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Vacuolar H+-ATPases (V-ATPases; V1Vo-ATPases) are rotary-motor proton pumps that acidify intracellular compartments and, in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo. H is required for MgATPase activity in holo-VATPase but also for stabilizing the MgADP-inhibited state in membrane-detached V1. However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full-length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunitH (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full-length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysis- driven conformational changes in V1 destabilized the interaction of H or HNT with V1ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation thatHbinding is affected by MgATP hydrolysis in V1 points to H's role in the mechanism of reversible disassembly. [ABSTRACT FROM AUTHOR]

Determination of cellular ATP levels, a key indicator of metabolic status, is essential for the quantitative analysis of metabolism. The biciliate green alga Chlamydomonas reinhardtii is an excellent experimental organism to study ATP production pathways, including photosynthesis and respiration, particularly because it can be cultured either photoautotrophically or heterotrophically. Additionally, its cellular ATP concentration, [ATP], is reflected in the beating of its cilia. However, the methods currently used for quantifying the cellular ATP levels are time consuming or invasive. In this study, we established a rapid method for estimating cytosolic [ATP] from the ciliary beating frequency in C. reinhardtii. Using an improved method of motility reactivation in demembranated cell models, we obtained calibration curves for [ATP]-ciliary beating frequency over a physiological range of ATP concentrations. These curves allowed rapid estimation of the cytosolic [ATP] in live wild-type cells to be ∼2.0 mM in the light and ∼1.5 mM in the dark: values comparable to those obtained by other methods. Furthermore, we used this method to assess the effects of genetic mutations or inhibitors of photosynthesis or respiration quantitatively and noninvasively. This sensor-free method is a convenient tool for quickly estimating cytosolic [ATP] and studying the mechanism of ATP production in C. reinhardtii or other ciliated organisms., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F(0)F(1) ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.

The rsmA gene of Streptomyces coelicolor lies directly upstream of the gene encoding the group 3 sigma factor sigma(M). The RsmA protein is a putative member of the HATPase_c family of anti-sigma factors but is unusual in that it contains seven cysteine residues. Bacterial two-hybrid studies demonstrate that it interacts specifically with sigma(M), and in vitro studies of the purified proteins by native PAGE and transcription assays confirmed that they form a complex. Characterization of RsmA revealed that it binds ATP and that, as isolated, it contains significant quantities of iron and inorganic sulfide, in equal proportion, with spectroscopic properties characteristic of a [2Fe-2S] cluster-containing protein. Importantly, the interaction between RsmA and sigma(M) is dependent on the presence of the iron-sulfur cluster. We propose a model in which RsmA regulates the activity of sigma(M). Loss of the cluster, in response to an as yet unidentified signal, activates sigma(M) by abolishing its interaction with the anti-sigma factor. This represents a major extension of the functional diversity of iron-sulfur cluster proteins.

We demonstrated previously in a liver perfusion system that agmatine increases oxygen consumption as well as the synthesis of N-acetylglutamate and urea by an undefined mechanism. In this study our aim was to identify the mechanism(s) by which agmatine up-regulates ureagenesis. We hypothesized that increased oxygen consumption and N-acetylglutamate and urea synthesis are coupled to agmatine-induced stimulation of mitochondrial fatty acid oxidation. We used 13C-labeled fatty acid as a tracer in either a liver perfusion system or isolated mitochondria to monitor fatty acid oxidation and the incorporation of 13C-labeled acetyl-CoA into ketone bodies, tricarboxylic acid cycle intermediates, amino acids, and N-acetylglutamate. With [U-13C16] palmitate in the perfusate, agmatine significantly increased the output of 13C-labeled beta-hydroxybutyrate, acetoacetate, and CO2, indicating stimulated fatty acid oxidation. The stimulation of [U-13C16]palmitate oxidation was accompanied by greater production of urea and a higher 13C enrichment in glutamate, N-acetylglutamate, and aspartate. These observations suggest that agmatine leads to increased incorporation and flux of 13C-labeled acetyl-CoA in the tricarboxylic acid cycle and to increased utilization of 13C-labeled acetyl-CoA for synthesis of N-acetylglutamate. Experiments with isolated mitochondria and 13C-labeled octanoic acid also demonstrated that agmatine increased synthesis of 13C-labeled beta-hydroxybutyrate, acetoacetate, and N-acetylglutamate. The current data document that agmatine stimulates mitochondrial beta-oxidation and suggest a coupling between the stimulation of hepatic beta-oxidation and up-regulation of ureagenesis. This action of agmatine may be mediated via a second messenger such as cAMP, and the effects on ureagenesis and fatty acid oxidation may occur simultaneously and/or independently.

Long-chain fatty acids (FAs) are the predominant energy substrate utilized by the adult heart. The heart can utilize unesterified FA bound to albumin or FA obtained from lipolysis of lipoprotein-bound triglyceride (TG). We used heart-specific lipoprotein lipase knock-out mice (hLpL0) to test whether these two sources of FA are interchangeable and necessary for optimal heart function. Hearts unable to obtain FA from lipoprotein TG were able to compensate by increasing glucose uptake, glycolysis, and glucose oxidation. HLpL0 hearts had decreased expression of pyruvate dehydrogenase kinase 4 and increased cardiomyocyte expression of glucose transporter 4. Conversely, FA oxidation rates were reduced in isolated perfused hLpL0 hearts. Following abdominal aortic constriction expression levels of genes regulating FA and glucose metabolism were acutely up-regulated in control and hLpL0 mice, yet all hLpL0 mice died within 48 h of abdominal aortic constriction. Older hLpL0 mice developed cardiac dysfunction characterized by decreased fractional shortening and interstitial and perivascular fibrosis. HLpL0 hearts had increased expression of several genes associated with transforming growth factor-beta signaling. Thus, long term reduction of lipoprotein FA uptake is associated with impaired cardiac function despite a compensatory increase in glucose utilization.

Acyl chain-labeled NBD-phosphatidylcholine (NBD-PC) has been used to identify three gene products (Lem3p, Dnf1p, and Dnf2p) that are required for normal levels of inward-directed phospholipid transport (flip) across the plasma membrane of yeast. Although the head group structure of acyl chain-labeled NBD phospholipids has been shown to influence the mechanism of flip across the plasma membrane, the extent to which the acyl chain region and the associated fluorophore affect flip has not been assessed. Given the identification of these proteins required for NBD-PC flip, it is now possible to determine whether the fluorophore attached to a phospholipid acyl chain influences the mechanism of flip. Thus, flip of phosphatidylcholine molecules with three different Bodipy fluorophores (Bodipy FL, Bodipy 530, and Bodipy 581) was tested and compared with that of NBD-PC in strains carrying deletions in LEM3, DNF1, and DNF2. Deletion of these genes significantly reduced the flip of NBD-PC and Bodipy FL-PC but had no effect on that of Bodipy 581-PC and Bodipy 530-PC. These data, in combination with comparisons of the effect of ATP depletion, collapse of the proton electrochemical gradient across the plasma membrane, and culture density led to the conclusion that at least three different flip pathways exist in yeast that are selective for the structure of the fluorophore attached to the acyl chain of phosphatidylcholine molecules.

Niemann-Pick type C1 (NPC1) disease is an autosomal recessive, fatal disorder characterized by a defect in cholesterol trafficking and progressive neurodegeneration. The disease is predominantly caused by mutations in the NPC1 gene; however, it has been assumed that heterozygous NPC1 mutations do not cause any symptoms. Here we demonstrate that cholesterol accumulation does not occur in young mouse brains; however, it does in aged (104-106-week-old) NPC1+/- mouse brains. In addition, Purkinje cell loss was observed in aged NPC1+/- mouse cerebellums. Immunoblot analysis using anti-phospho-tau antibodies (AT-8, AT-100, AT-180, AT-270, PHF-1, and SMI-31) demonstrates the site-specific phosphorylation of tau at Ser-199, Ser-202, Ser-212, and Thr-214 in the brains of aged NPC1+/- mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases, including glycogen synthase kinase 3beta, cyclin-dependent kinase 5, or stress-activated protein kinase/c-Jun N-terminal kinase were not activated. Cholesterol level in the lipid raft isolated from the cerebral cortices, ATP level, and ATP synthase activity in the cerebral cortices significantly decreased in the aged NPC1+/- brains compared with those in the NPC1+/+ brains. All of these changes observed in NPC1+/- brains were determined to be associated with aging and were not observed in the age-matched NPC1+/+ brains. These results clearly demonstrate that heterozygous NPC1 impairs neuronal functions and causes neurodegeneration in aged mouse brains, suggesting that human heterozygous NPC1 mutations may be a risk factor for neurodegenerative disorders, such as tauopathy, in the aged population.

Our goal was to investigate whether leucine culture affects beta-cell glucose sensing. One-day culture of rat islets with 10 mM leucine had no effect on glucose-induced insulin secretion. One-week leucine culture decreased the threshold for glucose-induced insulin secretion and increased maximal insulin secretion at 30 mM glucose. Glucose-induced cytosolic free Ca(2+) was increased at 1 week but not at 1 day of leucine culture. Without glucose, ATP content was not different with or without leucine culture for 1 week. With 20 mM glucose, ATP content was higher by 1.5-fold in islets cultured for 1 week with leucine than those without leucine. Microarray experiments indicated that culture of RINm5F cells with leucine increased expression of ATP synthase beta subunit 3.2-fold, which was confirmed by real time reverse transcription-PCR analysis (3.0- +/- 0.4-fold) in rat islets at 1 week but not after 1 day with leucine culture. Down-regulation of ATP synthase beta subunit by siRNA decreased INS1 cell ATP content and insulin secretion with 20 mM glucose. Overexpression of ATP synthase beta subunit in INS1 cell increased insulin secretion in the presence of 5 and 20 mM glucose. In conclusion, one-week leucine culture of rat islets up-regulated ATP synthase and increased ATP content, which resulted in elevated [Ca(2+)] levels and more insulin exocytosis by glucose. Depletion of ATP synthase beta subunit with siRNA produced opposite effects. These data reveal the fuel-sensing role of mitochondrial ATP synthase in the control of ATP production from glucose and the control of glucose-induced insulin secretion.

Previous studies showed that adenosine triphosphate (ATP) concentrations in Escherichia coli changed during certain growth transitions and directly controlled the rate of rRNA transcription initiation at those times. The relationship between ATP concentration and rRNA transcription during steady-state growth is less clear, however. This is because two commonly employed methods for measuring ATP concentrations in bacteria, both of which rely on physical extraction followed by chromatographic separation of small molecules, resulted in dramatically different conclusions about whether ATP concentration changed with steady-state growth rate. Extraction with formic acid indicated that ATP concentration did not change with growth rate, whereas formaldehyde treatment followed by extraction with alkali indicated that ATP concentration increased proportionally to the growth rate. To resolve this discrepancy, we developed a bioassay for ATP based on the expression of a variant of the firefly luciferase enzyme in vivo and measurement of luminescence in cells growing in different conditions. We found that the available ATP concentration did not vary with growth rate, either in wild-type cells or in cells lacking guanosine 5'-diphosphate, 3'-diphosphate, providing insight into the regulation of rRNA transcription. More broadly, the luciferase bioassay described here provides a general method for evaluating the ATP concentration available for biochemical processes in E. coli and potentially in other organisms.

ATP is released from astrocytes and is involved in the propagation of calcium waves among them. Neuronal ATP secretion is quantal and calcium-dependent, but it has been suggested that ATP release from astrocytes may not be vesicular. Here we report that, besides the described basal ATP release facilitated by exposure to calcium-free medium, astrocytes release purine under conditions of elevated calcium. The evoked release was not affected by the gap-junction blockers anandamide and flufenamic acid, thus excluding purine efflux through connexin hemichannels. Sucrose-gradient analysis revealed that a fraction of ATP is stored in secretory granules, where it is accumulated down an electrochemical proton gradient sensitive to the v-ATPase inhibitor bafilomycin A(1). ATP release was partially sensitive to tetanus neurotoxin, whereas glutamate release from the same intoxicated astrocytes was almost completely impaired. Finally, the activation of metabotropic glutamate receptors, which strongly evokes glutamate release, was only slightly effective in promoting purine secretion. These data indicate that astrocytes concentrate ATP in granules and may release it via a regulated secretion pathway. They also suggest that ATP-storing vesicles may be distinct from glutamate-containing vesicles, thus opening up the possibility that their exocytosis is regulated differently.

A mev-1(kn1) mutant of the nematode Caenorhabditis elegans is defective in the cytochrome b large subunit (Cyt-1/ceSDHC) in complex II of the mitochondrial electron transport chain. We have previously shown that a mutation in mev-1 causes shortened life span and rapid accumulation of aging markers such as fluorescent materials and protein carbonyls in an oxygen-dependent fashion. However, it remains unclear as to whether this hypersensitivity is caused by direct toxicity of the exogenous oxygen or by the damage of endogenous reactive oxygen species derived from mitochondria. Here we report important biochemical changes in mev-1 animals that serve to explain their abnormalities under normoxic conditions: (i) an overproduction of superoxide anion from mitochondria; and (ii) a reciprocal reduction in glutathione content even under atmospheric oxygen. In addition, unlike wild type, the levels of superoxide anion production from mev-1 mitochondria were significantly elevated under hyperoxia. Under normal circumstances, it is well known that superoxide anion is produced at complexes I and III in the electron transport system. Our data suggest that the mev-1(kn1) mutation increases superoxide anion production at complex II itself rather than at complexes I and III. The mev-1 mutant also had a lactate level 2-fold higher than wild type, indicative of lactic acidosis, a hallmark of human mitochondrial diseases. These data indicate that Cyt-1/ceSDHC plays an important role not only in energy metabolism but also in superoxide anion production that is critically involved in sensitivity to atmospheric oxygen.

Rapid exchange of high energy carrying molecules between intracellular compartments is essential in sustaining cellular energetic homeostasis. Adenylate kinase (AK)-catalyzed transfer of adenine nucleotide beta- and gamma-phosphoryls has been implicated in intracellular energy communication and nucleotide metabolism. To demonstrate the significance of this reaction in cardiac energetics, phosphotransfer dynamics were determined by [(18)O]phosphoryl oxygen analysis using( 31)P NMR and mass spectrometry. In hearts with a null mutation of the AK1 gene, which encodes the major AK isoform, total AK activity and beta-phosphoryl transfer was reduced by 94% and 36%, respectively. This was associated with up-regulation of phosphoryl flux through remaining minor AK isoforms and the glycolytic phosphotransfer enzyme, 3-phosphoglycerate kinase. In the absence of metabolic stress, deletion of AK1 did not translate into gross abnormalities in nucleotide levels, gamma-ATP turnover rate or creatine kinase-catalyzed phosphotransfer. However, under hypoxia AK1-deficient hearts, compared with the wild type, had a blunted AK-catalyzed phosphotransfer response, lowered intracellular ATP levels, increased P(i)/ATP ratio, and suppressed generation of adenosine. Thus, although lack of AK1 phosphotransfer can be compensated in the absence of metabolic challenge, under hypoxia AK1-knockout hearts display compromised energetics and impaired cardioprotective signaling. This study, therefore, provides first direct evidence that AK1 is essential in maintaining myocardial energetic homeostasis, in particular under metabolic stress.

To investigate residues involved in the formation of the noncatalytic nucleotide binding sites of the vacuolar proton-translocating adenosine triphosphatase (V-ATPase), cysteine scanning mutagenesis of the VMA2 gene that encodes the B subunit in yeast was performed. Replacement of the single endogenous cysteine residue at position 188 gave rise to a Cys-less form of the B subunit (Vma2p) which had near wild-type levels of activity and which was used in the construction of 16 single cysteine-containing mutants. The ability of adenine nucleotides to prevent reaction of the introduced cysteine residues with the sulfhydryl reagent 3-(N-maleimidopropionyl)biocytin (biotin-maleimide) was evaluated by Western blot. Biotin-maleimide labeling of the purified V-ATPase from the wild-type and the mutants S152C, L178C, N181C, A184C, and T279C was reduced after reaction with the nucleotide analog 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (BzATP). These results suggest the proximity of these residues to the nucleotide binding site on the B subunit. In addition, we have examined the level of endogenous nucleotide bound to the wild-type V-ATPase and to a mutant (the A subunit mutant R483Q) which is postulated to be altered at the noncatalytic site and which displays a marked nonlinearity in ATP hydrolysis (MacLeod, K. J., Vasilyeva, E., Baleja, J. D., and Forgac, M. (1998) J. Biol. Chem. 273, 150-156). The R483Q mutant contained 2.6 mol of ATP/mol of V-ATPase compared with the wild-type enzyme, which contained 0.8 mol of ATP/mol of V-ATPase. These results suggest that binding of additional ATP to the noncatalytic sites may modulate the catalytic activity of the enzyme.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a key enzyme in the protection of biomembranes exposed to oxidative stress. We investigated the role of mitochondrial PHGPx in apoptosis using RBL2H3 cells that overexpressed mitochondrial PHGPx (M15 cells), cells that overexpressed non-mitochondrial PHGPx (L9 cells), and control cells (S1 cells). The morphological changes and fragmentation of DNA associated with apoptosis occurred within 15 h in S1 and L9 cells upon exposure of cells to 2-deoxyglucose (2DG). The release of cytochrome c from mitochondria was observed in S1 cells after 4 h and was followed by the activation of caspase-3 within 6 h. Overexpression of mitochondrial PHGPx prevented the release of cytochrome c, the activation of caspase-3, and apoptosis, but non-mitochondrial PHGPx lacked the ability to prevent the induction of apoptosis by 2DG. An ability to protect cells from 2DG-induced apoptosis was abolished when the PHGPx activity of M15 cells was inhibited by diethylmalate, indicating that the resistance of M15 cells to apoptosis was indeed due to the overexpression of PHGPx in the mitochondria. The expression of members of the Bcl-2 family of proteins, such as Bcl-2, Bcl-xL, Bax, and Bad, was unchanged by the overexpression of PHGPx in cells. The levels of hydroperoxides, including hydrogen and lipid peroxide, in mitochondria isolated from S1 and L9 cells were significantly increased after the exposure to 2DG for 2 h, while the level of hydroperoxide in mitochondria isolated from M15 cells was lower than that in S1 and L9 cells. M15 cells were also resistant to apoptosis induced by etoposide, staurosporine, UV irradiation, cycloheximide, and actinomycin D, but not to apoptosis induced by Fas-specific antibodies, which induces apoptosis via a pathway distinct from the pathway initiated by 2DG. Our results suggest that hydroperoxide, produced in mitochondria, is a major factor in apoptosis and that mitochondrial PHGPx might play a critical role as an anti-apoptotic agent in mitochondrial death pathways.

We have shown that attachment to a fibronectin substrate stimulates two pathways of lipid biosynthesis in cultured human fibroblasts. Detachment of these cells (mechanically, with trypsin, or by RGDS peptides) caused a significant decrease in their 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and in their incorporation of [3H]acetate into fatty acids. This inhibition was substantially reversed by the reattachment of cells to fibronectin substrates, but not to poly-L-lysine substrates or to fibronectin in solution. Inhibiting phosphoprotein phosphatase activity with okadaic acid blocked the recovery of both biosynthetic activities. Both 3-hydroxy-3-methylglutaryl-coenzyme A reductase and fatty acid biosynthesis are known to be inhibited by the action of 5'-AMP-activated protein kinase, which is activated by an increase in the level of AMP relative to ATP. For example, in our system, sodium azide and 2-deoxy-D-glucose increased the ratio of cellular AMP to ATP and caused a decrease in lipid biosynthesis. We then verified the prediction that detachment of cells from substrates also caused an increase in the AMP/ATP ratio. We therefore conclude that the attachment of cells to fibronectin promotes lipid biosynthesis, presumably in coordination with the cellular growth response evoked by attachment to the extracellular matrix.

In a previous study, we showed that the molecular chaperone HSC70 self-associates in solution in a reversible and likely unlimited fashion. Here, we examine the influence of nucleotides, nucleotide analogs, peptides, and unfolded proteins on the self-association properties of this protein. Whereas in the presence of ADP, HSC70 exists as a slow, concentration- and temperature-dependent monomer-oligomer equilibrium, in the presence of ATP, the protein is essentially monomeric, indicating that ATP shifts this equilibrium toward the monomer by stabilizing the monomer. Dissociation of oligomers into monomers is also obtained with the slowly hydrolyzable ATP analogs, adenosine 5'-O-(thiotriphosphate) and 5'-adenylyl-beta,gamma-imidodiphosphate, or the complex between ADP and the phosphate analog, BeF3, indicating that binding but not hydrolysis of ATP is necessary and sufficient for the stabilization of HSC70 monomer. Furthermore, binding of short peptides or permanently unfolded proteins to the peptide binding site of HSC70 promotes the dissociation of oligomers into monomers, suggesting that protein substrates are able to compete with HSC70 for the same binding site. Because the release of peptides or unfolded proteins from HSC70 has also been shown to require ATP binding, these results indicate that dissociation of oligomers is controlled by a mechanism similar to that of release of protein substrates and suggest that binding of HSC70 to itself occurs via the peptide binding site and mimics binding of HSC70 to protein substrates.

In isolated rat hepatocytes, several tyrosine protein kinase inhibitors (tyrphostins) reduced the autophagic sequestration of electroinjected [3H]raffinose by 40-75% at doses that did not significantly affect cellular ATP levels or plasma membrane integrity. Tyrphostin 46 specifically inhibited autophagy, whereas tyrphostins 1, 25 and 51 also suppressed the receptor-mediated endocytic uptake of 125I-tyramine-cellobiose-asialoorosomucoid, 125I-TC-AOM, by 20-30% and its degradation by 70-90%. Tyrphostins 1 and 51, and the microtubule inhibitor vinblastine, inhibited an early endocytic step (endosome maturation/multivesiculation?), causing accumulation of endocytosed 125I-TC-AOM in a recycling compartment that corresponded to light endosomes (1.10-1.11 g/ml) in sucrose density gradients. In the electron microscope, these endosomes could be recognized as small, peripheral endocytic vesicles and tubules accumulating endocytosed AOM-gold. The serine/threonine protein phosphatase inhibitor okadaic acid inhibited an intermediate endocytic step (detachment of multivesicular endosomes from the tubulovesicular network?), causing accumulation of 125I-TC-AOM in a recycling compartment corresponding to light endosomes (1.10-1.11 g/ml), but with a multivesicular rather than a tubulovesicular morphology. Tyrphostin 25 inhibited endocytosis at a late step (endosome-lysosome fusion?), causing accumulation of 125I-TC-AOM in a non-recycling compartment corresponding to dense, multivesicular endosomes (1.14 g/ml) that had probably detached from the light endosomal network.

We have examined three strains of Azotobacter vinelandii, which contain defined deletions within the nifH, nifB, or nifE genes. All three strains accumulate inactive FeMo cofactor-deficient forms of the MoFe protein of nitrogenase. These forms can be activated in vitro by addition of isolated FeMo cofactor in N-methylformamide. Although the phenotypes of these strains are superficially the same, our characterizations demonstrate that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is quite different from that synthesized by either the delta nifB or delta nifE strains. These differences include the following: 1) the activation of the delta nifH protein requires MgATP, whereas the activation of the delta nifB and delta nifE proteins does not; 2) the delta nifH extracts can be activated with FeMo cofactor to wild-type levels of activity, whereas delta nifB and delta nifE extracts cannot; 3) the delta nifH protein is markedly less heat stable than the delta nifB and delta nifE proteins; and 4) the migration of the delta nifH protein on native gels is very different when compared with delta nifB and delta nifE, which look like each other. These data can be explained if the nifB and nifE gene products are only involved in FeMo cofactor biosynthesis, whereas the nifH gene product is involved in both the initial synthesis of FeMo cofactor and in the insertion of preformed FeMo cofactor into the MoFe protein. A model is presented that suggests that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is the one that normally participates in MoFe protein assembly in wild-type cells.

In this work we report an unusual pattern of activation by calmodulin on the (Ca2+ + Mg2+)-ATPase from basolateral membranes of kidney proximal tubule cells. The activity of the ATPase depleted of calmodulin is characterized by a high Ca2+ affinity (Km = 2.2-3.4 microM) and a biphasic dependence on ATP concentration. The preparation responded to the addition of calmodulin by giving rise to a new Ca2+ site of very high affinity (Km less than 0.05 microM). Calmodulin antagonists had diverse effects on ATPase activity. Compound 48/80 inhibited calmodulin-stimulated activity by 70%, whereas calmidazolium did not modify this component. In the absence of calmodulin, 48/80 still acted as an antagonist, increasing the Km for Ca2+ to 5.7 microM and reducing enzyme turnover by competing with ATP at the low affinity regulatory site. Calmidazolium did not affect Ca2+ affinity, but it did displace ATP from the regulatory site. At fixed Ca2+ (30 microM) and ATP (5 mM) concentrations, Pi protected against 48/80 and potentiated inhibition by calmidazolium. At 25 microM ATP, Pi protected against calmidazolium inhibition. We propose that the effects of ATP and Pi arise because binding of the drugs to the ATPase occurs mainly on the E2 forms.

Energy deprivation, induced in thyroid slices by incubation with an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone) or inhibitors of respiration (N2 or antimycin A), led to a disturbance in oligosaccharide-lipid metabolism which was characterized by a pronounced depletion of the glucosylated (Glc3Man9GlcNAc2) dolichyl pyrophosphoryl saccharide with an attendant accumulation of the Man9GlcNAc2 and, to a lesser extent, the Man8GlcNAc2 lipid-linked species. A concomitant decrease in the N-glycosylation of proteins was furthermore observed. The distribution of lipid-derived oligosaccharides observed by thin layer chromatographic separation was similar whether radiolabeling was achieved by a metabolic ([14C]glucose or [2-3H]mannose incubation) or chemical ([3H]NaBH4) procedure. The latter method proved useful for measuring the levels of individual oligosaccharide-lipids in unincubated tissue, and in this way it was found that unless thyroid was rapidly frozen or immediately immersed in oxygenated medium a marked decrease of glucose-containing oligosaccharide-lipids occurred which could, however, be reversed by a short incubation. The addition of glucose to the slice incubations did not prevent the Man8-9GlcNAc2 accumulation brought about by the inhibitors of energy production, nor did it alter the oligosaccharide-lipid pattern of uninhibited slices. The effect of the inhibitors on metabolically labeled liver slices was similar to that observed in thyroid. The size of the total chloroform/methanol/water (10:10:3)-extractable oligosaccharide-lipid pool (Glc3-Man9GlcNAc2 to Man5GlcNAc2) of thyroid remained quite constant (about 2 nmol/g) regardless of the energy state, and no substantial change in the level of smaller oligosaccharide-lipids, primarily represented by Man2GlcNAc2 (0.5 nmol/g) was evident. Moreover, no change in the total pool sizes was observed in puromycin-treated slices. The possible mechanisms by which energy deprivation leads to an accumulation of the glucose-free oligosaccharide-lipids are evaluated. While it is likely that a selective impairment of glucosylation of newly formed molecules occurs, it is also possible that an imbalance occurs in a postulated glucosyltransferase-glucosidase shuttle with a trapping of the oligosaccharide-lipid in its unglucosylated form.

Flagellar motors of some alkalophilic Bacillus strains have been suggested to be powered by the electrochemical potential gradient of Na+, namely the (formula: see text) (Hirota, N., Kitada, M., and Imae, Y. (1981) FEBS Lett. 132, 278-280). In the present study, we quantitatively measured the (formula: see text) and motility of one of the strains, YN-1. Swimming speed of YN-1 cells increased linearly with a logarithmic increase of Na+ concentration in the medium up to 100 mM. The intracellular Na+ concentration and the membrane potential of the cell were about 30 mM and -170 mV, respectively, and stayed constant irrespective of Na+ concentration in the medium. Thus, the swimming speed changed as a function of the chemical potential difference of Na+ across the cell membrane. When the membrane potential of YN-1 cells was decreased by a combination of valinomycin and various concentrations of K+ in the medium, the swimming speed of the cells decreased linearly and reached zero at around -90 mV. Under the condition, the intracellular Na+ concentration stayed constant. Thus, the membrane potential was also a determinant of the swimming speed. Furthermore, the chemical potential of Na+ and the membrane potential were found to be equivalent as the energy source for motility. Therefore, it is concluded that the (formula: see text) is the energy source for the flagellar motors of YN-1 cells. Threshold value of the (formula: see text) for motility was about -100 mV.

The correlation between the time courses of actin polymerization under continuous sonication and the associated ATP hydrolysis has been studied. ATP hydrolysis was not mechanistically coupled to polymerization, i.e. not necessary for polymerization, but occurred on F-actin in a subsequent monomolecular reaction. Under sonication, polymerization was complete in 10 s while hydrolysis of ATP on the polymer required 200 s. A value of 0.023 s-1 was found for the first order rate constant of ATP hydrolysis on the polymer at 25 degrees C, pH 7.8, in the presence of 0.2 mM ATP, 0.1 mM CaCl2, and 1 mM MgCl2, independent of the F-actin concentration. The conversion of ATP X F-actin to ADP X F-actin was accompanied by an increase in fluorescence of a pyrenyl probe covalently attached to actin, consistent with a 2-fold greater fluorescence for ADP X F-actin than for ATP X F-actin, with a rate constant of 0.022 s-1. In contrast, the fluorescence of F-actin labeled with 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole did not change significantly when ATP or ADP was bound. The direct consequence of the uncoupling between polymerization and ATP hydrolysis is the formation of an ATP cap at the ends of the filaments, which maintains the stability of the polymer, while most of the filament contains bound ADP. The heterogeneity of the filament with respect to ATP and ADP results in a nonlinear relationship between the rate of elongation and the concentration of G-actin with a discontinuity at the critical concentration, where the rate of growth is zero. In this respect, F-actin in ATP behaves similarly to microtubules in GTP.

The elemental and water content of cultured bovine adrenal chromaffin cells and their secretory chromaffin granules have been measured and compared with isolated chromaffin granules using quick freezing, ultracryomicrotomy, and electron microprobe analysis methods. In units of millimole/kilogram dry weight (+/- S.E.) granules in situ contained: P, 523 +/- 32; K+, 124 +/- 9; S, 82 +/- 3; Cl-, 74 +/- 9; Ca2+, 13 +/- 2; Mg2+, 6 +/- 2; and Na+, -2 +/- 2. Following routine isolation in isotonic sucrose buffer, granule K and Cl- had decreased while granule Na+ increased. Cl- exhibited a consistent decrease to 35-40 mmol/kg dry weight. Granule Na+ and K+ concentrations ranged from 43 to 12 mmol/kg and 28 to 60 mmol/kg dry weight, respectively, depending on the Na+ and K+ content of the buffer. Despite the redistribution of monovalent ions, granule Ca2+, granule P, being in the form of ATP, and granule S, being in the form of protein, were not significantly changed. The stability of these elements is consistent with the existence of a stable storage complex for Ca2+, ATP, and protein. Using the granule as an internal standard with a water content of 66%, the water contents of external space, nucleus, cytoplasm, and mitochondria were estimated to be 89, 88, 82, and 70%, respectively. Wet weight concentrations for each element were calculated for granules and cytoplasm from which the transgranular concentration gradients for K+, Cl-, and Na+ were determined. Cl-, a permeant anion, was 2-fold higher in the granule than in the cytoplasm while K+, a slightly permeant cation, had an opposite distribution ratio slightly less than two. Together, the K+ and Cl- data suggest the presence of an inside-positive granule membrane potential of approximately 10-16 mV. The surprising lack of Na+ from the granule matrix suggests a hugh inward gradient for Na+ even though the Na+ content of chromaffin cell cytoplasm is low at 5 mmol/kg water. The lack of an outward Na+ gradient is important in that it indicates that the previously described electroneutral Na+-Ca2+ exchange system, by which isolated granules accumulate Ca2+, does not operate in mature granules in situ. Consequently, if chromaffin granules regulate internal calcium during stimulus secretion coupling, a mechanism other that Na+-Ca2+ exchange is necessary.

The effect of anoxia and substrate removal on cytosolic free calcium (Ca2+i), cell calcium, ATP content, and calcium efflux was determined in cultured monkey kidney cells (LLC-MK2) exposed to 95% N2, 5% CO2 for 60 min. In the control period, the basal Ca2+i level was 70.8 +/- 9.4 nM. During 1 h of anoxia without substrate, ATP content decreased 70%, Ca2+i and calcium efflux increased 2.5-fold, while the total cell calcium did not change. When the cells were perfused again with O2 and 5 mM glucose, the ATP concentration, Ca2+i, and calcium efflux returned to control levels within 15-20 min. In the presence of 20 mM glucose, anoxia did not produce any change in ATP, in Ca2+i or in calcium efflux. An important source of calcium contributing to the rise in Ca2+i induced by anoxia appears to be extracellular because the rate of rise in Ca2+i is proportional to the extracellular calcium concentration, and because La3+ which blocks calcium influx greatly reduces the rise in Ca2+i. Mitochondria appear to control Ca2+i as well since the early rise in Ca2+i cannot be blocked by La3+ during the initial phase of anoxia, and since the mitochondrial inhibitor carbonyl cyanide p-trifluoromethoxyphenylhydrazone increases Ca2+i further during reoxygenation and slows the return of Ca2+i to control levels.

31P NMR spectra of isolated rabbit bladder and uterus were obtained under steady-state arterial perfusion in vitro at rest and while stimulated. The spectra contained seven major peaks: phosphoethanolamine, sn-glycero(3)phosphocholine, inorganic phosphate (Pi), phosphocreatine, and the gamma, alpha, and beta peaks of ATP. Chemical analyses, high-pressure liquid chromatography, and NMR spectroscopy of aqueous extracts of bladders identified a number of other components that also made contributions to, but were not resolved in, the spectra of the intact tissues: UTP, GTP, UDP-Glc, NAD+, phosphocholine, and sn-glycero(3)phosphoethanolamine. Intracellular pH of unstimulated bladders and uteri, measured from the chemical shift of the Pi peak, was 7.10 +/- 0.09 S.D. and 7.01 +/- 0.12 S.D., respectively. The chemical shift of the beta-ATP peak in the smooth muscles was significantly upfield (-0.3 ppm) compared to the chemical shift observed in striated muscles (cat biceps and rat myocardium). An ADP peak was identified in stimulated and ischemic bladders. The chemical shifts of the nucleotides observed in perfused bladders were calibrated as a function of free Mg2+ concentration in solutions containing phosphocreatine, Pi, ADP, and ATP at an ionic strength of 180 mM. We derived the following estimates for the intracellular free Mg2+ concentration: uterus, 0.40 mM; unstimulated bladder, 0.46 mM; stimulated and ischemic bladder, 0.50 mM (from the ATP chemical shift) and 0.45 (from the ADP chemical shift); cat biceps, 1.5 mM; and rat myocardium, 1.4 mM.

The stereochemical course of the ribosome-dependent GTPase reaction of elongation factor G from Escherichia coli has been determined. Guanosine 5'-(gamma-thio)triphosphate stereospecifically labeled with 17O and 18O in the gamma-position was hydrolyzed in the presence of the elongation factor and ribosomes. The configuration of the product, inorganic [16O, 17O, 18O]thiophosphate ws analyzed by 31P NMR after its stereospecific incorporation into adenosine 5'-(beta-thio)triphosphate. The analysis showed that the hydrolysis proceeds with inversion of configuration at the transferred phosphorus atom. It is therefore likely that the hydrolysis occurs in a single step by direct, in-line transfer of the phosphorus from GDP to a water oxygen, without a phosphoenzyme intermediate.

Quantitative aspects of de novo pyrimidine biosynthesis in rat hepatocytes were monitored. A reduction of intracellular UTP contents by different concentrations of D-galactosamine led to a dose-dependent increase of 14CO2 incorporation into the sum of all acid-soluble uracil nucleotides. In controls the rate of de novo synthesis which was calculated from the incorporation rate of 14CO2 into the sum of all acid-soluble uracil nucleotides was 0.014 mumol X h-1 X g-1 compared to 0.056 mumol X h-1 X g-1 wet weight of liver in situations of a maximally stimulated de novo synthesis. Incubation of hepatocytes with uridine led to a dose-dependent reduction of 14CO2 incorporation to less than 25% of the amount incorporated in the controls. Alterations of the CTP content had no influence on the 14CO2 incorporation. In the presence of high D-galactosamine concentrations the increase of the total amount of acid-soluble uracil nucleotides exceeded the rate of the de novo synthesis derived from the incorporation of 14CO2 into the sum of the acid-soluble uracil nucleotide pool. It was also greater than the increase of the total amount of intra- and extracellular orotate after acidic hydrolysis--even in the presence of 6-azauridine, which stimulated de novo pyrimidine biosynthesis by itself.

The unusual bis(5'-nucleosidyl)oligophosphates: Ap4A, Ap4G, Ap3A, and Ap3G, have been measured in cultures of Drosophila cells. Exponentially growing cells contain concentrations of 0.25, 0.31, 0.87, and 2.25 microM, respectively. These nucleotides have been followed after stressing the cells either by CdCl2 addition or by heat-shock treatment. Their concentrations are not affected by exposure to 500 microM CdCl2 during 6 h. Beyond this threshold of cadmium concentration, the nucleotides increase. With 5 mM CdCl2, an enhancement by 2 orders of magnitude of all the dinucleoside tri- and tetra-phosphates is observed. Upon heat-shock from 19 to 37 degrees C, Ap4A, Ap3A, and Ap3G increase up to 2.2, 3, and 3.3 times their initial levels, respectively. The increase is achieved within 1 h.

The purity of several preparations of adenyl-5'-yl imidodiphosphate (AMP-PNP) was analyzed using thin layer chromatography and the luciferin-luciferase assay. Three contaminants were identified: adenyl-5'-yl phosphoramidate, phosphorylated AMP-PNP, and ATP. The level of ATP contamination ranged from 0.02% to 0.3% in commercially obtained AMP-PNP preparations, and rose to 10% following incubation of AMP-PNP at 37 degrees C for 3 weeks in aqueous solution. The chemistry of the phosphoramidate bond is reviewed briefly, and evidence for a simple mechanism for the spontaneous formation of ATP from AMP-PNP is presented.

We have purified an ATP-dependent protease with protein-dependent ATPase activity from bovine adrenal cortex mitochondria to near homogeneity. The subunit molecular weight is 108,000 and the enzyme appears to be a hexamer with approximately identical subunits. Based on the experiments using various nucleoside triphosphates and their related compounds, it is concluded that hydrolysis of the high-energy bond in nucleoside triphosphates is not an absolute requirement for proteolysis. Nucleotide specificity of this enzyme varies, depending on the protein or peptide substrates used. When casein was the substrate, ATP and dATP were quite effective, but other nucleotides were not. When insulin and angiotensinogen were used as substrate, ATP, other nucleoside triphosphates, ADP, inorganic triphosphate, pyrophosphate, and phosphate were effective. One of the cleaving linkages hydrolyzed by this enzyme was revealed to be the Leu-Leu bond of angiotensinogen. However, the specificity appears to be broad in view of the hydrolysis pattern of glucagon.