Pi binding by the F1-ATPase of beef heart mitochondria and of the Escherichia coli plasma membrane

Pi binding by the F(1)-ATPase of beef heart mitochondria and of the Escherichia coli plasma membrane (E. coli F(1)) was examined by two methods: the centrifuge column procedure [Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891-2899] and the Paulus pressure dialysis cell [Paulus, H. (1969) Anal. Biochem. 32, 91-100]. The latter is an equilibrium dialysis-type procedure. Pi binding by beef heart F(1) could be determined by either procedure. However, direct binding of Pi to E. coli F(1) could be determined adequately only in the Paulus cell which indicated more than two binding sites per mol of enzyme with a K(d) in the range of 0.1 mM. It is concluded that previous failure to observe Pi binding to E. coli F(1) with the centrifuge column procedure is due to a rapid rate of dissociation of Pi from the E. coli enzyme which results in loss of Pi during transit of the enzyme-Pi complex through the column.     

Effect of acclimation temperature on the concentration of the mitochondrial 'uncoupling' protein measured by radioimmunoassay in mouse brown adipose tissue

The effect of acclimation temperature on the concentration of the mitochondrial 'uncoupling' protein (Mr 32000) from brown adipose tissue of mice has been investigated. The uncoupling protein was measured by a specific radioimmunoassay. Between 33 degrees C (thermoneutrality) and -2 degrees C there was a progressive increase with decreasing environmental temperature in the amount of uncoupling protein. For mice at -2 degrees C the mitochondrial concentration of the protein was 9-times higher than at 33 degrees C, while the total amount of the protein in interscapular brown adipose tissue was estimated to be nearly 80-times greater at -2 degrees C compared to 33 degrees C.     

Selective and reversible inhibition of the ATPase of Micrococcus denitrificans by 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole

The covalent inhibitor of the beef heart mitochondrial ATPase 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole inhibits the ATPase of phosphorylating particles prepared from Micrococcus denitrificans. Inhibition of both ATP synthesis and ATP hydrolysis occurs at similar rates, with a similar pH dependence, and in each case the inhibition is relieved by treatment with dithiothreitol. These results are compared with those previously obtained with the mitochondrial ATPase.     

The synthesis and properties of a functional fluorescent cholesterol analog

The synthesis of a new fluorescent cholesterol analog is described. The analog contains a cholesterol nucleus attached via a hydrophilic spacer to N-4-nitrobenzo-2-oxa-1,3-diazole. Since the cholesterol moiety is not perturbed this molecule probably interacts with lipid bilayers in much the same way as cholesterol itself does. The compound can be readily incorporated into small unilamellar vesicles by sonicating a mixture of it with egg yolk phosphatidylcholine in a buffer. Furthermore, the analog can be incorporated into preformed membranes either by exchange from vesicles containing the analog or by uptake from sonicated micelles of the analog. Thus this analog shows potential as a useful tool for studying the interactions of cholesterol with cell membranes.     

Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Modification of a tyrosine protected by phosphate

Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg²⁺-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca²⁺-ATPase activity of the soluble coupling factor from chromatophores (R. rubrum F₁) was inactivated by NBD-Cl with kinetics resembling those described for Mg²⁺-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD-R. rubrum F₁ fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca²⁺-ATPase activity of R. rubrum F₁ were fully protected by 5 mM P_i against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca²⁺-ATPase activity by P_i indicated that NBD-Cl and P_i are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F₁ underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.     

2,4,6-Trinitrobenzenesulfonate labels an essential amino group near the bound phosphate at the catalytic site of mitochondrial F1-ATPase

The amino reagent 2,4,6-trinitrobenzenesulfonate (TNBS) was found to inactivate mitochondrial F₁-ATPase through covalent labeling, which was not reversed by dithiothreitol. The observed rate of inactivation was retarded by inorganic phosphate, but enhanced by prior labeling of F₁ with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1). These observations are consistent with the presence of an essential amino group near the bound inorganic phosphate at the catalytic site of F₁. A comparison of the observed protection of F₁ from NBD-C1 and 5′-p-fluorosulfonyl-benzoyladenosine (FSBA) respectively by inorganic phosphate and by 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate (TNP-AMP) suggests that NBD-C1 labels an essential Tyr residue in the positively charged locus for binding the polyphosphate end of ATP, and that FSBA labels an essential Tyr residue in the more hydrophobic locus for binding the adenosine moiety of ATP at the catalytic site of F₁.     

Involvement of ATP synthase residues alphaArg-376, betaArg-182, and betaLys-155 in Pi binding

alphaArg-376, betaLys-155, and betaArg-182 are catalytically important ATP synthase residues that were proposed to be involved in substrate Pi binding and subsequent steps of ATP synthesis [Senior, A.E., Nadanaciva, S. and Weber, J. (2002) Biochim. Biophys. Acta 1553, 188-211]. Here, it was shown using purified Escherichia coli F(1)-ATPase that whereas Pi protected wild-type from reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, mutations betaK155Q, betaR182Q, betaR182K, and alphaR376Q abolished protection. Therefore, in ATP synthesis initial binding of substrate Pi in open catalytic site betaE is supported by each of these three residues.     

Nanosecond dynamics of charged fluorescent probes at the polar interface of a membrane phospholipid bilayer

Molecular relaxation fluorescence methods were applied to analyze the nature and characteristic times of motions of amphiphilic molecules absorbed in the polar region of a phospholipid bilayer. The fluorescence probes 2-toluidinonaphthalene-6-sulfonate and 1-anilinonaphthalene-8-sulfonate in egg phosphatidylcholine vesicles were studied. The methods of edge excitation fluorescence red shifts, nanosecond time-resolved spectroscopy, fluorescence quenching by hydrophilic and hydrophobic quenchers and emission wavelength dependence of polarization were used. The structural (dipolar) relaxation is shown to be a very rapid (subnanosecond) process. The observed nanosecond phenomena are related to translational movement of the chromophore itself towards a more polar environment and its rotation. The polar surface area of the phospholipid membrane appears to be a highly mobile liquid-like system.     

Discrimination of three parallel pathways of lactate transport in the human erythrocyte membrane by inhibitors and kinetic properties

The transmembrane movements of lactate and other monocarboxylate anions in mammalian erythrocytes have been claimed, by virtue of their sensitivity to SH-reagents, to involve a transfer system different from the classical anion system (Deuticke, B., Rickert, I. and Beyer, E. (1978) Biochim. Biophys. Acta 507, 137–155). Inhibition of monocarboxylate transfer by SH-reagents, however, was incomplete to an extent varying for different monocarboxylates. The transport component insensitive to SH-reagents has now been shown to involve (a) the classical anion-exchange system, as demonstrated by sensitivity to specific disulfonate inhibitors, and (b) nonionic diffusion, as indicated by the characteristic pH- and concentration dependency of this component and its stimulation by aliphatic alcohols. Under physiological conditions about 90% of total lactate movement proceed via the specific system, 5% via the classical anion-transfer system, 5% by nonionic diffusion. These three components of lactate exchange differ in their activation energies. The specific lactate system mediates net fluxes almost as fast as exchange fluxes, in marked contrast to the classical anion-exchange system which mediates halide exchange much faster than halide net movements. The underlying mechanism, for maintenance of electroneutrality, is an OH^?-antiport or an H⁺-symport as indicated by the particular response of lactate net fluxes to changes of intra- or extracellular pH.     

Inactivation and the site of labeling of F1-ATPase from Mycobacterium phlei by dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and quinacrine mustard

The F₁-ATPase from Mycobacterium phlei is inactivated by dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and quinacrine mustard. The inactivation is both time-and concentration-dependent and in the case of DCCD being more pronounced at acidic pH. The minimum inactivation half-time ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for DCCD, NBD-Cl and quinacrine mustard was observed to be 14, 6 and 7 min, respectively. Inactivation of F₁-ATPase resulted in the incorporation of [¹⁴C]DCCD as well as [¹⁴C]NBD-Cl into α and γ subunits. The incorporation of label into α and γ subunits, utilizing [¹⁴C]NBD-Cl, was reversible by dithiothreitol. Complete inactivation, by linear extrapolation to zero activity, revealed that 4 mol [¹⁴C]DCCD and 4 mol [¹⁴C]NBD-Cl bind per mol F₁-ATPase. Kinetic and binding studies show that these probes bind to site(s) distinct from ATP-binding site in F₁-ATPase from M. phlei.     

Increasing levels of cardiolipin differentially influence packing of phospholipids found in the mitochondrial inner membrane

It is essential to understand the role of cardiolipin (CL) in mitochondrial membrane organization given that changes in CL levels contribute to mitochondrial dysfunction in type II diabetes, ischemia–reperfusion injury, heart failure, breast cancer, and aging. Specifically, there are contradictory data on how CL influences the molecular packing of membrane phospholipids. Therefore, we determined how increasing levels of heart CL impacted molecular packing in large unilamellar vesicles, modeling heterogeneous lipid mixtures found within the mitochondrial inner membrane, using merocyanine (MC540) fluorescence. We broadly categorized lipid vesicles of equal mass as loosely packed, intermediate, and highly packed based on peak MC540 fluorescence intensity. CL had opposite effects on loosely versus highly packed vesicles. Exposure of loosely packed vesicles to increasing levels of CL dose-dependently increased membrane packing. In contrast, increasing amounts of CL in highly packed vesicles decreased the packing in a dose-dependent manner. In vesicles that were categorized as intermediate packing, CL had either no effect or decreased packing at select doses in a dose-independent manner. Altogether, the results aid in resolving some of the discrepant data by demonstrating that CL displays differential effects on membrane packing depending on the composition of the lipid environment. This has implications for mitochondrial protein activity in response to changing CL levels in microdomains of varying composition.     

Monitoring the looping up of acyl chain labeled NBD lipids in membranes as a function of membrane phase state

Lipids that are labeled with the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group are widely used as fluorescent analogues of native lipids in biological and model membranes to monitor a variety of processes. The NBD group of acyl chain labeled NBD lipids is known to loop up to the membrane interface in fluid phase membranes. However, the organization of these lipids in gel phase membranes is not resolved. In this paper, we monitored the influence of the membrane phase state on the looping up behavior of acyl chain labeled NBD lipids utilizing red edge excitation shift (REES) and other sensitive fluorescence approaches. Interestingly, our REES results indicate that NBD group of lipids, which are labeled at the fatty acyl region, resides in the more hydrophobic region in gel phase membranes, and complete looping of the NBD group occurs only in the fluid phase. This is supported by other fluorescence parameters such as polarization and lifetime. Taken together, our results demonstrate that membrane packing, which depends on temperature and the phase state of the membrane, significantly affects the localization of acyl chain labeled NBD lipids. In view of the wide ranging use of NBD-labeled lipids in cell and membrane biology, these results could have potentially important implications in future studies involving these lipids as tracers.     

Lysosomal degradation of membrane lipids

The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes.     

A peptide derived from the putative transmembrane domain in the tail region of E. coli toxin hemolysin E assembles in phospholipid membrane and exhibits lytic activity to human red blood cells: Plausible implications in the toxic activity of the protein

Hemolysin E (HlyE), a pore-forming protein-toxin and a potential virulence factor of Escherichia coli, exhibits cytotoxic activity to mammalian cells. However, very little is known about how the different individual segments contribute in the toxic activity of the protein. Toward this end, the role of a 33-residue segment comprising the amino acid region 88 to 120, which contains the putative transmembrane domain in the tail region of HlyE has been addressed in the toxic activity of the protein-toxin by characterizing the related wild type and mutant peptides and the whole protein. Along with the 33-residue wild type peptide, H-88, two mutants of the same size were synthesized; in one mutant a conserved valine at 89^th position was replaced by aspartic acid and in the other both glycine and valine at the 88^th and 89^th positions were substituted by aspartic acid residues. These mutations were also incorporated in the whole toxin HlyE. Results showed that only H-88 but not its mutants permeabilized both lipid vesicles and human red blood cells (hRBCs). Interestingly, while H-88 exhibited a moderate lytic activity to human red blood cells, the mutants were not active. Drastic reduction in the depolarization of hRBCs and hemolytic activity of the whole toxin HlyE was also observed as a result of the same double and single amino acid substitution in it. The results indicate an important role of the amino acid segment 88-120, containing the putative transmembrane domain of the tail region of the toxin in the toxic activity of hemolysin E.     

Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin

Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0 M TMAO increased the free energy change (ΔG⁰) significantly from 2.1 to 3.8 kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.     

Species-specific modulation of the mitochondrial permeability transition by norbormide

In the present study, we show that norbormide stimulates the opening of the permeability transition pore (PTP) in mitochondria from various organs of the rat but not of guinea pig and mouse. Norbormide does not affect the basic parameters that modulate the PTP activity since the proton electrochemical gradient, respiration, phosphorylation and Ca²⁺ influx processes are only partially affected. On the other hand, norbormide induces rat-specific changes in the fluidity of the lipid interior of mitochondrial membranes, as revealed by fluorescence anisotropy of various reporter molecules. Such changes increase the PTP open probability through the internal Me²⁺ regulatory site. The lack of PTP opening by norbormide is matched by a negligible perturbation of internal lipid domains in guinea pig and mouse, suggesting that the drug does not gain access to the matrix in the mitochondria from these species. Consistent with this interpretation, we demonstrate a preferential interaction of norbormide with the mitochondrial surface leading to alterations of the Me²⁺ binding affinity for the external PTP regulatory site. Our findings indicate that norbormide affects Me²⁺ binding to the regulatory sites of the PTP, and suggest that the drug could be taken up by a mitochondrial transport system unique to the rat. The characterization of the norbormide target may lead to a better understanding of the mechanisms underlying the mitochondrial PTP as well as to the identification of species-specific drugs that affect mitochondrial function.     

Oxidatively modified fatty acyl chain determines physicochemical properties of aggregates of oxidized phospholipids

In vivo oxidation of glycerophospholipid generates a variety of products including truncated oxidized phospholipids (tOx-PLs). The fatty acyl chains at the sn-2 position of tOx-PLs are shorter in length than the parent non-oxidized phospholipids and contain a polar functional group(s) at the end. The effect of oxidatively modified sn-2 fatty acyl chain on the physicochemical properties of tOx-PLs aggregates has not been addressed in detail, although there are few reports that modified fatty acyl chain primarily determines the biological activities of tOx-PLs. In this study we have compared the properties of four closely related tOx-PLs which differ only in the type of modified fatty acyl chain present at the sn-2 position: 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC). Aggregates of individual tOx-PL in aqueous solution were characterized by fluorescence spectroscopy, size exclusion chromatography, native polyacrylamide and agarose gel electrophoresis. The data suggest that aggregates of four closely related tOx-PLs form micelle-like particles of considerably different properties. Our result provides first direct evidence that because of the specific chemical composition of the sn-2 fatty acyl chain aggregates of particular tOx-PL possess a distinctive set of physicochemical properties.     

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)