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The ethylmaleimide-sensitive phosphate-transport system of heart mitochondria was isolated and the activity reconstituted in liposomes. The identification of the phosphate-carrier protein was complicated by several factors. 1 The phosphate-carrier fraction, isolated through different procedures in different laboratories, contains 4–5 protein components of very similar apparent molecular weight, as shown by gradient dodecylsulfate gel electrophoresis. 2 The amino acid composition of protein 1, 2 and 3 (apparent Mr= 34500, 34000 and 33000, respectively) is quite similar although not identical. 3 Including several protease inhibitors during isolation of the phosphate-carrier fraction does not influence the protein pattern. 4 Labeled N-ethylmaleimide binds only protein 1 and 3, labeled N,N′-dicyclohexylcarbodiimide bonds only to protein 1 and 2. 5 N,N′-Dicyclohexylcarbodiimide had no effect on the reconstituted phosphate-exchange activity after incubation with intact mitochondria or proteoliposomes. 6 In the presence of protecting mersalyl concentrations N-ethylmaleimide inhibits the phosphate transport in intact mitochondria. Under these conditions no label ethylmaleimide was bound to the phosphate-carrier fraction. 7 Comparison of the reconstituted transport activity with the protein pattern of fractions, isolated with or without cardiolipin, shows correlation of transport with the amount of protein 2 but not with that of protein 1, which binds ethylmaleimide. A model of the mitochondrial phosphate carrier is presented which suggests a proteolytic degradation of the phosphate-carrier protein during its isolation. The model explains the contradictory results of this investigation.

Recent studies have suggested that parts of the hepatic activities of diacylglycerol acyltransferase and acyl cholesterol acyltransferase are expressed in the lumen of the endoplasmic reticulum (ER). However the ER membrane is impermeable to the long-chain fatty acyl-CoA substrates of these enzymes. Liver microsomal vesicles that were shown to be at least 95% impermeable to palmitoyl-CoA were used to demonstrate the membrane transport of palmitoylcarnitine and free L-carnitine - processes that are necessary for an indirect route of provision of ER luminal fatty acyl-CoA through a luminal carnitine acyltransferase (CAT). Experimental conditions and precautions were established to permit measurement of the transport of [14C]palmitoylcarnitine into microsomes through the use of the luminal CAT and acyl-CoA:ethanol acyltransferase as a reporter system to detect formation of luminal [14C]palmitoyl-CoA. Rapid, unidirectional transport of free L-[3H]carnitine by microsomes was measured directly. This process, mediated either by a channel or a carrier, was inhibited by mersalyl but not by N-ethylmaleimide or sulfobetaine - properties that differentiate it from the mitochondrial inner membrane carnitine/acylcarnitine exchange carrier. These findings are relevant to the understanding of processes for the reassembly of triacylglycerols that lipidate very low density lipoprotein particles as part of a hepatic triacylglycerol lipolysis/re-esterification cycle.

The accumulation of carnitine was measured in cerebral cortex neurons isolated from adult rat brain. This process was found to be lowered by 40% after preincubation with ouabain and with SH-group reagents (N-ethylmaleimide and mersalyl). The initial velocity of carnitine transport was found to be inhibited by 4-aminobutyrate (GABA) in a competitive way (Ki = 20.9 +/- 2.4 mM). However, of various inhibitors of GABA transporters, only nipecotic acid and very high concentrations of 1-[2-([(diphenylmethylene)amino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO-711) acid decreased carnitine accumulation while betaine, taurine and beta-alanine had no effect. The GABA transporters expressed in Xenopus laevis oocytes did not transport carnitine. Moreover, carnitine was not observed to diminish the accumulation of GABA in cerebral cortex neurons, which further excluded a possible involvement of the GABA transporter GAT1 in the process of carnitine accumulation, despite the expression of this protein in the cells under study. The absence of carnitine transporter OCTN2 in rat cerebral cortex neurons (K. A. Nalecz, D. Dymna, J. E. Mroczkowska, A. Broer, S. Broer, M. J. Nalecz and R. Cecchelli, unpublished results), together with the insensitivity of carnitine accumulation towards betaines, implies that a novel transporting protein is present in these cells.

Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin Km and Vmax values were 4.4 ± 1.3 µm and 35 ± 5 pmol·min−1·(mg protein)−1, respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent Km and apparent Vmax values for FMN were 5.4 ± 0.9 µm and 22.9 ± 1.4 pmol·min−1·(mg matrix protein)−1, respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.

Substrate hydrolysis by the H+-pyrophosphatase (pyrophosphate phosphohydrolase, H+-PPase) of the photosynthetic bacterium Rhodospirillum rubrum follows a two-pathway reaction scheme in which preformed 1:1 and 1:2 . enzyme . Mg2+ complexes (EMg and EMg2) convert dimagnesium pyrophosphate (the substrate). This scheme is applicable to isolated enzyme, uncoupled chromatophores and chromatophores energized by a K+/valinomycin diffusion potential. Tris and other amine buffers exert a specific effect on the bacterial H+-PPase by increasing the Michaelis constant for substrate binding to EMg by a factor of 26-32, while having only small effect on substrate binding to EMg2. Formation of EMg requires a basic group with pKa of 7.2-7.7 and confers resistance against inactivation by mersalyl and N-ethylmaleimide to H+-PPase. The dissociation constants governing EMg and EMg2 formation, as estimated from the mersalyl-protection assays and steady-state kinetics of PPi hydrolysis, respectively, differ by an order of magnitude. Comparison with the data on soluble PPases suggests that, in spite of gross structural differences between H+-PPase and soluble PPases and the added ability of H+-PPase to act as a proton pump, the two classes of enzyme utilize the same reaction mechanism in PPi hydrolysis.

Metabolic control analysis was applied to describe the control of mitochondrial oxidative phosphorylation in calcium (approximately 2 microM free calcium) activated saponin-skinned rat musculus soleus fibers oxidizing glutamate and malate. Under these circumstances approximately 80% of mitochondrial active-state respiration was reached due to the activation of ATP turnover by actomyosin ATPase. The flux control coefficients of H(+)-ATPase, adenine-nucleotide translocase, phosphate transporter, NADH:ubiquinone oxidoreductase and cytochrome-c oxidase were determined to be equal to 0.16 +/- 0.08 (n = 6), 0.34 +/- 0.12 (n = 5), 0.08 +/- 0.03 (n = 5), 0.01 +/- 0.006 (n = 4) and 0.09 +/- 0.03 (n = 3) using inhibitor titrations with the specific inhibitors oligomycin, carboxyatractyloside, mersalyl, rotenone and cyanide, respectively, and applying non-linear regression of the entire titration curve. The flux control coefficient of actomyosin ATPase was determined with vanadate to be equal to 0.50 +/- 0.09 (n = 6), measuring independently the vanadate-caused inhibition of fiber respiration and ATP-splitting activity. In contrast to results with isolated rat skeletal muscle mitochondria reconstituted with soluble F1-ATPase the decrease in phosphate concentration from 10 mM to 1 mM only slightly affected the distribution of flux control coefficients. This difference is caused by different kinetic properties of soluble F1-ATPase and actomyosin ATPase. Therefore, phosphate seems to be in skeletal muscle in vivo only a modest modulator of control of oxidative phosphorylation.

An NADPH-dependent membrane-bound flavoprotein dehydrogenase, assayed as a catalyst of electron transfer from NADPH to cytochrome c, was extracted from membranes of rabbit peritoneal neutrophils with Triton X-100 and sodium deoxycholate in the presence of diisopropylfluorophosphate as antiprotease, and purified to electrophoretic homogeneity. The purified enzyme in detergent was able to enhance the rate of formation of the superoxide anion O2- in a cell-free system, consisting of membrane and cytosolic fractions from resting neutrophils complemented with arachidonic acid, guanosine 5'-[gamma- thio]triphosphate and Mg2+. This suggested that the NADPH dehydrogenase was a component of the rabbit neutrophil oxidase complex. The purification factor of the enzyme with respect to the membrane fraction was close to 1000 and the recovery of activity was 33%. FMN and FAD were associated with the enzyme in a molar ratio close to 1. On SDS/PAGE, the enzyme migrated with a molecular mass of 77 kDa. A similar mass was determined by filtration on a molecular sieve. The isoelectric point of this enzyme was 4.7 +/- 0.1. Its activity was maximal between pH 7.5 and pH 8.5, and depended on the ionic strength of the medium, with a maximum at an ionic strength of 0.5. Reduction of cytochrome c by NADPH obeyed Michaelis-Menten kinetics with a KM value of 15 microM for cytochrome c. When NADPH was the variable substrate, a KM value of 1.9 microM for NADPH was found, but a significant deviation from Michaelis-Menten kinetics was observed at high concentrations of NADPH. Mersalyl strongly inhibited the reductase activity when added to the enzyme prior to NADPH; preincubation of the enzyme with NADPH considerably reduced the inhibitory efficiency of mersalyl. A partially proteolyzed water-soluble, active, form of enzyme with a molecular mass of 67 kDa was prepared. The proteolyzed enzyme exhibited the same specificity, and kinetic behavior with respect to NADPH, and the same dependency on the ionic strength, as the native enzyme.

Recent studies have suggested that parts of the hepatic activities of diacylglycerol acyltransferase and acyl cholesterol acyltransferase are expressed in the lumen of the endoplasmic reticulum (ER). However the ER membrane is impermeable to the long-chain fatty acyl-CoA substrates of these enzymes. Liver microsomal vesicles that were shown to be at least 95% impermeable to palmitoyl-CoA were used to demonstrate the membrane transport of palmitoylcarnitine and free L-carnitine - processes that are necessary for an indirect route of provision of ER luminal fatty acyl-CoA through a luminal carnitine acyltransferase (CAT). Experimental conditions and precautions were established to permit measurement of the transport of [14C]palmitoylcarnitine into microsomes through the use of the luminal CAT and acyl-CoA:ethanol acyltransferase as a reporter system to detect formation of luminal [14C]palmitoyl-CoA. Rapid, unidirectional transport of free L-[3H]carnitine by microsomes was measured directly. This process, mediated either by a channel or a carrier, was inhibited by mersalyl but not by N-ethylmaleimide or sulfobetaine - properties that differentiate it from the mitochondrial inner membrane carnitine/acylcarnitine exchange carrier. These findings are relevant to the understanding of processes for the reassembly of triacylglycerols that lipidate very low density lipoprotein particles as part of a hepatic triacylglycerol lipolysis/re-esterification cycle.

We have used site-directed chemical labelling to demonstrate the membrane topology and to identify neighbouring subunits of subunit 8 (Y8) in yeast mitochondrial ATP synthase (mtATPase). Unique cysteine residues were introduced at the N or C-terminus of Y8 by site-directed mutagenesis. Expression and targeting to mitochondria in vivo of each of these variants in a yeast Y8 null mutant was able to restore activity to an otherwise nonfunctional ATP synthase complex. The position of each introduced cysteine relative to the inner mitochondrial membrane was probed with thiol-specific nonpermeant and permeant reagents in both intact and lysed mitochondria. The data indicate that the N-terminus of Y8 is located in the intermembrane space of mitochondria whereas the C-terminus is located within the mitochondrial matrix. The proximity of Y8 to other proteins of mtATPase was tested using heterobifunctional cross-linking reagents, each with one thiol-specific reactive group and one nonspecific, photoactivatible reactive group. These experiments revealed the proximity of the C-terminal domain of Y8 to subunits d and f, and that of the N-terminal domain to subunit f. It is concluded that Y8 possesses a single transmembrane domain which extends across the inner membrane of intact mitochondria. As subunit d is a likely component of the stator stalk of mitochondrial ATP synthase, we propose, on the basis of the observed cross-links, that Y8 may also be part of the stator stalk.

Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I-sensitive component(s). In the presence of a concentration of Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2- production by Ph2I. The O2(-)-generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2- production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.

The ornithine/citrulline carrier from rat liver mitochondria, solubilized with Triton X-100 and partially purified on hydroxyapatite, was identified and completely purified by PD-10, DEAE-Sephacel and celite chromatography. On SDS/polyacrylamide gel electrophoresis, the purified ornithine/citrulline carrier consisted of a single protein band with an apparent molecular mass of 33.5 kDa. When reconstituted into liposomes the ornithine carrier protein catalyzed an active mersalyl sensitive ornithine/ornithine exchange. It was purified 438-fold with a recovery of 38% and a protein yield of 0.09% with respect to the extract derived from mitoplasts. The purified and reconstituted protein did not catalyze a significant unidirectional transport of ornithine. Citrulline was found to be the best countersubstrate for the transport of ornithine, followed by lysine and arginine. The exchange activity was inhibited by several sulphydryl reagents.

Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD+-dependent and NADP+-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca2+-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The K(m) for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The V(max) was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 μM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation. (Less)

1Pyruvate equilibrates, like other carboxylic acids, across the membrane of rat-liver mitochondria according to the transmembrane pH difference. 2Comparison of the rate of pyruvate, acetoacetate and acetate efflux from mitochondria as well as a study of the effect of mersalyl and N-ethylmaleimide on this process, show that the diffusion of pyruvic and acetoacetic acid across the mitochondrial membrane is carrier-mediated. The process might consist of pyruvate (acetoacetate) hydroxyl ion exchange-diffusion or of pyruvic acid (acetoacetic acid) uniport. On the contrary acetic acid appears to diffuse freely across the membrane. 3The initial rate of the net pyruvate uptake by rat liver mitochondria follows saturation kinetics. The increase of the external pH of the medium as well as the addition of acetoacetate inhibit competitively pyruvate uptake. Pyruvate uptake is inhibited by mersalyl and N-ethylmaleimide. 4Mitochondria1 [14C]pyruvate exchanges with unlabelled pyruvate and acetoacetate but not with other monocarboxylates, Pi and citric-acid cycle intermediates. 5The pyruvate-pyruvate and pyruvate-acetoacetate exchanges follow saturation kinetics and exhibit the same V but different Km values. Both the exchanges are inhibited by mersalyl, but not by N-ethylmaleimide. 6It is concluded that pyruvate translocation across the mitochondrial membrane is mediated by a specific transport system.

A specific labeling by radioactive N-ethylmaleimide of a protein involved in phosphate transport was obtained by protecting one of the two -SH groups of the transport system with low concentrations of mersalyl. Subsequently, the other free -SH groups were blocked with excess N-ethylmaleimide. Removal of mersalyl by cysteine and subsequent incubation with labeled N-ethylmaleimide results in a “specific” binding of N-ethylmaleimide to one - SH group functionally involved in phosphate transport. The isolated inner membrane fraction of the labeled mitochondria was subjectcd to dodecylsulfate gel electrophoresis. The following results were obtained. 1 The difference of the radioactivity pattern on the dodecylsulfate-polyacrylamide gel of inner membrane proteins, labeled with N-[14C]ethylmaleimide in the absence and with N-[3H]ethylmaleimide in the presence of mersalyl during preincubation of mitochondria, shows only one main labeled peak. The same labeled peak is obtained from the difference of labeling after preincubation with a constant low concentration of mersalyl at 32 °C and at 0 °C. 2 The position of the labeled peak on the dodecylsulfate-polyacrylamide gel corresponds to a protein of molecular weight of 26500 ± 800. 3 The amount of one of the two - SH groups, involved in phosphate transport, was estimated to be 30 nmol per g of mitochondrial protein.

The phosphate transport protein (PTP) has been isolated from beef heart mitochondria in the presence of cardiolipin and reconstituted in asolectin and phosphatidylcholine vesicles. As expected, the activity of the reconstituted PTP is inhibited by N-ethylmaleimide and mersalyl. It is also inhibited by adriamycin and Br-daunomycin. 1 Storage of isolated mitochondria at - 80 °C prior to the isolation of the PTP decreases the maximal activity of the unidirectional transport of phosphate (Pi) in reconstituted vesicles. The sensitivity of the system to the four inhibitors remains the same. 2 The inhibition of the PTP by adriamycin is reversed by KCl and prevented by MgSO4. Since the interaction between adriamycin and cardiolipin is of ionic type, KCl apparently replaces adriamycin on the negative charges of cardiolipin. By contrast, MgSO4 complexes adriamycin directly, eliminating its inhibitory effect on the PTP. 3 No PTP activity is found after reconstitution in phosphatidylcholine vesicles in the absence of cardiolipin. Addition of buffer-dispersed cardiolipin to the vesicles restores the Pi-transport activity. The addition of adriamycin to the vesicles together with cardiolipin removes the reactivation. 4 Succinylation of the PTP at pH 8.0 eliminates the inhibitory effect of adriamycin in the reconstituted system. The effects of N-ethylmaleimide and mersalyl are not modified. The succinylated preparation reconstituted in phosphatidylcholine vesicles is reactivated by cardiolipin, but in this case the reactivation is not counteracted by adriamycin. 5 Succinylation of the PTP at pH 9.0 results in its complete inhibition. 6 The results indicate, (a) that the sites of interaction of MalNEt/mersalyl and adriamycin with the PTP · cardiolipin complex are different, and (b) that the adriamycin-binding site in the complex is probably on cardiolipin molecules essential to the PTP activity. It is postulated that succinylation of the PTP prevents the interaction of adriamycin with these molecules.

1 A rat olfactroy preparation, suitable for biochemical studies in vitro on olfactory mechanisms, is described. 2 The effects of the impermeant modification reagents mersalytl (a thiol reagent) and enzymatic iodination, on the amplitude of the electroolfactogram (EOG) responses elicited from rat olfactory mucosa by pulses of odorant vapours was studied using 12 odorants differing widely in odour qualituy and molecular structure: amyl acetate, carvone, decanal, butylamine, cineole, citronello, cresol, diacetyl, dimethylethylpyrazine, naphthalene, octanethiol and valeric acid. 3 Both reagents irreversibly reduced the EOG amplitude to all odorants to an extent dependent on the reagent concentration. Two subpopulations of animal preparations could be distinguished on the basis of the extent to which they survived the iodination whereas mersalyl appeared to sample a single population of preparations. 4 Small but statistically significant differences werer observed between the responses of each odorant with each reagent but no simple correlation between either the molecular structure or odour quality of the odorants and the reagent effect is apparent for the case of mersalyl. With iodination the responses from the three flexible-aliphatic-chain odorants were reduced to a greater extent than the other odorants, all of which had a dissimilar molecular structure. 5 The ability of three odorants amyl acetate, carvone and decanal, to protect the receptors for the odoransts against chemical modification was investigated. the protecting odorants were applied directly to the mucosa as a dilute solution in Ringer's medium. No specific odorant protection effects were observed. 6 The results are discussed in relation to a model of the olfactory mechanism involving relatively non-specific rece3ptor proteins. Each receoptor type is envisaged as interacting weakly with a number of odorants and each odorant interacts with a numnber of receptors.

Uptake of the hepatodiagnostic dye bromosulfophthalein into isolated hepatocytes was studied with special regard to the kinetics of transport. The following results were obtained. 1 The uptake of bromosulfophthalein follows Michaelis-Menten kinetics only at low substrate concentrations with an apparent Km= 7 ± 2 μM and V= 2.6 ± 1.7 nmol × mg protein−1× min−1′. At higher bromosulfophthalein concentrations a second mechanism of uptake is observed as indicated by the deviation from linearity in the Lineweaver-Burk plot. 2 The activation energy of uptake was found to be 11 kcal/mol at 10 μM bromosulfophthalein. 3 Uptake is independent of metabolic energy and of the Na+ gradient across the membrane. 4 Taurocholate does not inhibit uptake while indocyanine green inhibits competitively at low bromosulfophthalein concentrations and activates uptake at high bromosulfophthalein concentrations (> 201 μM). 5 Amino acid reagents, such as dinitrofluorobenzene, mersalyl, N-ethylmaleimide, and dithionitrobenzene, which modify specific functional groups, did not affect uptake at a concentration of 100 μM. 6 No pH optimum for bromosulfophthalein uptake was observed in the physiological pH range. 7 Adsorption of bromosulfophthalein to the liver cell membrane has two distinguishable site, with affinities K1= 5.7 × 10−6 M and K2=7 × 10−5 M and binding capacities n1= 1.2 nmol/mg protein and n2= 7 nmol/mg protein. Adsorption is inhibited by indocyanine green. The results do not indicate the mediation of bromosulfophthalein uptake by a carrier protein and are consistent with the hypothesis that bromosulfophthalein is bound in an energy-consuming process to a translocating site, possibly in the undissociated form or as ion pair. The consecutive transfer across the membrane appears to require little additional energy.

Previous investigation showed two distinct ATP-dependent proton-transporting systems in microsomal vesicle from radish seedlings, one inhibited by vanadate and one inhibited by NO-3. On the bases of the effects of these inhibitors we could discriminate two distinct ATPase activities in the same material. The NO-3 sensitive activity was separated from the vanadate-sensitive activity and partially purified by a single-step chromatographic method, which lead to approx 35-fold purification from the microsomes and to a specific activity of 2.3 mumol Pi X min-1 X mg protein-1, at 30 degrees C. The partially purified activity was specific for ATP, some activity being observed toward GTP, and even less toward CTP, UTP and ITP. No significant Pi hydrolysis was found with ADP, AMP, p-nitrophenylphosphate and glucose 6-phosphate. ADP but not AMP was inhibiting in the presence of ATP. The activity was dependent on divalent cations in the order of preference: Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+ greater than Zn2+. The activity was unaffected by monovalent cations, strongly activated by Cl-, inhibited by 90% by 50 mM NO-3, virtually unaffected by oligomycin and NaN3. At least 90% of the activity was abolished in the presence of each: 10 microM N,N'-dicyclohexylcarbodiimide, 10 microM erythrosin B, 10 mu mersalyl, 100 microM trimethyltin, 100 microM diethylstilbestrol, 100 microM N-ethylmaleimide. No inhibition has been found in the presence of Ca2+, at a concentration blocking the vanadate-sensitive activity. Nigericin, gramicidin and carbonylcyanide p-trifluoromethoxyphenylhydrazone stimulated the activity of this preparation after it was incubated in the presence of sonicated phospholipids, suggesting the capacity of the ATPase to function as a H+-transporting system. All characteristics mentioned were closely similar to those described in the vacuolar ATPases.

1 A highly specific labeling of a phosphate-transporting protein from rat-liver mitochondria was obtained with [203Hg]mersalyl under the following conditions: (a) labeling of mitoplasts, isolated by the French-Press procedure; (b) washing of the isolated inner membranes with NaCl/octylglucoside. 2 The concentration-dependent labeling of the protein with mersalyl showed a biphasic saturation which correlated well with the protection and inhibition of phosphate transport. 3 The molecular weight (Mr 31000) and the amount of labeled carrier protein (24 nmol/g mitochondrial protein) at 100 protection correlated well with published data from the literature. 4 Gel filtration of the labeled protein in the presence of non-ionic detergents showed a molecular weight of at least twice of that obtained by gel electrophoresis in the presence of sodium dodecylsulfate.

Ehrlich ascites tumour cells contain a neutral protease, capable of solubilising fluorescein-labelled telopeptides from fluorescein-labelled polymeric collagen fibrils. The cells also contain an inhibitor for this enzyme and for trypsin. The enzymically inactive enzyme-inhibitor complex can be dissociated with the mercurial thiol agent, mersalyl, with the consequent regain of enzymic activity. The reactivated neutral protease and also trypsin can be inhibited by addition of thiols such as cysteine, mercaptoethanol and dithiothreitol. Trypsin can be protected from inactivation by the tumor inhibitor by addition of cystine or L-1-tosylamido-2-phenylethyl chloromethyl ketone(TosPheCH2Cl)-inactivated chymotrypsin. The evidence suggests that the inhibitor contains a reactive thiol group which exchanges with one or more significant disulphide bridges in trypsin and the neutral protease, resulting in enzyme-inhibitor complex formation and loss of activity. Similarly, thiols interact with these enzymes resulting in a corresponding loss of enzymic activity. The evidence obtained with Tos-PheCH2Cl-inactivated chymotrypsin, which reactivated previously inhibited trypsin and neutral protease, demonstrates that the active site of the enzyme is not involved in the interaction with the thiol of the inhibitor but that the significant disulphide bond in the enzyme is required for the maintenance of the active site conformation. This disulphide exchange mechanism is therefore a form of reversible allosteric control of proteolytic activity and has been shown to be distinct from the mechanism by which soya bean trypsin inhibitor interacts with trypsin.

The adenylate cyclase activity from a rat liver plasma membrane preparation was inhibited by low concentrations (1-10 muM) of the mercurial diuretic mersalyl. Complete inhibition was obtained with 0.1 mM mersalyl. Similar effects were observed whether the adenylate cyclase preparation was assayed in the presence of 10 muM GTP, 0.1 muM glucagon, 10 mM NaF or without any addition. The effect of mersalyl was not due to inhibition of the regenerating system present in the incubation medium, since the effect of mersalyl was preserved and even enhanced in its absence. The inhibition brought about by mersalyl was due to both a decrease of the maximal velocity of the reaction and of the affinity of the enzyme for the substrate. It was immediate, and irreversible spontaneously, but it was reversed by the simultaneous additions of 2-mercaptoethanol, in a dose-dependent fashion. Other -SH reagents were found to have an effect equal to, or lower than, that of mersalyl. Mersalyl had no effect upon Mg2+-ATPase, although it inhibited the (Na+-K+) activated ATPase. Since mersalyl is known to be a 'non-penetrant' reagent, it is postulated that a catalytically important, mercurial-sensitive, part of adenylate cyclase is at the surface of the plasma membrane. This view is supported by the following facts: (a) mersalyl acted with a similar dose-response curve upon an intact as well as a detergent-dispersed cyclase preparation while no effect was observed upon a solubilized Mg2+-ATPase preparation; (b) a covalent p-chloromercuribenzoate-Sephadex preparation (but not its supernatant) inhibited the cyclase from intact membranes. It is proposed that mercurial derivatives, by their relative specificity of action (no effect on Mg2+-ATPase), can serve as useful probes in the elucidation of the multicomponent structure of the cyclase system.

A membrane-bound peroxidase (EC 1.11.1.7) from rat stomach has been solubilized by 0.2% cetyltrimethylammonium bromide in the presence of 1.2 M NH4Cl. The enzyme was purified 3355-fold to apparent homogeneity as judged by acid polyacrylamide gel electrophoresis and appears to be a cationic protein. In sodium dodecyl sulfate gel electrophoresis, the enzyme shows single polypeptide band of Mr 45,000. In gel permeation, the Mr has been estimated as 47,000. Spectral properties indicate the presence of Soret band at 412 nm which shifts to 425 nm on complexation with CN- and to 430 nm on reduction with dithionite. The velocity constant, k1 for the reaction of the peroxidase with H2O2 is 1.38 X 10(7) M-1 s-1 and Km for H2O2 is 0.1 mM. The enzyme contains active sulphydryl groups and is inhibited by sulphydryl reagents of which p-hydroxymercuribenzoate is more reactive than mersalyl or N-ethylmaleimide. The enzyme is very resistant to thermal denaturation up to 65 degrees C and also to chaotropic reagents at least up to 2 M above which it is inactivated. The enzyme shows similarity with the intestinal eosinophil peroxidase as regards the molecular mass, spectral, kinetic and some of the catalytic properties. However, they differ significantly in terms of their interaction with fluoride ion, sulphydryl reagents, chaotropic reagent and also with the antiserum against the gastric peroxidase. Histochemically, the gastric peroxidase is shown to be localised in the gastric gland proper of the fundic stomach, rich in parietal and chief cells.

The kinetics of the exchange reactions between 2-oxoglutarate, malate and malonate have been measured at 4 °C in preparations of rat-heart mitochondria under conditions where the oxoglutarate translocator is operating exclusively. This was possible since no activity of the tricarboxylate translocator can be measured in such preparations and since the activities of the phosphate and dicarboxylate translocators may be blocked by mersalyl, to which the oxoglutarate translocator is insensitive. The metabolism of the substrates was prevented by addition of rotenone and arsenite. Measurements were made at three different external and three different internal concentrations of the substrates. The results show that the affinity of the oxoglutarate translocator is very much higher for external substrates than for the corresponding internal substrates. The Michaelis constants increase in the following order: external oxoglutarate < external malate < external malonate < internal oxoglutarate < internal malate < internal malonate. The results also show that the affinity of the translocator for a given substrate, either external or internal, is independent of the nature of the counter-ion. This is not true of the maximal velocities of the exchange reactions. Of the various mechanism described by Cleland for two-substrate-two-product enzyme-catalysed reactions, only the so-called mechanism of rapid-equilibrium random bi-bi is compatible with our experimental results. This implies that the Michaelis constants determined are the dissociation constants of the various translocator-substrate(s) complexes.

1 Flavoproteins of intact and fractionated mitochondria have been studied by means of spectrophotometric and fluorimetric techniques. 2 The high and low redox potential fluorescent flavoproteins of rat liver mitochondria previously assigned to the respiratory chain are located in the soluble fraction obtained on ultrasonic disintegration. 3 In intact mitochondria, the low potential species has a redox potential of −279 mV at pH 7.2 and 25° measured by fluorescence or absorbance, and undergoes a 2 equivalent reduction. After ultrasonic disruption of mitochondria, its redox potential is -294 m V at pH 7.2 and 25°. 4 In submitochondrial particles, low potential flavoprotein reducible by NADH in the presence of rotenone is not fluorescent, appears to have a redox potential more positive than −209 mV and is attributed mainly to cytochrome b5 reductase and cytochrome b5. Mersalyl can be used to selectively inhibit cytochrome b5 reductase, and it has been possible to estimate the concentration of the low redox potential flavoprotein free from cytochrome b5 interference. 5 The high redox potential flurescent flavoprotein is recovered in the soluble fraction after ultrasonic disruption of rat liver mitochondria and can be reduced by succinate in the presence of antimycin A inhibited particulate fraction. This fluorescent species is also found in rat kidney mitochondria but not in blowfly mitochondria. 6 In submitochondrial particles, a non-fluorescent high redox potential species is found which may be flavoprotein or non-heme iron. This species has a redox potential of + 30 m V at pH 7.2 and 25° and undergoes 1 equivalent reduction. 7 Flavoprotein fluorescence measurements are subject to significant errors caused by absorption of the exciting light, and conseuently do not always measure flavoproteins free from interference by other respiratory carriers, e. q. cytochromes and non-heme iron proteins.

Mutants of Saccharomyces cerevisiae were isolated which supported two unlinked nuclear mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial one conferring paromomycin sensitivity. Mitochondria isolated from such a mutant exhibited modifications of several phosphate-requiring functions: (a) kinetic parameters of the phosphate dependence of ATP synthesis were modified; (b) in the absence of phosphate the inner mitochondrial membrane exhibited a high proton leakage; (c) mutant mitochondria always exhibited a poor respiratory control and required tenfold more phosphate to reach a maximal state 3 of respiration; (d) phosphate transport, as measured by swelling experiments, was mersalyl-insensitive and, consequently, state 3 of the respiration and ATP synthesis remained less mersalyl-sensitive than in wild-type mitochondria. Analysis of the mitochondrial metabolism of diploid and segregant strains indicates that these modifications are related to the cryosensitive phenotype; however, at present, a cooperative effect of the mitochondrial mutation cannot be eliminated. It is proposed that the phosphate carrier itself or a regulatory element was modified.

A study is presented on the effect of chemical modification of thiol groups on proton conduction by the H+-ATPase complex in 'inside out' submitochondrial particles, before and after removal of the F1 moiety, and by F0 liposomes. The results obtained show that modification with monofunctional reagents [N-ethylmaleimide, 2,2'-dithiobispyridine, mersalyl and N-(7-dimethylamino-4-methyl-coumarinyl)-maleimide] of thiol residues in membrane integral proteins of F0 results in inhibition of proton conduction. Comparison of the inhibitory effects with the binding of [14C]N-ethylmaleimide to the various F0 polypeptides indicates that the inhibition of proton conduction by thiol reagents was correlated with modification of the 25-kDa, 11-kDa and 9-kDa (N,N'-dicyclohexylcarbodiimide-binding protein) proteins. Involvement of the last component is supported by the observation that modification by thiol reagents depressed the binding of N,N'-dicyclo[14C]hexylcarbodiimide to the 9-kDa protein.

Melittin, the main constituent of honeybee venom, is derived from promelittin. In the amino acid sequence of the 'pro' region of this precursor, every second residue is either proline or alanine. The possibility has been investigated that activation of promellitin might proceed via sequential liberation of dipeptides catalyzed by a dipeptidylpeptidase IV. As substrates we used promelittin isolated from queen bees fed with radioactive proline, and enzymatic fragments of prepromelittin which contained the entire pro part and the NH2-terminal hexapeptide of melittin. It could first be demonstrated that pig kidney of dipeptidyleptides catalyzed by a dipeptidylpeptidase IV. As substrates we used promelittin isolated from queen bees fed with radioactive proline, and enzymatic fragments of prepromelittin which contained the entire pro part and the NH2-terminal hexapeptide of melittin. It could first be demonstrated that pig kidney of dipeptidylpeptidase IV releases dipeptides from the pro part. An enzyme of this type could then be detected in extracts from venom glands of q bees, which also contain a dipeptidase. After inhibiting the latter enzyme with mersalyl, a stepwise cleavage of dipeptides, starting at the amino end of the pro region could be demonstrated. This hydrolysis did not proceed into the melittin sequence. Furthermore, fragments with an extra residue at the amino end, which therefore had the wrong 'reading frame' for the dipeptidylpeptidase, were not hydrolyzed. With intact promelittin as substrate the rate of hydrolysis was always lower than with the fragments. The results presented in this paper suggest a new type of precursor-product conversion proceeding via stepwise cleavage of dipeptide units. Our experimental evidence also ascribes a biological function to a dipeptidylpeptidase IV, a type of enzyme widely distributed in animals tissues. The evidence, that the observed reaction in vitro reflects the mechanism of promelittin activation in vivo is discussed.

The mitochondrial inner membrane anion channel catalyzes the electrophoretic transport of a wide variety of anions and is inhibited by matrix divalent cations and protons. In this paper, evidence is provided that mersalyl and p-chloromercuribenzene-sulfonate each interact with this uniporter at two distinct sites. Binding to site 1 causes a shift in the pH dependence of transport, characterized by a decrease in the pIC50 for protons from about 7.8 to about 7.3, and leads to substantial stimulation of transport in the physiological pH range. This effect is not reversed by addition of thiols such as thioglycolate. Binding of mersalyl and p-chloromercuribenzenesulfonate to site 2 inhibits the transport of most anions including Pi, citrate, malonate, sulfate and ferrocyanide. The transport of Cl− is inhibited about 60% by mersalyl, but is not inhibited by p-chloromercuribenzenesulfonate. These data suggest that inhibition is a steric effect dependent on the size of the anion and the size of the R group of the mercurial. This inhibition is reversed by thioglycolate. Dose/response curves indicate that mersalyl binds to site 1 as the dose increased from 7 to 13 nmol/mg, whereas it binds to site 2 as the dose is increased from 10 to 18 nmol/mg. Thus, at certain pH values both stimulatory and inhibitory phases can be seen in the same dose/response curve. It is suggested that these sites may contain thiol groups and that physiological regulators may exist which can effect changes in activity of the inner membrane anion uniporter similar to those exerted by mercurials.

A latent form of collagenase had been isolated from crude extracts of the insoluble, fibrous material from Walker tumor homogenates. Purified preparations of this enzyme yielded a major unit of Mr approximately 62000, as determined by gel filtration on AcA 54 Ultrogel. In its activated form collagenase had been purified to apparent homogeneity with an approximate Mr of 42000. The active enzyme cleaved soluble collagen into three-quarter and one-quarter length fragments in the manner of vertebrate collagenases. Latent collagenase from culture media eluted with an apparent Mr of 53000 and was thus slightly larger in size than its activated form that eluted at 42000. Extracted latent collagenase and latent collagenase from culture media could be activated enzymatically by trypsin or chymotrypsin and non-enzymatically by mersalyl, an organic mercurial compound. We suggest that latent collagenase from Walker tumors are complexes of active enzyme with inhibitor(s) of low molecular weight(s) and are not true zymogens.

1. Trypsin has been reacted with dithiothreitol and with a naturally occurring thiol-containing trypsin inhibitor to form enzyme-inhibitor complexes. This complex formation is known to be via a reversible intermolecular disulphide linkage. 2. These latent forms of trypsin have been re-activated with mersalyl [N-(O-carboxymethylsalicyloyl)-3-hydroxymercuric-2-methoxypropylamine], 4-aminophenylmercuric acetate and with cystine. 3. Active-site titration analysis of trypsin in the presence of incremental additions of dithiothreitol demonstrated the simultaneous inhibition and modification of the enzyme active site, demonstrating a direct involvement of a significant disulphide controlling the conformation of the active site of the enzyme. 4. Mersalyl addition to the dithiothreitol-reduced trypsin resulted in a regain of enzymic activity and a corresponding regain of availability of the active sites for titration. 5. Mersalyl and 4-aminophenylmercuric acetate were shown to re-activate the trypsin-inhibitor complex. 6. A molecular mechanism for the organomercurial re-activation of latent enzymes of this particular type (involving disulphide exchange) has been proposed.

The effects of sulphydryl reagents on glutaminase activity in rat-liver mitochondria were studied. The following results were obtained. 1 Mersalyl and N-ethylmaleimide strongly inhibited glutaminase activity in intact and disrupted mitochondria. The inhibition by both these reagents in intact mitochondria was not relieved by preincubation with phosphate. 2 The addition of dithioerythritol to intact mitochondria reversed the effect of mersalyl but not of N-ethylmaleimide. Dithioerythritol did not restore gllutaminase activity when added to mitochondria which had first been exposed to mersalyl and then to N-ethylmaleimide. 3 It was found that N-ethylmaleimide penetrates the inner mitochondrial membrane whereas mersalyl does not under incubation conditions similar to those used for measuring glutaminase activity [cf. Gaudemer, Y and Latruffe, N.(1975) FEBS Lett. 54, 30-34]. 4 Preincubation of mitochondria with organic mercurials completely prevented the entry of 1 mM[U-14C]- glutamine into the mitochondrial matrix. N-Ethylmaleimide, at a concentration which maximally inhibits glutaminase activity, was without effect. Similar observations were made when glutamine transport was measured by the swelling of mitochondria in isotonic L-glutamine. 5 The concentration of intramitochondrial glutamine and glutamate were measured in mitochondria metabolizing 10mM [U-14C] glutamine. Mersalyl and N-ethylmaleimide, added to the mitochondria without preincubation, increased the concentration of glutamine and decreased the concentration of glutamate. 6 The glutaminase activity of sonicated extracts prepared from mitochondria which had been washed after preincubation with mersalyl was found to be inhibited when compared with similar extracts from untreated mitochondria. 7 It is concluded that N-ethylmaleimide directly inhibits glutaminase activity and mersalyl inhibits both the transport of glutamine and itssssss hydrolysis. The inhibitory effects of mersalyl and N-ethylmaleimide are due to reaction with at least two different -SH groups. The results support the view that glutaminase is attached to the inner membrane.

Mersalyl, 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) and fluorescent Thiolyte DB react with SH groups in the H+ channel (SHc) of the uncoupling protein of brown adipose tissue mitochondria, as inferred from their inhibition of H+ transport. Cl- transport by the uncoupling protein was unaffected. Using these modifiers and N-ethylmaleimide (MalNEt), distinct SH groups (SHB) in the purine nucleotide binding site were identified. Nbs2 reacts more readily with the SHB than with the SHc groups, but mersalyl and Thiolyte DB are more reactive with the SHc groups. MalNEt reacts exclusively with the SHB. GDP inhibition is fully prevented after sufficient modification of the SHB. Pretreatment with p-diazobenzenesulfonate (N2PhSO2) suppresses only 20-25% of fluorescence of Thiolyte-DB-labeled uncoupling protein on SDS/PAGE gels, while MalNEt suppresses 66% and Nbs2 80-90%. Since N2PhSO2 also affects the GDP binding site, these results demonstrate that the N2PhSO2-reactive residue is not identical with the SHB.

Efflux of taurocholate from isolated rat hepatocytes was studied to characterize the mechanism of bile acid secretion. Cells were incubated with taurocholate for 15 min. The amount of the intracellularly accumulated bile acid was directly related to the concentration in the medium. Transfer of the loaded cells from the incubation medium to a medium without taurocholate led to taurocholate efflux. Efflux was saturable, its activation energy amounted to 12 kcal/mol (50 kJ). It was strongly inhibited by the metabolic inhibitor antimycin A and to a lesser extend by the uncoupler carbonyl-cyanide-m-chlorophenylhydrazone. Dinitrofluorobenzene and mersalyl, reagents which react with amino acids, inhibited efflux by about 30% when applied at concentrations of 50 μM. Ouabain increased the rate of efflux. The observations indicate that secretory functions are maintained in isolated liver cells.

1 The isolated and liposome-reconstituted mitochondrial phosphate carrier exhibits a sigmoidal inhibition curve by mersalyl, similar to that found with intact mitochondria. In contrast a hyperbolic inhibition curve is found (a) by titration of the soluble carrier with mersalyl before reconstitution in liposomes and (b) by titration of the reconstituted carrier with mersalyl after successively pretreatment of the mitochondria with low, non-inhibitory, concentrations of mersalyl, excess N-ethylmaleimide and dithiothreitol. 2 The inhibition of the reconstituted, but not of the soluble, phosphate carrier by mersalyl can be reversed by dithiothreitol. 3 Cupric di(1,10-phenanthroline) inhibits the soluble but not the reconstituted phosphate carrier. The inhibited phosphate carrier can be reactivated by dithiothreitol in the soluble state but not after reconstitution in liposomes. 4 The data support the previously suggested model of the phosphate carrier, assuming a dimer of two identical subunits for the active unit.