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PubMed1995

Interaction of SecB with intermediates along the folding pathway of maltose-binding protein.

SecB, a molecular chaperone involved in protein export in Escherichia coli, displays the remarkable ability to selectively bind many different polypeptide ligands whose only common feature is that of being nonnative. The selectivity is explained in part by a kinetic partitioning between the folding of a polypeptide and its association with SecB. SecB has no affinity for native, stably folded polypeptides but interacts tightly with polypeptides that are nonnative. In order to better understand the nature of the binding, we have examined the interaction of SecB with intermediates along the folding pathway of maltose-binding protein. Taking advantage of forms of maltose-binding protein that are altered in their folding properties, we show that the first intermediate in folding, represented by the collapsed state, binds to SecB, and that the polypeptide remains active as a ligand until it crosses the final energy barrier to attain the native state.

ATP-Binding Cassette Transporters↗

PubMed2001

Mechanisms for experimental buprenorphine hepatotoxicity: major role of mitochondrial dysfunction versus metabolic activation.

BACKGROUND/AIMS: Although sublingual buprenorphine is safely used as a substitution drug in heroin addicts, large overdoses or intravenous misuse may cause hepatitis. Buprenorphine is N-dealkylated to norbuprenorphine by CYP3A. METHODS: We investigated the mitochondrial effects and metabolic activation of buprenorphine in isolated rat liver mitochondria and microsomes, and its toxicity in isolated rat hepatocytes and treated mice. RESULTS: Whereas norbuprenorphine had few mitochondrial effects, buprenorphine (25-200 microM) concentrated in mitochondria, collapsed the membrane potential, inhibited beta-oxidation, and both uncoupled and inhibited respiration in rat liver mitochondria. Both buprenorphine and norbuprenorphine (200 microM) underwent CYP3A-mediated covalent binding to rat liver microsomal proteins and both caused moderate glutathione depletion and increased cell calcium in isolated rat hepatocytes, but only buprenorphine also depleted cell adenosine triphosphate (ATP) and caused necrotic cell death. Four hours after buprenorphine administration to mice (100 nmol/g body weight), hepatic glutathione was unchanged, while ATP was decreased and serum transaminase increased. This transaminase increase was attenuated by a CYP3A inducer and aggravated by a CYP3A inhibitor. CONCLUSIONS: Both buprenorphine and norbuprenorphine undergo metabolic activation, but only buprenorphine impairs mitochondrial respiration and ATP formation. The hepatotoxicity of high concentrations or doses of buprenorphine is mainly related to its mitochondrial effects.

Adenosine Triphosphate↗

PubMed2002

Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin.

Oedema factor, a calmodulin-activated adenylyl cyclase, is important in the pathogenesis of anthrax. Here we report the X-ray structures of oedema factor with and without bound calmodulin. Oedema factor shares no significant structural homology with mammalian adenylyl cyclases or other proteins. In the active site, 3'-deoxy-ATP and a single metal ion are well positioned for catalysis with histidine 351 as the catalytic base. This mechanism differs from the mechanism of two-metal-ion catalysis proposed for mammalian adenylyl cyclases. Four discrete regions of oedema factor form a surface that recognizes an extended conformation of calmodulin, which is very different from the collapsed conformation observed in other structures of calmodulin bound to effector peptides. On calmodulin binding, an oedema factor helical domain of relative molecular mass 15,000 undergoes a 15 A translation and a 30 degrees rotation away from the oedema factor catalytic core, which stabilizes a disordered loop and leads to enzyme activation. These allosteric changes provide the first molecular details of how calmodulin modulates one of its targets.

Adenylyl Cyclases↗

PubMedDate not supplied

Transmembrane phospholipid distribution in blood cells: control mechanisms and pathophysiological significance.

This review deals with current concepts on the regulation and function of phospholipid asymmetry in biological membranes. This ubiquitous phenomenon is characterized by a distinctly different lipid composition between the inner and outer leaflet of the membrane bilayer. Transbilayer asymmetry is controlled by different membrane proteins that function as lipid transporters, catalyzing uni- or bi-directional transbilayer movement of lipids. Under normal conditions, an ATP-dependent protein (aminophospholipid translocase) generates and maintains phospholipid asymmetry by promoting unidirectional transport of aminophospholipids from the outer- to the inner leaflet. The membrane lipid asymmetry may be compromised during cellular activation by a Ca2+-dependent transporter (lipid scramblase) that facilitates rapid bi-directional movement of all major phospholipid classes. A major consequence of this collapse of lipid asymmetry is the exposure of phosphatidylserine (PS) at the outer membrane surface. Surface exposure of PS has important physiological and pathological implications for blood coagulation, apoptosis, and cell-cell recognition.

Animals↗

PubMed2002

Inhibition of mitochondrial respiration by endogenous nitric oxide: a critical step in Fas signaling.

We have found that activation of human adult T cell leukemia (Jurkat) cells with anti-Fas Ab leads, in a concentration-dependent manner, to an early burst of production of nitric oxide (NO), which inhibits cell respiration. This results in mitochondrial hyperpolarization, dependent on the hydrolysis of glycolytic ATP by the F1F(o)-ATPase acting in reverse mode. During this early phase of activation, there is a transient release of superoxide anion. All these processes can be prevented by an inhibitor of NO synthase. Approximately 2 h after stimulation with anti-Fas Ab, a distinct second phase can be detected. This comprises a concentration-dependent collapse in mitochondrial membrane potential, a second wave of free radical production, and activation of caspase-8 leading to apoptosis. This second phase is abolished by an inhibitor of caspase activation. In contrast, inhibition of NO synthesis leads to an enhancement and acceleration of these latter processes, suggesting that the early NO-dependent phase represents a protective mechanism. The significance of the two phases in relation to cell survival and death remains to be studied.

Apoptosis↗

PubMed1993

The regulation of bidirectional mitochondrial transport is coordinated with axonal outgrowth.

Although small molecules such as ATP diffuse freely in the cytosol, many types of cells nonetheless position their mitochondria in regions of intense ATP consumption. We reasoned that in the highly elongated axonal processes of growing neurons in culture, the active growth cone would form a focus of ATP consumption so distant from the cell body as to require the positioning of mitochondria nearby via regulated axonal transport. To test this hypothesis, we quantified the distribution and transport behavior of mitochondria in live, aerobically respiring chick sympathetic neurons. We found that in the distal region of actively growing axons, the distribution of mitochondria was highly skewed toward the growth cone, with a sevenfold higher density in the region immediately adjacent to the growth cone than in the region 100 microns away. When axonal outgrowth was blocked by substratum-associated barriers or mild cytochalasin E treatment, the gradient of mitochondrial distribution collapsed as mitochondria exited retrogradely from the distal region, becoming uniformly distributed along the axon within one hour. Analysis of individual mitochondrial behaviors revealed that mitochondrial movement everywhere was bidirectional but balanced so that net transport was anterograde in growing axons and retrograde in blocked axons. This reversal in net transport derived from two separate modulations of mitochondrial movement. First, moving mitochondria underwent a transition to a persistently stationary state in the region of active growth cones that was reversed when growth cone activity was halted. Second, the fraction of time that mitochondria spent moving anterogradely was sharply reduced in non-growing axons. Together, these could account for the formation of gradients of mitochondria in growing axons and their dissipation when outgrowth was blocked. This regulated transport behavior was not dependent upon the ability of mitochondria to produce ATP. Our data indicate that mitochondria possess distinct motor activities for both directions of movement and that mitochondrial transport in axons is regulated by both recruitment between stationary and moving states, and direct regulation of the anterograde motor.

Anaerobiosis↗

PubMed2002

Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite.

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of diseases with excess bone resorption. Recent studies have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R(2) side chain. Alendronate, like other nitrogen-containing bisphosphonates, inhibits bone resorption and causes apoptosis of osteoclasts and other cells in vitro by preventing post-translational modification of GTP-binding proteins with isoprenoid lipids. Clodronate, a bisphosphonate that lacks a nitrogen, does not inhibit protein isoprenylation but can be metabolized intracellularly to a beta-gamma-methylene (AppCp-type) analog of ATP, which is cytotoxic to macrophages in vitro. The detailed molecular basis for the cytotoxic effects of adenosine-5'-[beta,gamma-dichloromethylene]triphosphate (AppCCl(2)p) has not been determined yet. We addressed this question by studying the effects of alendronate, clodronate, and the clodronate metabolite AppCCl(2)p on isolated mitochondria, mitochondrial fractions, and mitochondrial membrane potential in isolated human osteoclasts. We found that AppCCl(2)p inhibits mitochondrial oxygen consumption by a mechanism that involves competitive inhibition of the ADP/ATP translocase. Alendronate or the native form of clodronate did not have any immediate effect on mitochondria. However, longer treatment with liposome-encapsulated clodronate caused collapse of the mitochondrial membrane potential, although prominent apoptosis was a late event. Hence, inhibition of the ADP/ATP translocase by the metabolite AppCCl(2)p is a likely route by which clodronate causes osteoclast apoptosis and inhibits bone resorption.

Adenine↗

PubMed2001

Collapse of extracellular glutamate regulation during epileptogenesis: down-regulation and functional failure of glutamate transporter function in rats with chronic seizures induced by kainic acid.

We used northern and western blotting to measure the quantity of glutamate and GABA transporters mRNA and their proteins within the hippocampal tissue of rats with epileptogenesis. Chronic seizures were induced by amygdalar injection of kainic acid 60 days before death. We found that expression of the mRNA and protein of the glial glutamate transporters GLAST and GLT-1 were down-regulated in the kainic acid-administered group. In contrast, EAAC-1 and GAT-3 mRNA and their proteins were increased, while GAT-1 mRNA and protein were not changed. We performed in vivo microdialysis in the freely moving state. During the interictal state, the extracellular glutamate concentration was increased, whereas the GABA level was decreased in the kainic acid group. Following potassium-induced depolarization, glutamate overflow was higher and the recovery time to the basal release was prolonged in the kainic acid group relative to controls. Our data suggest that epileptogenesis in rats with kainic acid-induced chronic seizures is associated with the collapse of extracellular glutamate regulation caused by both molecular down-regulation and functional failure of glutamate transport.

ATP-Binding Cassette Transporters↗

PubMed1990

Na-H exchange in myocardium: effects of hypoxia and acidification on Na and Ca.

Historically, increase in cell Na content during ischemic and hypoxic episodes were thought to result from impaired ATP production causing decreased Na(+)-K(+)-ATPase activity. Here we report the results of testing the alternate hypothesis that hypoxia-induced Na uptake is 1) the result of increased entry, as opposed to decreased extrusion 2) via Na-H exchange operating in a pH regulatory capacity and that cell Ca accumulation occurs via Na-Ca exchange secondary to collapse of the Na gradient. We used 23Na-, 19F-, and 31P-nuclear magnetic resonance (NMR) to measure intracellular Na content (Nai), Ca concentration [( Ca]i), pH (pHi), and high-energy phosphates in Langendorff-perfused rabbit hearts. When the Na(+)-K(+)-ATPase was inhibited by ouabain and/or K-free perfusion, hearts subjected to hypoxia gained Na at a rate greater than 10 times that of normoxic controls [during the first 12.5 min Nai increased from 7.9 +/- 5.8 to 34.9 +/- 11.0 (SD) meq/kg dry wt compared with 11.1 +/- 16.3 to 13.6 +/- 9.0 meq/kg dry wt, respectively]. When normoxic hearts were acidified using a 20 mM NH4Cl prepulse technique, pHi rapidly fell from 7.27 +/- 0.24 to 6.63 +/- 0.12 but returned to 7.07 +/- 0.10 within 20 min, while Na uptake was similar in rate and magnitude to that observed during hypoxia (24.5 +/- 13.4 to 132.1 +/- 17.7 meq/kg dry wt). During hypoxia and after NH4Cl washout, increases in [Ca]i were similar in time course to those observed for Na.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1993

Effect of Iron Deficiency on the Respiration of Sycamore (Acer pseudoplatanus L.) Cells.

The effects of iron deficiency on cell culture growth, cell respiration, mitochondrial oxidative properties, and the electron transport chain were studied with suspension-cultured sycamore (Acer pseudoplatanus L.) cells. Iron deprivation considerably decreased the initial growth rates and limited the maximum density of the cells. Under these conditions, the cells remained swollen throughout their growth. The absence of iron led to a steady decline in the uncoupled rate of O2 consumption. When the uncoupled rate of O2 uptake closely approximated the respiratory rate, the cells began to collapse. At this stage, the level of all the cytochromes and electron paramagnetic resonance-detectable Fe-S clusters of the mitochondrial inner membrane were dramatically decreased. Nevertheless, it appeared from substrate oxidation measurements that this overall depletion in iron-containing components solely disturbed the functioning of complex II, whereas neither complexes I, III, or IV, nor the machinery involved in ATP synthesis, was apparently impaired in iron-deficient mitochondria. However, our results suggest that the impairment of complex II resulted in a strong reduction of the overall capacity of the mitochondrial electron transport chain, which was responsible for determining the rate of endogenous respiration in sycamore cells. Finally, this situation led to a depletion of various energy metabolites that could contribute to the premature cell death.

Journal Article↗

PubMed2006

Developmental changes of CaMKII localization, activity and function during postembryonic CNS remodelling in Manduca sexta.

Insect metamorphosis is a compelling example of postembryonic remodelling of neuronal structure and synaptic connectivity as larval and adult behaviours place distinct demands on the CNS. Holometabolous insects such as the moth Manduca sexta have long served as suitable models for the study of steroid effects on CNS remodelling, but activity and calcium-dependent mechanisms have been found to act in concert with hormonal signals. This study examines developmental changes in the localization and the activational state of CaMKII during postembryonic Manduca CNS remodelling. Western blotting, CaMKII purification and autophosphorylation with gamma(32)P-ATP indicate that the lepidopteran CNS may contain only one CaMKII isoform. In situ immunohistochemistry reveals developmental changes in the expression patterns of CaMKII in different types of thoracic neurons and in different neuronal compartments. Early pupal life is characterized by an increase in postsynaptic CaMKII localization, which coincides with a developmental increase in CaMKII activation. Both events correlate temporally with motoneuron dendritic filopodia collapse and rapid synaptogenesis, indicating a possible functional role for CaMKII for the postembryonic development of invertebrate motor circuitry. Substrate phosphorylation assays demonstrate that CaMKII activity in the ventral nerve cord reflects changes in calcium influx through voltage-activated channels as occurring in vivo during normal development.

Animals↗

PubMed1983

31P nuclear magnetic resonance study of the recovery characteristics of high energy phosphate compounds and intracellular pH after global ischaemia in the perfused guinea-pig heart.

The recovery of high energy phosphate compounds in perfused guinea-pig heart at 20 degrees C after a 12 min period of global ischaemia was examined using 31P-NMR with a time resolution of 12 s. This time resolution was achieved by overlaying the data acquired from five successive ischaemic periods by arresting and restoring the flow of perfusion fluid to the heart in synchrony with the data acquisition sequence. The rate of creatine phosphate resynthesis after the ischaemic period proceeded 14 times faster than its rate of loss during the ischaemic period. ATP levels did not decrease during ischaemia and ADP was undetectable at any time. Estimates of intracellular pH from the chemical shift of the inorganic phosphate peak were impossible in normal guinea-pigs since the inorganic phosphate peak was not clearly defined. During the ischaemic period the inorganic phosphate peak increased in size and shifted upfield. On restoration of flow, the inorganic phosphate peak collapsed in a complex way following a different path to its formation during ischaemia.

Acid-Base Equilibrium↗

PubMed2001

The KATP channel opener diazoxide protects cardiac myocytes during metabolic inhibition without causing mitochondrial depolarization or flavoprotein oxidation.

1. The K(ATP) channel opener diazoxide has been proposed to protect cardiac muscle against ischaemia by opening mitochondrial K(ATP) channels to depolarize the mitochondrial membrane potential, DeltaPsi(m). We have used the fluorescent dye TMRE to measure DeltaPsi(m) in adult rat freshly isolated cardiac myocytes exposed to diazoxide and metabolic inhibition. 2. Diazoxide, at concentrations that are highly cardioprotective (100 or 200 microM), caused no detectable increase in TMRE fluorescence (n=27 cells). However, subsequent application of the protonophore FCCP, which should collapse DeltaPsi(m), led to large increases in TMRE fluorescence (>300%). 3. Metabolic inhibition (MI: 2 mM NaCN+1 mM iodoacetic acid (IAA) led to an immediate partial depolarization of DeltaPsi(m), followed after a few minutes delay by complete depolarization which was correlated with rigor contracture. Removal of metabolic inhibition led to abrupt mitochondrial repolarization followed in many cells by hypercontracture, indicated by cell rounding and loss of striated appearance. 4. Prior application of diazoxide (100 microM) reduced the number of cells that hypercontracted after metabolic inhibition from 63.7+/-4.7% to 24.2+/-1.8% (P< 0.0001). 5-hydroxydeanoate (100 microM) reduced the protection of diazoxide (46.8+/-2.7% cells hypercontracted, P< 0.0001 vs diazoxide alone). 5. Diazoxide caused no detectable change in flavoprotein autofluorescence (n=26 cells). 6. Our results suggest that mitochondrial depolarization and flavoprotein oxidation are not inevitable consequences of diazoxide application in intact cardiac myocytes, and that they are also not essential components of the mechanism by which it causes protection.

Animals↗

PubMed1999

Increase in glutamate-aspartate transporter (GLAST) mRNA during kanamycin-induced cochlear insult in rats.

Kanamycin (KM)-induced changes in expression of the gene for glutamate-aspartate transporter (GLAST) in the rat cochlea were analyzed by Northern blotting. With the administration of KM (600 mg/kg/day) once daily for 20 days, the expression of GLAST mRNA gradually increased and reached a peak on day 20. Although the expression of GLAST mRNA remained at a high level until 12 days after the completion of the KM treatment, it then fell to the normal level within 2 months. Such KM treatment resulted in loss of both inner and outer hair cells and a concomitant profound permanent threshold shift. The present findings suggest that during KM administration, high concentrations of extracellular glutamate released by collapsing hair cells induced GLAST mRNA expression. Increased GLAST mRNA might play an important role in the prevention of the secondary death of spiral ganglion neurons from glutamate neurotoxicity.

ATP-Binding Cassette Transporters↗

PubMed2005

ABCA5 resides in lysosomes, and ABCA5 knockout mice develop lysosomal disease-like symptoms.

ABCA5 is a member of the ABC transporter A subfamily, and a mouse orthologue (mABCA5) in newborn mouse brain and neural cells was identified by reverse transcription-PCR. Full-length cDNA cloning revealed that mABCA5 consists of 1,642 amino acid residues and that its putative structure is that of a full-type ABC transporter having two sets of six transmembrane segments and a nucleotide binding domain. Immunohistochemical studies revealed that mABCA5 is expressed in brain, lung, heart, and thyroid gland. A subcellular localization analysis showed that mABCA5 is a resident of lysosomes and late endosomes. Abca5(-)(/)(-) mice exhibited symptoms similar to those of several lysosomal diseases in heart, although no prominent abnormalities were found in brain or lung. They developed a dilated cardiomyopathy-like heart after reaching adulthood and died due to depression of the cardiovascular system. In addition, Abca5(-)(/)(-) mice also exhibited exophthalmos and collapse of the thyroid gland. Therefore, ABCA5 is a protein related to a lysosomal disease and plays important roles, especially in cardiomyocytes and follicular cells.

ATP-Binding Cassette Transporters↗

PubMed1981

Proton: substrate stoichiometries during active transport of biogenic amines in chromaffin ghosts.

The relationship between the electrochemical proton gradient (delta mu-H+) and the electrochemical gradient for biogenic amines (delta mu-A) was investigated in isolated chromaffin ghosts free of endogenous components and gradients. The addition of ATP to a ghost suspension resulted in the generation of a large proton concentration gradient (delta pH), acidic inside (measured by [14C]-methylamine distribution), and a large proton electrical gradient (delta psi), positive inside (measured by [14C]-thiocyanate distribution). In the presence of this large electrochemical proton gradient, the accumulation of [14C]5-hydroxytryptamine (serotonin) and other biogenic amines rapidly reached an apparent steady state level. Collapse of the proton gradients after steady state levels were achieved resulted in the efflux of the accumulated amines. Uptake in the presence of a delta psi alone produced an amine gradient equal to the magnitude of the delta psi, while in the presence of a delta pH alone biogenic amine distribution was equal to twice the magnitude of the delta pH. Using additions of ammonia or thiocyanate, it was possible to vary the magnitude of the electrochemical proton gradient over a wide range of values; the driving force for amine accumulation under these conditions was found to be equal to delta psi--2Z delta pH (where Z = 2.3 RT/F). The results, which provide unequivocal evidence for the primary role of the electrochemical proton gradient in the active transport of biogenic amines, are consistent with a model based on the chemiosmotic hypothesis, of an obligatory coupling of amine influx and proton efflux, mediated via a putative reserpine-sensitive translocator. Measurement of the stoichiometry of this coupling of H+ and amines permits conclusions concerning the molecular mechanism of amino accumulation.

Ammonia↗

PubMed2003

Ion motive force dependence of protease secretion and phage transduction in Vibrio cholerae and Pseudomonas aeruginosa.

Vibrio cholerae is known to secrete a large number of proteins into the extracellular milieu, including the important virulence factor cholera toxin (CT). However, one of the most abundant proteins found in V. cholerae supernatants is the zinc-metalloprotease HA/protease (HAP). Whereas efficient protein secretion in Escherichia coli requires ATP hydrolysis and the proton motive force (pmf), little is known about the energy requirements for protein secretion in V. cholerae. To analyze some of the energy requirements for protein secretion in V. cholerae, HAP accumulation in culture supernatants following growth in the presence of various ionophores was assayed. Extracellular production of HAP was strongly reduced in the presence of monensin, an artificial Na(+)/H(+) antiporter that collapses the DeltapNa(+) across the membrane without affecting Deltapsi, whereas the protonophore CCCP had no significant effect on the extracellular accumulation of HAP. In contrast, extracellular protease production in Pseudomonas aeruginosa was affected by CCCP, but not monensin. Furthermore, extracellular protease production of V. cholerae, but not P. aeruginosa, was increased in increasing amounts of NaCl in the culture medium. Together these results indicate that the V. cholerae HAP requires an intact sodium motive force (smf) for its efficient translocation across the membranes, whereas extracellular protease production by P. aeruginosa requires only pmf. As the entry of some bacteriophage genomes has been reported to require pmf, the effects of ionophores on the efficiency of tranduction of V. cholerae by the CTXPhi phage were analyzed. CTXPhi transduction was strongly affected by CCCP, but not monensin, suggesting that phage entry requires pmf but not smf. Understanding the energy requirements for these potentially important virulence aspects of pathogens might lead to novel intervention strategies.

Bacteriophages↗

PubMed1981

Nitrogenase activity and membrane electrogenesis in the cyanobacterium Anabaena variabilis Kütz.

Relationships between nitrogenase activity and individual components of the proton electrochemical potential gradient (delta microH+) in Anabaena variabilis have been investigated. The ionophore nigericin was found to collapse delta pH in favour of the membrane potential (delta psi); hyperpolarization of delta psi was correlated with an increase in nitrogenase activity. A positive relationship between nitrogenase activity and membrane potential was also observed using the ionophore valinomycin and the uncoupler carbonylcyanide m-chlorophenylhydrazone. Furthermore, using the energy transfer inhibitor N,N'-dicyclohexylcarbodiimide, nitrogenase activity appeared to be limited by the supply of reductant rather than ATP. The data suggest, for intact filaments and isolated heterocysts, that delta psi may be important in regulating nitrogenase activity in vivo.

Cell Membrane↗