Stoichiometry of chargerin II (A6L) in the H(+)-ATP synthase of rat liver mitochondria

Previous studies suggested that the hydrophobic protein chargerin II, which is encoded in the A6L of mitochondrial DNA, may have a key role in the energy transduction by mitochondrial H(+)-ATP synthase because an antibody against chargerin II inhibited ATP synthesis and ATP-Pi exchange, in an energy-dependent fashion. In the present work, the contents of chargerin II in the H(+)-ATP synthase purified from rat liver mitochondria and in submitochondrial particles were determined by radioimmunoassay. Results showed that the H(+)-ATP synthase contained chargerin II in a molar ratio of one to one. This is the first report on the stoichiometry of the A6L-product in mitochondrial H(+)-ATP synthase.     

Stoichiometry of subunit e in rat liver mitochondrial H(+)-ATP synthase and membrane topology of its putative Ca(2+)-dependent regulatory region

Previous studies have revealed that residues 34-65 of subunit e of mitochondrial H(+)-ATP synthase are homologous with the Ca(2+)-dependent tropomysin-binding region for troponin T and have suggested that subunit e could be involved in the Ca(2+)-dependent regulation of H(+)-ATP synthase activity. In this study, we determined the content of subunit e in H(+)-ATP synthase purified from rat liver mitochondria, and we also investigated the membrane topology of a putative Ca(2+)-dependent regulatory region of subunit e using an antibody against peptide corresponding to residues 34-65 of subunit e. Quantitative immunoblot analysis of subunit e in the purified H(+)-ATP synthase revealed that 1 mol of H(+)-ATP synthase contained 2 mol of subunit e. The ATPase activity of mitoplasts, in which the C-side of F(0) is present on the outer surface of the inner membrane, was significantly stimulated by the addition of the antibody, while the ATPase activity of submitochondrial particles and purified H(+)-ATP synthase was not stimulated. The antibody bound to mitoplasts but not to submitochondrial particles. These results suggest that the putative Ca(2+)-dependent regulatory region of subunit e is exposed on the surface of the C-side of F(0) and that subunit e is involved in the regulation of mitochondrial H(+)-ATP synthase activity probably via its putative Ca(2+)-dependent regulatory region.     

Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells

Extracellular ATP synthesis on human umbilical vein endothelial cells (HUVECs) was examined, and it was found that HUVECs possess high ATP synthesis activity on the cell surface. Extracellular ATP generation was detected within 5 s after addition of ADP and inorganic phosphate and reached a maximal level at 15 s. This type of ATP synthesis was almost completely inhibited by mitochondrial H(+)-ATP synthase inhibitors (e.g., efrapeptins, resveratrol, and piceatannol), which target the F(1) catalytic domain. Oligomycin and carbonyl cyanide m-chlorophenylhydrazone, but not potassium cyanide, also inhibited extracellular ATP synthesis on HUVECs, suggesting that cell surface ATP synthase employs the transmembrane electrochemical potential difference of protons to synthesize ATP as well as mitochondrial H(+)-ATP synthase. The F(1)-targeting H(+)-ATP synthase inhibitors markedly inhibited the proliferation of HUVECs, but intracellular ATP levels in HUVECs treated with these inhibitors were only slightly affected, as shown by comparison with the control cells. Interestingly, piceatannol inhibited only partially the activation of Syk (a nonreceptor tyrosine kinase), which has been shown to play a role in a number of endothelial cell functions, including cell growth and migration. These findings suggest that H(+)-ATP synthase-like molecules on the surface of HUVECs play an important role not only in extracellular ATP synthesis but also in the proliferation of HUVECs. The present results demonstrate that the use of small molecular H(+)-ATP synthase inhibitors targeting the F(1) catalytic domain may lead to significant advances in potential antiangiogenic cancer therapies.     

A simple, rapid method for purification of epsilon-subunit, coupling factor 6, subunit d, and subunit e from rat liver H(+)-ATP synthase and determination of the complete amino acid sequence of epsilon-subunit.

The rat liver mitochondrial epsilon-subunit, coupling factor 6, subunit d, and subunit e of H(+)-ATP synthase, which are all extra subunits with no counterparts in Escherichia coli, were purified by reverse-phase high performance liquid chromatography. The complete amino acid sequence of the rat epsilon-subunit was determined by automated Edman degradation of the whole protein and derived peptides. The protein contains 50 amino acids and has a molecular mass of 5635 kDa. It is a basic hydrophilic protein with an isoelectric point of 10.5. The sequence of the rat epsilon-subunit is highly homologous with that of the epsilon-subunit of bovine heart and slightly similar to those of the epsilon-subunit of the yeast and sweet potato mitochondria. However, it has no homology with any subunit of bacterial or chloroplast H(+)-ATP synthase.     

Complete amino acid sequence of subunit e of rat liver mitochondrial H(+)-ATP synthase.

Subunit e of H(+)-ATP synthase from rat liver mitochondria was isolated from the purified enzyme by reverse-phase high-performance liquid chromatography. The amino acid sequence of the subunit was determined by automated Edman degradation of the whole protein and derived peptides. The nucleotide sequence of the import precursor of subunit e of rat liver H(+)-ATP synthase was determined from a recombinant cDNA clone isolated by screening a rat hepatoma cell line H4TG cDNA library with a probe DNA. The sequence was composed of 289 nucleotides including a coding region for the import precursor of subunit e and noncoding regions on the 5'- and 3'-sides. The possible import precursor of subunit e and its mature polypeptide deduced from the open reading frame consisted of 71 and 70 amino acid residues with molecular weights of 8254 and 8123, respectively. Subunit e is a basic hydrophilic protein with an isoelectric point of 9.78. The sequence of the rat subunit e is highly homologous with that of subunit e of bovine heart, but has no homology with any subunit of bacterial or chloroplast H(+)-ATP synthase. The function of subunit e is unknown. However, a homology search in the database of the National Biomedical Research Foundation revealed that residues 34-65 of subunit e are homologous with residues 90-117 of troponin T, and with residues 529-561 of h-caldesmon and residues 289-319 of l-caldesmon, which are the homologous sequences corresponding to the Ca(2+)-dependent tropomyosin-binding region of troponin T.     

Structural and functional characterization of F(o)F(1)-ATP synthase on the extracellular surface of rat hepatocytes

Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-F(o)F(1)-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F(1) (alpha/beta and gamma) and F(o) (F(o)I-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF(1) is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H(+) and are inhibited by F(1) and F(o)-targeting inhibitors. We hypothesise that ecto-F(o)F(1)-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.     

Role of F0 and F1 subunits in the gating and coupling function of mitochondrial H(+)-ATP synthase. The effect of dithiol reagents.

A study is presented on the role of F0 and F1 subunits in oligomycin-sensitive H+ conduction and energy transfer reactions of bovine heart mitochondrial F0F1 H(+)-ATP synthase. Mild treatment with azodicarboxylic acid bis(dimethylamide) (diamide) enhanced oligomycin-sensitive H+ conduction in submitochondrial particles containing F1 attached to F0. This effect was associated with stimulation of the ATPase activity, with no effect on its inhibition by oligomycin, and depression of the 32Pi-ATP exchange. The stimulatory effect of diamide on H+ conduction decreased in particles from which F1 subunits were partially removed by urea. The stimulatory effect exerted by diamide in the submitochondrial particles with F1 attached to F0 was directly correlated with a decrease of the original electrophoretic bands of a subunit of F0 (F0I-PVP protein) and the gamma subunit of F1, with corresponding formation of their cross-linking product. In F0 liposomes, devoid of gamma subunit, diamide failed to stimulate H+ conduction and to cause disappearance of F0I-PVP protein, unless purified gamma subunit was added back. The addition to F0 liposomes of gamma subunit, but not that of alpha and beta subunits, caused per se inhibition of H+ conduction. It is concluded that F0I-PVP and gamma subunits are directly involved in the gate of the F0F1 H(+)-ATP synthase. Data are also presented indicating contribution to the gate of oligomycin-sensitivity conferral protein and of another protein subunit of F0, F6.     

Halotolerant cyanobacterium Aphanothece halophytica contains an Na+-dependent F1F0-ATP synthase with a potential role in salt-stress tolerance

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium that can grow in media of up to 3.0 m NaCl and pH 11. Here, we show that in addition to a typical H(+)-ATP synthase, Aphanothece halophytica contains a putative F(1)F(0)-type Na(+)-ATP synthase (ApNa(+)-ATPase) operon (ApNa(+)-atp). The operon consists of nine genes organized in the order of putative subunits β, ε, I, hypothetical protein, a, c, b, α, and γ. Homologous operons could also be found in some cyanobacteria such as Synechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017. The ApNa(+)-atp operon was isolated from the A. halophytica genome and transferred into an Escherichia coli mutant DK8 (Δatp) deficient in ATP synthase. The inverted membrane vesicles of E. coli DK8 expressing ApNa(+)-ATPase exhibited Na(+)-dependent ATP hydrolysis activity, which was inhibited by monensin and tributyltin chloride, but not by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The Na(+) ion protected the inhibition of ApNa(+)-ATPase by N,N'-dicyclohexylcarbodiimide. The ATP synthesis activity was also observed using the Na(+)-loaded inverted membrane vesicles. Expression of the ApNa(+)-atp operon in the heterologous cyanobacterium Synechococcus sp. PCC 7942 showed its localization in the cytoplasmic membrane fractions and increased tolerance to salt stress. These results indicate that A. halophytica has additional Na(+)-dependent F(1)F(0)-ATPase in the cytoplasmic membrane playing a potential role in salt-stress tolerance.     

Fatty Acid 2-Hydroxylase Mediates Diffusional Mobility of Raft-associated Lipids,GLUT4 Level,and Lipogenesis in 3T3-L1 Adipocytes

Straight chain fatty acid α-oxidation increases during differentiation of 3T3-L1 adipocytes, leading to a marked accumulation of odd chain length fatty acyl moieties. Potential roles of this pathway in adipocyte differentiation and lipogenesis are unknown. Mammalian fatty acid 2-hydroxylase (FA2H) was recently identified and suggested to catalyze the initial step of straight chain fatty acid α-oxidation. Accordingly, we examined whether FA2H modulates adipocyte differentiation and lipogenesis in mature adipocytes. FA2H level markedly increases during differentiation of 3T3-L1 adipocytes, and small interfering RNAs against FA2H inhibit the differentiation process. In mature adipocytes, depletion of FA2H inhibits basal and insulin-stimulated glucose uptake and lipogenesis, which are partially rescued by the enzymatic product of FA2H, 2-hydroxy palmitic acid. Expression of fatty-acid synthase and SCD1 was decreased in FA2H-depleted cells, and levels of GLUT4 and insulin receptor proteins were reduced. 2-Hydroxy fatty acids are enriched in cellular sphingolipids, which are components of membrane rafts. Accelerated diffusional mobility of raft-associated lipids was shown to enhance degradation of GLUT4 and insulin receptor in adipocytes. Consistent with this, depletion of FA2H appeared to increase raft lipid mobility as it significantly accelerated the rates of fluorescence recovery after photobleaching measurements of lipid rafts labeled with Alexa 488-conjugated cholera toxin subunit B. Moreover, the enhanced recovery rates were partially reversed by treatment with 2-hydroxy palmitic acid. In conclusion, our findings document the novel role of FA2H in adipocyte lipogenesis possibly by modulation of raft fluidity and level of GLUT4.     

The stoichiometry of the chloroplast ATP synthase oligomer III in Chlamydomonas reinhardtii is not affected by the metabolic state

The chloroplast H(+)-ATP synthase is a key component for the energy supply of higher plants and green algae. An oligomer of identical protein subunits III is responsible for the conversion of an electrochemical proton gradient into rotational motion. It is highly controversial if the oligomer III stoichiometry is affected by the metabolic state of any organism. Here, the intact oligomer III of the ATP synthase from Chlamydomonas reinhardtii has been isolated for the first time. Due to the importance of the subunit III stoichiometry for energy conversion, a gradient gel system was established to distinguish oligomers with different stoichiometries. With this methodology, a possible alterability of the stoichiometry in respect to the metabolic state of the cells was examined. Several growth parameters, i.e., light intensity, pH value, carbon source, and CO(2) concentration, were varied to determine their effects on the stoichiometry. Contrary to previous suggestions for E. coli, the oligomer III of the chloroplast H(+)-ATP synthase always consists of a constant number of monomers over a wide range of metabolic states. Furthermore, mass spectrometry indicates that subunit III from C. reinhardtii is not modified posttranslationally. Data suggest a subunit III stoichiometry of the algae ATP synthase divergent from higher plants.     

Plant 14-3-3 proteins assist ion channels and pumps

Turgor pressure is a cellular parameter, important for a range of physiological processes in plants, like cell elongation, gas exchange and gravitropic/phototropic bending. Regulation of turgor pressure involves ion and water transport at the expense of metabolic energy (ATP). The primary pump in the plasma membrane (the H(+)-ATPase) is a key player in turgor regulation since it provides the driving force for ion uptake, followed by water influx through osmosis. Using the phytotoxin fusicoccin (a well-known activator of the ATPase) as a tool, 14-3-3 proteins were identified as regulators of the H(+)-ATPase. Since fusicoccin has a dramatic effect on K(+) accumulation and cellular respiration as well, we studied whether 14-3-3 proteins play a role in the regulation of the mitochondrial F(0)F(1)-ATP synthase and ion channels in the vacuolar and plasma membranes. Besides the plasma membrane H(+)-ATPase, we have identified thus far at least four other transport proteins that are regulated by 14-3-3 proteins. The mechanism of regulation will be described and the possibility that 14-3-3 proteins act as coordinators of ion transporters with varied but interdependent functions will be discussed.     

Adrenergic regulation of adipocyte metabolism

Adipocytes can be readily isolated from intact adipose tissue. In adipocytes from hamster and human white adipose tissue it is possible to demonstrate beta, alpha 1, and alpha 2 adrenoceptors. Alpha 2 adrenoceptor activation inhibits while beta adrenoceptor activation stimulates cyclic AMP accumulation and lipolysis. The effects of catecholamines on cyclic AMP accumulation are mediated through regulation of adenylate cyclase activity, which is activated through beta adrenoceptors and inhibited through alpha 2 adrenoceptors. Activation of alpha 1 adrenergic receptors has been shown to be associated with elevations of cytosol calcium and increased turnover of phosphatidylinositol. In white adipocytes, the only known alpha 1 adrenergic effects are inhibition of glycogen synthase and stimulation of glycogen phosphorylase via mechanisms distinct from those by which cyclic AMP produces similar end effects. In brown adipocytes, alpha 1 adrenoceptor activation stimulates respiration. Thyroid hormones primarily regulate the sensitivity of adipocytes to beta-adrenergic amines while having little effect on alpha adrenoceptor sensitivity.     

Regulation of hepatic glycogen phosphorylase and glycogen synthase by calcium and diacylglycerol

Incubation of rat hepatocytes with angiotensin II (1 nM) produced a time-dependent accumulation of 1, 2-diacylglycerol and inactivation of glycogen synthase with maximum effects at 10 min. The level of diacylglycerol then gradually declined and the activity of glycogen synthase I returned to control values at 30 min. In contrast, angiotensin II caused an increase in cytosolic Ca2+ and an activation of glycogen phosphorylase which were rapid and transient, reaching maximum values in less than 2 min and then returning to control levels at 15 min. There were excellent correlations between the changes in glycogen synthase I and diacylglycerol levels and between the changes in phosphorylase alpha and cytosolic Ca2+ in these time-course studies. However, there was no correlation between the changes in diacylglycerol and phosphorylase alpha or between the changes in cytosolic Ca2+ and glycogen synthase I. Norepinephrine also caused a slow increase in diacylglycerol and inactivation of glycogen synthase, and a rapid increase in cytosolic free Ca2+ and activation of glycogen phosphorylase. Addition of an alpha1-adrenergic blocker (prazosin or phentolamine) caused rapid decreases in cytosolic free Ca2+ and phosphorylase alpha, but only slowly reversed the inactivation of synthase and accumulation of diacylglycerol. The dose-response curves for norepinephrine and prazosin on glycogen synthase were well correlated with those on diacylglycerol. It is proposed that in liver cells, Ca2+-mobilizing hormones regulate phosphorylase a through a Ca2+-dependent mechanism and inactivate glycogen synthase through the generation of diacylglycerol, at least in part. The data provide additional support for the view that protein kinase C may be important in the regulation of glycogen synthase in liver.     

Importance for absorption of Na+ from freshwater of lysine, valine and serine substitutions in the alpha1a-isoform of Na,K-ATPase in the gills of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

In the gills of rainbow trout and Atlantic salmon, the alpha1a- and alpha1b-isoforms of Na,K-ATPase are expressed reciprocally during salt acclimation. The alpha1a-isoform is important for Na(+) uptake in freshwater, but the molecular basis for the functional differences between the two isoforms is not known. Here, three amino acid substitutions are identified in transmembrane segment 5 (TM5), TM8 and TM9 of the alpha1a-isoform compared to the alpha1b-isoform, and the functional consequences are examined by mutagenesis and molecular modeling on the crystal structures of Ca-ATPase or porcine kidney Na,K-ATPase. In TM5 of the alpha1a-isoform, a lysine substitution, Asn783 --> Lys, inserts the epsilon-amino group in cation site 1 in the E(1) form to reduce the Na(+)/ATP ratio. In the E(2) form the epsilon-amino group approaches cation site 2 to force ejection of Na(+) to the blood phase and to interfere with binding of K(+). In TM8, a Asp933 --> Val substitution further reduces K(+) binding, while a Glu961 --> Ser substitution in TM9 can prevent interaction of FXYD peptides with TM9 and alter Na(+) or K(+) affinities. Together, the three substitutions in the alpha1a-isoform of Na,K-ATPase act to promote binding of Na(+) over K(+) from the cytoplasm, to reduce the Na(+)/ATP ratio and the work done in one Na,K pump cycle of active Na(+) transport against the steep gradient from freshwater (10-100 microM: Na(+)) to blood (160 mM: Na(+)) and to inhibit binding of K(+) to allow Na(+)/H(+) rather than Na(+)/K(+) exchange.     

Delta mu Na+ drives the synthesis of ATP via an delta mu Na(+)-translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1.

Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.     

Impaired lipid accumulation by trans10, cis12 CLA during adipocyte differentiation is dependent on timing and length of treatment

Conjugated linoleic acids (CLAs) are a group of polyunsaturated fatty acids found in ruminant products, where the predominant isomers are cis9, trans11 (c9,t11) and trans10, cis12 (t10,c12) CLA. We have previously shown that t10,c12 CLA prevents lipid accumulation in mature adipocytes in part by acting as a peroxisome proliferator-activated receptor gamma (PPAR gamma) modulator. The objective of this study was to further establish the molecular mechanisms underlying the attenuating effect on lipid accumulation by t10,c12 CLA, with focus on time point and duration of treatment during adipogenesis. We have shown that t10,c12 CLA treatment has its most attenuating effect early (day (D) 0-6) during differentiation. Treatment during this period is sufficient to prevent lipid accumulation in mature adipocytes. The adipogenic marker genes PPAR gamma and CCAAT/enhancer binding protein alpha (C/EBP alpha) are both down-regulated after treatment within the period from D0-6, while additional treatment also down-regulates the expression of sterol regulatory element binding protein-1c (SREBP-1c), liver X receptor alpha (LXR alpha), fatty acid binding protein (aP2), fatty acid translocase (CD36) and insulin-sensitive glucose transporter 4 (GLUT4). These effects of t10,c12 CLA reflect the subsequent attenuation of lipid accumulation observed in mature adipocytes. Interestingly, the early B-cell factor (O/E-1), which is known to promote adipogenesis and to be involved in control of genes important for terminal adipocyte differentiation, is unaffected by treatment of t10,c12 CLA. Taken together, our data indicate that inhibition of lipid accumulation induced by t10,c12 CLA treatment during adipocyte differentiation is associated with a tight regulatory cross-talk between early (PPAR gamma and C/EBP alpha) and late (LXR alpha, aP2 and CD36) adipogenic marker genes.     

Purification and biochemical characterization of the F1Fo-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1

We describe here purification and biochemical characterization of the F(1)F(o)-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F(1)F(o)-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F(1) subunits alpha, beta, gamma, delta, and epsilon and F(o) subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F(1)F(o) holoenzyme or the isolated F(1) moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F(1). After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Deltapsi). A transmembrane proton gradient or sodium ion gradient in the absence of Deltapsi was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na(+)/H(+) antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na(+) ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.     

Thymidylate synthase inhibition in cells with arrested DNA synthesis is not due to an allosteric interaction in the replitase complex

Activity of thymidylate synthase was measured in situ in leukemia cells by tritium release from [5-3H]dUrd. Aphidicolin, an inhibitor of DNA polymerase alpha, but not thymidylate synthase, caused a time dependent inhibition of the enzyme when added to the cells after [5-3H]dUrd. Cells treated with hydroxyurea and aphidicolin in sequence before addition of [5-3H]dUrd had a high initial thymidylate synthase activity that decreased with time. This pattern indicates that thymidylate synthase activity is linked to DNA synthesis; however, its inhibition by drugs that inhibit DNA synthesis may be due to accumulation of thymidine nucleotide(s), rather than to an allosteric interaction in the replitase complex.     

Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis

Mitochondrial F₁F_o-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F₁F_o-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F₁F_o-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F₁F_o-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F₁ catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F₁ to the lipid monolayer and the F_o membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F_o by electron microscopy and AFM.