Inhibition of Na+-dependent phosphate transport by group-specific covalent reagents in rat kidney brush border membrane vesicles. Evidence for the involvement of tyrosine and sulfhydryl groups on the interior of the membrane

The effects of tyrosine- and sulfhydryl-specific reagents on the Na+-dependent transport of phosphate in brush border membrane vesicles prepared from rat renal cortex were investigated. This study is the first to show that the tyrosine-specific reagents 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and tetranitromethane inactivate the transporter in a concentration- and time-dependent fashion while the membrane impermeant tyrosine reagent, N-acetylimidazole, has no effect on phosphate uptake. The membrane permeant sulfhydryl reagent N-ethylmaleimide also caused a time- and concentration-dependent inactivation of this transport process but the membrane impermeant reagents 7-chloro-4-sulfobenzo-2-oxa-1,3-diazole and eosin-5-maleimide had little effect on phosphate uptake. The inhibitory effects of both tyrosine- and sulfhydryl-specific reagents were additive, but no protection from inactivation by tyrosine-specific reagents could be achieved by preincubation of the vesicles with the substrates of the transporter or with competitive inhibitors of the transport process. These results suggest that the amino acids modified by these agents are located either within the membrane or on the cytosolic surface of the transporter. These residues may not participate in substrate binding, but may be important for the conformational change of the transporter necessary for the translocation of phosphate across these membranes. This study also shows that Na+-dependent phosphate transport can be inactivated by other reagents which covalently modify histidine, carboxyl, and amino groups on proteins.     

Action of spermine on phosphate transport in liver mitochondria

Spermine, at concentrations similar to those normally present in the cytosol of liver cells, facilitates the transport of phosphate into mitochondria and thus its accumulation within the matrix space. Both mersalyl and N-ethylmaleimide (NEM) inhibit phosphate influx either in the absence or in the presence of spermine. These inhibitors also inhibit, but only partially, the efflux from mitochondria of phosphate generated within the matrix space by the hydrolysis of ATP induced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or the valinomycin-K+ system. The inhibition of phosphate efflux by both mersalyl and NEM is almost completely removed, unlike that of phosphate influx, by spermine. The possibility that spermine may induce phosphate efflux by damaging mitochondrial membranes and consequently inducing an unspecific permeability to phosphate is excluded by the full restoration of transmembrane potential once FCCP has been removed by albumin. Since spermine does not react with either thiol groups or thiol group reagents, the simplest explanation of the reported results is that the pathway of phosphate efflux is distinct from that of phosphate influx.     

Inhibition of mitochondrial phosphate transport by p-diazobenzenesulphonate (Short Communication)

The impermeant protein-modifying reagent p-diazobenzenesulphonate inhibits phosphate transport in rat liver mitochondria. The rate of inhibition is increased by the presence of oxidizable substrate, and decreased by uncoupling agents. These results are compared with the results on inhibition of phosphate transport by N-(N-acetyl-4-sulphamoylphenyl)maleimide (Klingenberg et al., 1974).     

Cyclosporin A inhibits the proliferative response and the generation of helper, suppressor and cytotoxic T-cell functions in the autologous mixed lymphocyte reaction

Chemical oxidation or reduction of lymphocyte cell surface thiol or disulfide groups, respectively, has been shown to alter the proliferative activity of murine T cells. S-2-(3-aminopropylamino)ethylphosphothioic acid, a compound containing no free thiol group until it is intracellularly dephosphorylated, did not enhance Con A-induced proliferation which suggested that thiols did not mediate proliferative enhancement via an intracellular mechanism. Glutathione, an impermeant thiol, enhanced T-cell proliferation 68% as effectively as 2-mercaptoethanol (2-ME), which suggested that the thiol-sensitive site was at the cell surface. A battery of structural analogs to 2-ME was employed to elucidate the chemical requirements for the biological activity of the thiols. The necessity for a hydrogen-binding moiety on the thiol reagent was determined by the use of non-hydrogen-binding analogs and by competitive inhibition of the thiol-enhancing activity of 2-ME by non-thiol-containing hydrogen-binding analogs. Pretreatment of cells with the copper:phenanthroline complex (CuP), an impermeant oxidant of thiol groups, reduced the Con A-induced response >79%; however, the presence of 2-ME in culture completely reversed the inhibitory effect of CuP pretreatment. Oxidation of T cells by high oxygen tension (17% O₂) also ablated the Con A response but did not alter the response to Con A + 2-ME. Protection from oxidative inhibition also was afforded T cells by sequential reduction and blockage of cell surface thiol groups. Finally, a model which correlates the chemical study of cell surface residues with T-lymphocyte responsiveness is presented.     

Asymmetrical distribution of thiol groups involved in ATP-32Pi exchange on mitochondrial membranes.

The use of mitoplasts, that is mitochondria devoid of outer membrane oriented as normal mitochondria, and of sonicated vesicles, the membrane of which is inside-out has shown that the thiol groups involved in the process of ATP synthesis are on the matrix face of the mitochondrial membrane: carboxypyridine disulfide (CPDS) a thiol reagent that cannot penetrate across hydrophobic membranes does not inhibit the ATP-³²P_i exchange catalyzed by mitoplasts, while 5,5′-dithio-$\text{bis}$-(2-nitrobenzoate), which penetrates more readily, can completely inhibit this exchange. In contrast, both reagents react similarly with inside-out vesicles. The nature of the component of the ATPase-ATP synthase complex to which this thiol group may belong is discussed.     

Photomodulation of the nucleating activity of a photocleavable crosslinked actin dimer.

The ability to generate substrate concentration jumps through photo-deprotection of amine, carboxyl and phosphate groups has been an important development for investigations of protein activity in complex systems. To broaden the versatility and applications of photo-deprotection techniques for the photomodulation of protein activity we describe the synthesis and characterisation of a reagent for generating free thiol from thioether groups and a related photocleavable, heterobifunctional crosslinking reagent. Chemical and spectroscopic studies of a model thiol protected derivative were used to show some features of thiol group photodeprotection. To demonstrate how the photocleavable crosslinking reagent may be used to modulate the activity of proteins we investigated the effect of light on the nucleating activity of crosslinked actin dimer; thus following near-ultraviolet irradiation of the actin dimer the crosslink was cleaved, presumeably at the thioether bond, resulting in the concomitant dissociation of dimer, loss of nucleating activity and creation of a concentration jump of polymerisable G-actin monomer. On the basis of this initial study we discuss applications and limitations of these reagents for the photomodulation of protein activity in vitro and in vivo.     

Production of thiol groups and retention of calcium ions by cardiac mitochondria.

Efflux of Ca2+ from previously Ca2+-loaded heart mitochondria was measured after inhibiting respiratory activity. The efflux was increased by p-chloromercuribenzoate, methylmercuric chloride, Cu2+, Fe2+, 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (uncoupler). 1,1,1-trifluoro-3-(2-thienylacetone and indomethacin; after such increase it could be diminished by dithiothreitol. The induced loss of the Ca2+ was accompanied by a loss of endogenous adenine nucleotide. Methylmercuric chloride was particularly effective, since it was active at ratios of about 1 nmol/mg of mitochondrial protein. The non-respiring mitochondria were found to regenerate bound thiol groups after their original complement had reacted with thiol-blocking reagent. This regeneration was diminished by the Ca2+-efflux stimulatig agents that were not themselves thiol-blocking reagents, such as thyroxine, uncoupler, trifluorothienylacetone and indomethacin. The external exposure of thiol groups was also diminished by thyroxine, uncoupler and trifluorothienylacetone. The results support the proposal made previously that the membrane is maintained in a state of low permeability by adenine nucleotide and Mg2+ being bound to thiol-dependent sites.     

Parallel efflux of Ca2+ and Pi in energized rat liver mitochondria.

Addition of Ruthenium Red to energized rat liver mitochondria that have previously accumulated Ca2+ and phosphate from the external medium induces a parallel efflux of both these ions. Mersalyl or dithioerythritol, which decrease Ruthenium Red-insensitive Ca2+ efflux, also decrease phosphate efflux to the same extent. Conversely diazenedicarboxylic acid bis(NN-dimethylamide) (DDBA), which increases the Ruthenium Red-induced Ca2+ efflux concurrently increases phosphate release. Dithioerythritol and DDBA, reducing and oxidizing agents of thiol groups respectively, modify Ca2+ and Pi efflux without penetrating the mitochondrial inner membrane. Under all the adopted conditions the membrane potential is preserved. The release of resting respiration and the parallel efflux of Mg2+ and adenine nucleotides, events closely correlated to Ca2+ cycling, are equally prevented either by mersalyl, which inhibits phosphate transport, or dithioerythritol; DDBA has the opposite effect. These findings and the observation that suggest that Ca2+ and phosphate transport in energized liver mitochondria are closely related and dependent on the redox state of membrane-bound thiol groups.     

Interaction between catalytic and regulatory sites of the pyruvate dehydrogenase from Escherichia coli studied by the ESR technique

The accessibility of sulfhydryl groups at the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was reinvestigated. Hydrophobic interactions appear to control the reactivity of an essential cysteine residue at the active site with thiol reagents. This explains why the essential cysteine residue reacts only with thiol reagents of minor polarity, like p-hydroxymercuribenzoate or phenylmercuric nitrate, but not with Ellman's reagent or jodoacetamide. The pyruvate dehydrogenase component was modified with a nitroxide derivative of p-hydroxymercuribenzoate. The ESR spectrum of the spin-labelled enzyme changed dramatically upon addition of the cofactors thiamine diphosphate and Mg2+. Obviously spin-spin interaction occurs under these conditions caused by a transition of an inactive to an active state of the enzyme. The same conformational change is observed when the allosteric activator AMP instead of the cofactors was bound to the enzyme. The implications of these results for the allosteric regulation of the pyruvate dehydrogenase complex are discussed.     

Abnormal lysosomal hydrolases excreted by cultured fibroblasts in I-cell disease (mucolipidosis II).

The characteristics of rat liver mitochondria swelling induced by diamide, an oxidizing agent for thiol groups, and by Ca ions are very similar. In both cases the swelling, which is initiated by addition of 0.5–1 mM phosphate or acetate, is prevented by FCCP, antimycin A, EGTA, Mg⁺⁺ and ruthenium red. Diamide potentiates the swelling action of Ca⁺⁺, while DTE potentiates that of Mg⁺⁺. The additive effects of calcium and diamide on rat liver mitochondria have been correlated with their synergic action in promoting the release of mitochondrial Mg⁺⁺. The results strongly indicate that some of the effects of diamide are mediated by a mobilization of endogenous divalent ions and that the antagonism between Ca⁺⁺ and Mg⁺⁺ is closely correlated with the redox state of membrane bound thiol groups.     

Phosphate transport protein of rat heart mitochondria: location of its SH-groups and exploration of their environment

(1) The properties of the SH groups of the phosphate transport protein of rat heart mitochondria were investigated on the basis of inhibition caused by SH reagents under different conditions. (2) The essential thiol groups are located near the external surface, as they are accessible to impermeable reagents from the external space. (3) The environment of the sulfhydryl groups influences their reactivity, as alteration of the external pH affects adversely their reactions with ionizable and non-ionizable SH reagents. (4) Intramitochondrial pH exerts a transmembrane effect: alkalinization augments and acidification diminishes the reaction rate of the sulfhydryl groups on the opposite surface of the membrane. (5) Changes of the concentration of the transported substrate occurring exclusively in the extramitochondrial space do not influence the reactivity of the essential SH groups. (6) It is concluded that in transport studies the phosphate transport protein of heart and liver mitochondria show basic similarity. It is suggested that the amino-acid sequence around the NEM-reactive cysteine (i.e., Lys-41 - Cys-42 - Arg-43) does not participate in substrate binding.     

The uncoupling protein dimer can form a disulfide cross-link between the mobile C-terminal SH groups

Isolated uncoupling protein (UCP) can be cross-linked, by various disulfide-forming reagents, to dimers. The best cross-linking is achieved with Cu2+-phenanthroline oxidation. Because cross-linking is independent of UCP concentration and prevented by SDS addition, a disulfide bridge must be formed between the two subunits of the native dimer. Cross-linking is prevented by SH reagent and reversed by SH-reducing reagents. In mitochondria, cross-linking of UCP with disulfide-forming agents is even more efficient than in isolated state. It proves that UCP is a dimer in mitochondria, before isolation. Disulfide-bridge formation does not inhibit GTP-binding to UCP. Cross-linked UCP re-incorporated in proteoliposomes either before or after cross-linking fully retains the H1-transport function. Rapid cross-linking by membrane impermeant reagents indicates a surface localization of the C-terminus in soluble UCP and projection to the outer surface in mitochondria. Intermolecular disulfide-bridge formation in a dimer requires juxtaposition of identical cysteines at the twofold symmetry axis. A rigid juxtaposition of cysteines is unlikely, unless intended for a native disulfide bridge. The absence of such a bridge in UCP suggests that juxtaposition of cysteines is generated by high mobility. In order to localize the cysteine involved, cross-linked UCP was cleaved by BrCN. The CB-7 C-terminal peptide, which contains cysteines at positions 287 and 304, disappears. Limited trypsinolytic cleavage, previously shown to occur at Lys-292, removed cross-linking in UCP both in the solubilized and mitochondrially bound state. The cleaved C-terminal peptide of 11 residues contains only cystein-304 which, thus, should be the only one (out of 7 cysteines in UCP) involved in the S-S bridge formation. Obviously, the C-terminal location of the cysteine, because of its high mobility, permits juxtapositioning for cross-linking. This agrees with predictions from hydrophobicity analysis that the last 14 residues in UCP protrude from the membrane.     

Evidence for a role of a vicinal dithiol in the transport of gamma-butyrobetaine in Agrobacterium sp

An Agrobacterium sp. isolated from soil is able to use gamma-butyrobetaine as its sole source of carbon and nitrogen. The involvement of thiol groups for active transport of gamma-butyrobetaine was investigated by use of the thiol alkylating reagent N-ethylmaleimide (NEM) and the dithiol specific reagent phenylarsine oxide (PAO). Both reagents strongly inhibited gamma-butyrobetaine uptake, but also induced the release of the accumulated substrate, suggesting that the transport system either contains a dithiol-dependent protein or that a small thiol-containing molecule is implicated in the uptake phenomenon.     

Thiols related to mitochondrial ATPase and transports: unmasking upon conformational changes supported by the comparative effects of ethacrynate and dihydroethacrynate.

Comparison between the effects on various rat liver mitochondrial functions of ethacrynate, a thiol reagent inhibitor of oxidative phosphorylations [3, 4] and those of dihydroethacrynate its saturated derivative which is not a thiol reagent, has been performed. Both, ethacrynate and dihydroethacrynate increase oxygen consumption by mitochondria in state 4 (succinate as substrate) in a concentration dependent way (from 1 to 5 X 10(-4) M EA or DHEA). This activation is followed, only with ethacrynate, by an inhibition appearing sooner with higher concentrations. After preincubation or mitochondria with ethacrynate (1 to 5 X 10(-4) M), the stimulation of respiration by (ADP + Pi) is completely inhibited whereas it is only weakly affected by dihydroethacrynate at the same concentrations. Ethacrynate and dihydroethacrynate provoke variations of intramitochondrial Mg2+ and K+ levels which need energy from the respiratory chain. These are affected by Pi or (Pi + ADP) in a different way with ethacrynate and with dihydroethacrynate. After preincubation with mitochondria, ethacrynate and to a smaller extent dihydroethacrynate, inhibit partially ADP translocation; ADP increases the inhibitory effect of EA on translocation and not that of dihydroethacrynate. Ethacrynate increases the oligomycin sensitive ATPase activity and dihydroethacrynate still more. After a ten minutes preincubation with mitochondria, ethacrynate and dihydroethacrynate hardly affect the 2.4 DNP stimulated ATPase activity. Preincubation with succinate or ADP strongly increases the ethacrynate inhibition whereas it decreases dihydroethacrynate inhibition. Ethacrynate and dihydroethacrynate do not affect the efflux of Pi produced by ATP hydrolysis but ethacrynate enforces the inhibitory effect of mersalyl (Mg2+ containing medium). After ten minutes of preincubation with mitochondria, ethacrynate binds 25 nmoles of -SH/mg protein (DTNB titration) and dihydroethacrynate has no effect. These results show an effect of ethacrynate on two types of thiols linked with energy conservation mechanisms and ADP translocation. These thiols could be unmasked or made accessible by conformational modifications of the inner membrane upon energization or addition of ADP.     

Extracellular phosphate requirement for insulin action on isolated rat hepatocytes

Isolated rat hepatocytes were prepared in KHB buffer, pH 7.4; were centrifuged and washed twice in KHB buffer containing various amounts of phosphate and calcium; and were incubated at 30 degrees in the presence of tracer [2,3-14C]succinate and a 0.5 mM concentration of each of the 20 natural amino acids. Hepatocytes washed and incubated in KHB buffer containing less than 0.1 mM phosphate failed to show any insulin stimulation of [2,3-14C]succinate oxidation or protein incorporation of tracer carbons. The absence or presence of extracellular phosphate did not alter the specific activity of 32P-adenine nucleotides; they remained the same in the presence or absence of insulin. The maximal insulin stimulatory effect on succinate oxidation and tracer incorporation into protein was observed in the presence of 1.18 mM phosphate and 1.9 mM calcium ion. The lack of external phosphate did not prevent the stimulation of succinate oxidation by either glucagon on epinephrine, whereas removal of calcium from the medium abolished their hormonal effects. The lack of medium calcium also prevented the insulin stimulation of succinate oxidation and protein synthesis. Our data indicate that a diminished insulin responsiveness in hypophosphatemic patients may be due to the insensitivity of mitochondria to insulin in the hypophosphatemic state.     

Oligomycin sensitivity of mitochondrial sulfhydryl groups

Dithionitrobenzoate has been used to titrate sulfhydryl groups of rat liver mitochondria in glutamate buffer, pH 7.4.Reaction with oligomycin and different SH reagents preceded the SH titration. Under these conditions it was found that 2-mercaptopropionylglycine and N-ethylmaleimide reacted in an oligomycin-sensitive manner, so that the control values (in the absence of SH reagent) were obtained.Similar concentrations of mersalyl and of N-(N-acetyl-4-sulfamoylphenyl) maleimide, in the presence of oligomycin, enhanced reactivity toward Nbs₂.The concentration range of oligomycin-sensitive SH groups was thus defined between approx. 5 and 9 nmol reagent/mg mitochondrial protein.In this way, a differentiation between SH groups, which are implicated in phosphate transport and those, which react in an oligomycin-sensitive manner, and which are probably connected with the coupling mechanism, was achieved.     

Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.     

The soluble carnitine palmitoyltransferase from bovine liver. A comparison with the enzymes from peroxisomes and from the mitochondrial inner membrane.

The properties of two carnitine acyltransferases (CPT) purified from bovine liver are compared to confirm that they are different proteins. The soluble CPT and the inner CPT from mitochondria differ in subunit Mr, native Mr, pI and reactivity with thiol reagents. All eight free thiol groups in soluble CPT react with 5,5'-dithiobis-(2-nitrobenzoate) in the absence of any unfolding reagent, and activity is gradually lost. The inner CPT activity is completely stable in the presence of 5,5'-dithiobis-(2-nitrobenzoate), and only one thiol group per molecule of subunit is modified in the native enzyme. Antisera to each enzyme inhibit that enzyme, but do not cross-react. CPT activity in subcellular fractions can now be identified by titration with these antibodies. The soluble CPT from bovine liver is probably peroxisomal in origin, but, although antigenically similar, it differs from the peroxisomal carnitine octanoyltransferase found in rat and mouse liver in its specificity for the longer-chain acyl-CoA substrates.     

Bromobimane crosslinking studies in oligomycin-sensitive ATPase from beef heart mitochondria. Mr 31 000 protein crosslinked

Using a bromobimane fluorescent label the Mr 31 000 protein band oligomycin-sensitive (OS)-ATPase from beef heart mitochondria is shown to become much intensified by 2-mercaptopropionylglycine. In the presence of 3.5 nmol/mg protein of the thiol reagent ATP-Pi exchange activity is increased by 90%. With the fluorescent crosslinking reagent dibromobimane (DB) we show that a new fluorescent peak appears between Mr 50 000 and 60 000. ATP-Pi exchange is very much decreased by DB. The results suggest that for regulation of ATP-synthetase activity sulfhydryl groups in the region of the Mr 31 000 protein(s) play an important role.