons to power the synthesis of ATP while the respiratory chain complexes replenish the gradient. Using the technique of electron cryo-tomography and subtomogram averaging, we are characterizing how the structure and spatial distribution of membrane proteins influence mitochondrial function. So far we have discovered: 1) the structure of the ATP synthase varies dramatically between different eukaryotic lineages, 2) that in all species studied so far, the ATP synthase form rows of dimers along the highly curved edges of the cristae membranes, 3) that the structure of the rows varies between protozoan and multicellular organisms, and 4) the purpose of the rows appears to be the maintenance of organized inner membrane invaginations. In parallel, we have shown that the proteins of the respiratory chain are located in the flat membrane regions of cristae and that they form species specific supercomplexes. Alongside the cryo-ET studies, we have determined a 6.2A structure of the mitochondrial ATP synthase of Polytomella sp. using single particle cryo-EM. Our structure revealed four highly tilted membrane-intrinsic helices (∼20° to the membrane plane) adjacent to the rotor ring which we assign to the elusive a-subunit. Based on this structure, we present a model describing how protons move through the ATP synthase.

The protective effect of dexpanthenol on testicular atrophy at 60th day following experimental testicular torsion

Despite the prompt diagnosis and treatment of testicular torsion (TT), there are problems with fertility and atrophy after testicular salvage. Dexpanthenol (Dxp) is the biologically active alcohol of pantothenic acid (PA). Dxp is converted to PA in tissues. PA increases the content of reduced glutathione (GSH), Coenzyme A and ATP synthesis in cells. GSH and glutathione-dependent peroxidases (GPX) are the major defense systems against oxidative stress. GPX-4 is the major antioxidant in testicular tissue. However, the activity of GPX-4 appeared and increased only after puberty. We investigated the effect of Dxp on testicular atrophy after TT at the 60th day. Rats were separated randomly into four groups. Group C: control group, group Td: torsion + detorsion, group Sal: torsion + saline + detorsion, group Dxp: torsion + Dxp + detorsion. The left testis was rotated 720 degrees for 2 h. In group Sal, normal saline and in group Dxp, Dexpanthenol were injected intraperitonally, 30 min before detorsion. After 60 days, the testicular weights and volumes were measured. Histopathology of the left testis was evaluated with mean seminiferous tubular diameter (MSTD) and mean testicular biopsy score (MTBS). The left (torsed) testicular weight and volume of groups Td and Sal were significantly lower compared to group Dxp. The MSTD and MTBS of group Td and Sal were significantly lower than group Dxp. Contralateral testicular weight and volume of groups Td, Sal and Dxp had no significant difference compared to the control group. Dxp significantly prevented testicular atrophy after 60 days of TT. Dxp has FDA approval, is safe, cost effective and readily available. Its relevance for clinical trials may especially be for the problem of testicular atrophy catastrophe, seen very frequently following testicular salvage.

The mtDNA T8993G (NARP) mutation results in an impairment of oxidative phosphorylation that can be improved by antioxidants.

A T8993G point mutation in the mtDNA results in a Leu156Arg substitution in the MTATP6 subunit of the mitochondrial F1F0-ATPase. The T8993G mutation causes impaired oxidative phosphorylation (OXPHOS) in two mitochondrial disorders, NARP (neuropathy, ataxia and retinitis pigmentosa) and MILS (maternally inherited Leigh's syndrome). It has been reported, in some studies, that the T8993G mutation results in loss of assembled F1F0-ATPase. Others reported that the mutation causes impairment of proton flow through F0. In addition, it was shown that fibroblasts from NARP subjects have a tendency to undergo apoptotic cell death, perhaps as a result of increased free radical production. Here, we show that the T8993G mutation inhibits oxidative phosphorylation and results in enhanced free radical production. We suggest that free radical-mediated inhibition of OXPHOS contributes to the loss of ATP synthesis. Importantly, we show that antioxidants restore respiration and partially rescue ATP synthesis in cells harboring the T8993G mutation. Our results indicate that free radicals might play an important role in the pathogenesis of NARP/MILS and that this can be prevented by antioxidants. The effectiveness of antioxidant agents in cultured NARP/MILS cells suggests that they might have a potential beneficial role in the treatment of patients with NARP.

Binding of the b-subunit in the ATP synthase from Escherichia coli.

The rotary mechanism of ATP synthase requires a strong binding within stator subunits. In this work we studied the binding affinity of the b-subunit to F(1)-ATPase of Escherichia coli. The dimerization of the truncated b-subunit without amino acids 1-33, b(34-156)T62C, was investigated by analytical ultracentrifugation, resulting in a dissociation constant of 1.8 microM. The binding of b-subunit monomeric and dimeric forms to the isolated F(1) part was investigated by fluorescence correlation spectroscopy and steady-state fluorescence. The mutants b(34-156)T62C and EF(1)-gammaT106C were labeled with several fluorophores. Fluorescence correlation spectroscopy was used to measure translational diffusion times of the labeled b-subunit, labeled F(1), and a mixture of the labeled b-subunit with unlabeled F(1). Data analysis revealed a dissociation constant of 0.2 nM of the F(1)b(2) complex, yielding a Gibbs free energy of binding of DeltaG(o)= -55 kJ mol(-1). In steady-state fluorescence resonance energy transfer (FRET) measurements it was found that binding of the b-subunit to EF(1)-gammaT106C-Alexa488 resulted in a fluorescence decrease of one-third of the initial FRET donor fluorescence intensity. The decrease of fluorescence was measured as a function of b-concentration, and data were described by a model including equilibria for dimerization of the b-subunit and binding of b and b(2) to F(1). For a quantitative description of fluorescence decrease we used two different models: the binding of the first and the second b-subunit causes the same fluorescence decrease (model 1) or only the binding of the first b-subunit causes fluorescence decrease (model 2). Data evaluation revealed a dissociation constant for the F(1)b(2) complex of 0.6 nM (model 1) or 14 nM (model 2), giving DeltaG(o)= -52 kJ mol(-1) and DeltaG(o)= -45 kJ mol(-1), respectively. The maximal DeltaG observed for ATP synthesis in cells is approximately DeltaG= 55 kJ mol(-1). Therefore, the binding energy of the b-subunit seems to be too low for models in which the free energy for ATP synthesis is accumulated in the elastic strain between rotor and stator subunits and then transduced to the catalytic site in one single step. Models in which energy transduction takes place in at least two steps are favored.

Mitochondrial ATP Synthesis in Permeabilized Cells: Assessment of the ATP/O Valuesin Situ

We have developed a method to assess the actual mitochondrial ATP synthesis in cells (fibroblasts and Ehrlich ascites tumor cells). This method relies on gentle permeabilization of the cells, inhibition of nonmitochondrial ATP synthesis systems (glycolysis and adenylate kinase), and the inhibition of all ATPase activities (ATP hydrolysis) by decreasing the cytosolic magnesium concentration with EDTA. We have simultaneously measured the rate of respiration, and the ATP/O values obtained with this method are similar to those obtained with isolated mitochondria using the same respiratory substrates. This method is highly appropriate for dealing with small amounts of tissue such as human biopsies.

The importance of the creatine kinase reaction

The creatine kinase reaction is traditionally viewed as providing an energy reserve in muscle. However, the physiological importance of this reaction (and the analogous invertebrate reaction catalyzed by arginine kinase) is better understood when viewed as providing metabolic capacitance. This capacitance allows reduction of peak rates of ATP synthesis in cells that alternate between periods of high and low energy consumption. Furthermore, the capacitance allows repayment of energy "debt" that is incurred during periods of high energy demand to occur during periods of low rates of energy consumption. The creatine kinase reaction provides facilitated diffusion of ATP and ADP, which leads to spatial buffering in addition to temporal buffering. Data are presented which suggest that the existence of the creatine kinase reaction allows muscle cells to maintain reduced mitochondrial volume, support larger diameter fibers, and express faster isoforms of myosin. These data lead to speculation that there may be a coupling in the expression of metabolic and contractile proteins.

The significance of hydrogenase activity for the energy metabolism of green algae: anaerobiosis favours ATP synthesis in cells of Chlorella with active hydrogenase

In cells of the green alga Chlorella fusca, which contain active hydrogenase(s), the concentration of ATP, NADH and NADPH were measured during a 5 h period of anaerobiosis in the dark and upon subsequent illumination with high light intensities (770 W/m2), conditions which favour optimal hydrogen photoproduction.