Proteins dealing with N-ethylmaleimide inhibition of pig heart mitochondrial phosphate transport system.

Our data clearly demonstrate that protective effect of phosphate and protective effect of mersalyl against NEM-inhibition of phosphate transport act at the level of two kinds of proteins. (1)Two major components are phosphate and nigericin NEM sensitive. According to our previous data [13] it has been also demonstrated that these two proteins components are valinomycin NEM sensitive (results not shown here) suggesting a relationship between these proteins and the energy linked proton translocation process. Relationships between these proteins and the phosphate translocation process are not evident and are under further investigations. (2) Two other insoluble major components localised at the level of the subparticular fraction are mersalyl NEM sensitive. We can suggest that these proteins are implicated in the translocation of phosphate in pig heart mitochondria.     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

Specific labeling of a phosphate-transporting protein from rat-liver mitochondria by [203Hg]mersalyl

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.     

Mitochondrial phosphate transport: Correlation between alkylation of membrane proteins with N-[3H]ethylmaleimide and inhibition of transport

N-ethylmaleimide inhibits the mitochondrial phosphate carrier. Mitochondria were titrated with N-[³H]ethylmaleimide, dissolved in dodecylsulfate-mercaptoethanol, and their proteins separated on dodecylsulfate-polyacrylamide gels. While the phosphate transport is essentially insensitive to low concentrations of N-ethylmaleimide, the six primary N-ethylmaleimide reactive inner membrane proteins are labeled in direct proportion to the amount of inhibitor added. The reaction of N-[³H]ethylmaleimide with proteins I and III is independent of the preincubation of the mitochondria with p-mercuribenzoic acid, a membrane impermeable inhibitor of the transport. Comparing the alkylation of proteins II, IV, V and VI with the inhibition of phosphate transport, it is found that only proteins IV (45,000 daltons) and V (32,000 daltons) are maximally labeled at the same N-[³H]ethylmaleimide concentration that maximally inhibits the transport.     

Biogenesis of the mitochondrial ATPase from sea urchin embryos

The mitochondrial rutamycin-sensitive ATPase from sea urchin eggs was purified to homogeneity. The subunit structure of the enzyme was characterized by SDS-gel electrophoresis. Eight polypeptides were identified with molecular weights of 55,000, 52,000, 39,000, 31,000, 28,000, 23,000, 17,000 and 10,000. Developing sea urchin embryos were incubated with [2H]leucine in the presence of emetine preferentially to label mitochondrially made proteins. Under these conditions sea urchin mitochondria synthesize eight different polypeptides. Two of these proteins, with molecular weights of 31,000 and 23,000, co-purify with the ATPase. Antibody directed against the pure rutamycin-sensitive ATPase precipitated only these two proteins. Therefore, two of the eight sea urchin ATPase subunits appear to be made by mitochondria.     

Re-evaluation of the H+/site ratio of mitochondrial electron transport with the oxygen pulse technique.

The number of protons ejected per pair of electrons passing each energy-conserving site in the electron transport chain (the H+/site ratio) has been investigated in rat liver mitochondria by means of the oxygen pulse technique introduced by Mitchell and Moyle (1967) (Biochem. J. 105, 1147-1162). The usual H+/site values of 2.0 observed by this method were found to be substantially underestimated as a result of the influx of phosphate into the mitochondria. This was shown by three different kinds of experiments. 1. Addition of N-ethylmaleimide or mersalyl, inhibitors of mitochondrial phosphate transport, increased the H+/site ratio from 2.0 to 3.0. The dependence of this effect on the concentration of either inhibitor was identical with that for inhibition of phosphate transport. Added phosphate diminished the H+/site ratio to values below 2.0 in the absence of N-ethylmaleimide. N-Ethylmaleimide protected the elevated H+/site ratio of 3.0 against the deleterious effect of added phosphate, but did not prevent a lowering effect of weak acid anions such as 3-hydroxybutyrate. 2. Prior washing of mitochondria to remove the endogenous phosphate that leaks out during the anaerobic preincubation led to H+/site ratios near 3.0, which were not increased by N-ethylmaleimide. Addition of low concentrations of phosphate to such phosphate-depleted mitochondria decreased the H+/site ratio to 2.0; addition of N-ethylmaleimide returned the ratio to 3.0. 3. Lowering the temperature to 5 degrees, which slows down phosphate transport, led to H+/site values of 3.0 even in the absence of N-ethylmaleimide. The H+/site ratio of 3.0 observed in the absence of phosphate movements was not dependent on any narrowly limited set of experimental conditions. It occurred with either Ca2+ or K+ (in the presence of valinomycin) as mobile permeant cation. It was independent of the concentration of succinate, oxygen, mitochondria, or rotenone, additions of Ca2+, Li+, or Na+ and was independent of medium pH between 6.5 and 7.5. Inhibitors of the transport of ions or acids other than phosphate did not affect the H+/site ratio. These results indicate that re-uptake of endogenous phosphate, lost from mitochondria during anaerobic preincubation, reduces the observed H+ ejection and leads to underestimated H+/site ratios of 2.0 in the oxygen pulse method. When phosphate movements are eliminated by the procedures described above, the observed H+/site ratio is about 3.0. This value appears to be closer to the true H+/site ratio for the primary H+ ejection process during electron transport.     

The transport of sulphate and sulphite in rat liver mitochondria

1. The mechanism of sulphite and sulphate permeation into rat liver mitochondria was investigated. 2. Extramitochondrial sulphite and sulphate elicit efflux of intramitochondrial phosphate, malate, succinate and malonate. The sulphate-dependent effluxes and the sulphite-dependent efflux of dicarboxylate anions are inhibited by butylmalonate, phenylsuccinate and mersalyl. Inhibition of the phosphate efflux produced by sulphite is caused by mersalyl alone and by N-ethylmaleimide and butylmalonate when present together. 3. External sulphite and sulphate cause efflux of intramitochondrial sulphate, and this is inhibited by butylmalonate, phenylsuccinate and mersalyl. 4. External sulphite and sulphate do not cause efflux of oxoglutarate or citrate. 5. Mitochondria swell when suspended in an iso-osmotic solution of ammonium sulphite; this is not inhibited by N-ethylmaleimide or mersalyl. 6. Low concentrations of sulphite, but not sulphate, produce mitochondrial swelling in iso-osmotic solutions of ammonium malate, succinate, malonate, sulphate, or phosphate in the presence of N-ethylmaleimide. 7. It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO(2) or an exchange of the sulphite anion for hydroxyl ion(s).     

Photoaffinity Labeling of Adenylate Cyclase-Linked Serotonin Receptors in Aplysia Neurons

Serotonin stimulated adenylate cyclase in Aplysia neurons with a Kact of 0.7 microM. Under the same conditions, 1-[2-(4-aminophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine stimulated adenylate cyclase with a Kact of 20 microM. The azido derivative of this compound, 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine, or of serotonin, (4-amino, 3-nitrophenylazido-serotonin), also stimulated the cyclase in the dark, but with lower efficiency (Kact greater than 10(-4) M). Irradiation of the membranes in the presence of 100 microM 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine abolished 75% of the cyclase activity stimulated by 5 microM serotonin. Under the same conditions, 100 microM 4-amino, 3-nitrophenylazido-serotonin did not inhibit serotonin-stimulated adenylate cyclase activity. When [3H]1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine (20 microM) was irradiated with membranes for 5 min at 4 degrees C, a dozen peptides were labeled, as revealed by a fluorogram of sodium dodecyl sulfate-polyacrylamide gels. Among them, the labeling of five polypeptides (molecular weights of 45,000, 55,000, 63,000, 80,000, and 94,000) was protected by the presence of 0.2 mM serotonin during photolysis. These peptides may be related to serotonin receptors.     

Special features of Pi transport in pig heart and rat liver mitochondria as revealed by 6,6'-dithiodinicotinic acid (CPDS).

CPDS (6,6'-dithiodinicotinic acid), a non permeant thiol agent which affects several mitochondrial functions in a way different to that of mersalyl [18-19] revealed striking differences between the phosphate translocating systems of pig heart and rat liver mitochondria. Pi entry was measured either by swelling in 0.12 M ammonium phosphate or by rapid centrifugation in 32Pi medium. Pi efflux was measured after preloading of mitochondria with 32Pi, by exchange against Pi or malate; the "ATP-FCCP" system has been tested previously [19]. In pig heart mitochondria, Pi entry seems to proceed exclusively via the Pi/OH- carrier; CPDS completely inhibits this transport and the energy-linked functions. In contrast n-butyl-malonate does not affect the Pi-entry and the energy-linked functions. The Pi efflux is not affected either by CPDS or mersalyl, which do not produce a swelling in the "ATP-uncoupler system". In rat liver mitochondria, CPDS inhibits only the Pi/OH- carrier; both CPDS and n-butylmalonate are necessary to inhibit completely Pi entry. CPDS as well as mersalyl provokes a swelling in the presence of the "APT-uncoupler system". The results suggest two distinct functions of phosphate transport in both types of mitochondria.     

Identification of 5-Hydroxytryptamine1A Receptor Proteins in Bovine Frontal Cortex

5-Hydroxytryptamine1A (5-HT1A) receptor proteins were identified by a novel approach in which photoaffinity labeling technique was used in conjunction with affinity column chromatography. 5-HT1A receptors were solubilized from bovine frontal cortical membranes with 0.3% digitonin and 0.1% Nonidet P-40, and bound effectively to 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP)-coupled Affi-Gel 10 in a time-dependent manner. PAPP was shown previously to be a selective ligand for the 5-HT1A receptor. Two protein bands with molecular masses of approximately 55,000 and 38,000 daltons revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were eluted from the affinity column with either 1 mM 5-HT or 1 microM [3H]1-[2-(4-azidophenyl)ethyl]-4-(3-trifluoromethyl-phenyl)piperazine ([3H]p-azido-PAPP). [3H]p-Azido-PAPP is a selective photoaffinity labeling probe for the 5-HT1A receptor. The intensity of these two protein bands and the incorporation of [3H]p-azido-PAPP into these two proteins decreased significantly when the solubilized fraction was preincubated with excess 5-HT or PAPP (saturating all 5-HT1A receptors) prior to affinity column chromatography. These results suggest strongly that these two proteins are related to the 5-HT1A receptor protein. The isoelectric points of the photolabeled 5-HT1A receptor proteins were 6.0 and 6.5.     

ATP- and coenzyme A-dependent fatty acid incorporation into proteins of cell-free extracts from mouse tissues

The incorporation of tritiated fatty acids into proteins has been studied in cell-free extracts from mouse tissues. Incubation of heart extracts with [3H]tetradecanoic or [3H]palmitic acid in the presence of ATP and CoA resulted in the time-dependent and selective labeling of proteins (Mr = 60,000, 47,000, 42,000, 31,000, 16,000, and 13,000) which could be detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Two polypeptides (Mr = 47,000 and 42,000) reached a maximum in fatty acid incorporation very rapidly and were mainly localized in the membrane subcellular fractions of the extract. These proteins underwent transient labeling with [3H] tetradecanoyl-CoA, the maximum incorporation being obtained within 1 min. The fatty acid-labeled proteins from tissue extracts had the same properties as other proteins known to be acylated in intact cells, i.e. the acyl moiety was resistant to delipidation with organic solvents but could be hydrolyzed by treatment with neutral hydroxylamine. Screening of different tissues showed that extracts from liver and kidney also catalyze the ATP- and CoA-dependent formation of a similar group of fatty acid-acylated proteins. The results provide evidence for a group of proteins in mammalian tissues which selectively incorporate fatty acids in vitro and should be of value for further studies on the biosynthesis of acylated proteins.     

Reactivity of the -SH groups of the mitochondrial phosphate carrier under native, solubilized and reconstituted conditions

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. The inhibition of the reconstituted, but not of the soluble, phosphate carrier by mersalyl can be reversed by dithiothreitol. 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. The data support the previously suggested model of the phosphate carrier, assuming a dimer of two identical subunits for the active unit.     

A cyanine dye tri-S-C7(5). Phosphate-dependent cationic uncoupler of oxidative phosphorylation in mitochondria

The trinuclear cyanine dye, tri-S-C₇(5), at about 10 μM stimulated State 4 respiration of rat liver mitochondria more than 6-fold and released oligomycin-inhibited respiration completely. Thus, the dye is concluded to be a very effective cationic uncoupler of oxidative phosphorylation in mitochondria. However, for exhibition of its uncoupling action, the presence of P_i (or arsenate) was necessary, and a phosphate-transport inhibitor, N-ethylmaleimide or mersalyl, inhibited its action. The stimulation of phosphate transport via the P_i carrier by the dye is suggested to be directly related to the uncoupling action.     

Thiol reactivity and the molecular individuality of α- and β-adrenoreceptors in rat liver plasma membranes

Whether or not α- and β-adrenoreceptors are non-identical binding sites on the same protein is still an open question. We investigated the effects of sulfhydryl reagents and dithiothreitol on the binding of [³H]dihydroalprenolol and [³H]dihydroergocryptine to β- and α-adrenoreceptors of rat liver plasma membranes. Dithiothreitol inhibited the binding of [³H]dihydroalprenolol to the β-adrenoreceptor, whereas it had no effect on the specific binding of [³H]dihydroergocryptine to the α-adrenoreceptor. In contrast, mersalyl, a mercurial SH reagent, readily blocked the α-adrenoreceptor and, although to a lesser extent, the β-adrenoreceptor. The interaction of mersalyl with the α-adrenoreceptors was almost instantaneous. In contrast, under the same experimental conditions, the inactivation of the β-adrenoreceptors was much slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{:7}\text{min}$). Finally, a marked difference in the accessibility of the SH groups to mersalyl was observed between the α- and β-adrenoceptors. The presence of 15 μM (?)-epinephrine or 1.5 μM phentolamine was sufficient to prevent the blockade of the α-adrenoreceptor by mersalyl, but inactivation of the β-adrenoreceptor by mersalyl was not modified by 500 μM (?)-epinephrine and was only slightly decreased by 50 μM (?)-propranolol. Thus, the α- and β-adrenoreceptors from rat liver plasma membranes exhibited biochemical differences which may be interpreted in favor of their molecular individuality.     

Uncoupling of oxidative phosphorylation by divalent cationic cyanine dye. Participation of phosphate transporter

The trinuclear cationic cyanine dye tri-S-C4(5) was found to be an uncoupler of oxidative phosphorylation. Its uncoupling required inorganic phosphate (Pi) or arsenate, which is transported into mitochondria via the Pi transport system, and was abolished by the Pi-transport inhibitor N-ethylmaleimide or mersalyl. The dye stimulated Pi uptake into mitochondria, and its uncoupling action was accompanied by swelling of the mitochondria. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of Pi, the dye did not cause uncoupling, but its uptake was much greater than in the presence of Pi. The cyanine dye is suggested to induce uncoupling by acting on the membrane, rather than after its electrophoretic transfer into the mitochondria.     

Identification of the 5-HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8-Methoxy-2-[N-n-Propyl, N-3-(2-Nitro-4-Azidophenyl)Aminopropyl]Aminotetralin

The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3'-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3'-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI. As expected of a possible correspondence between PI and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline- and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of PI were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).     

Proton translocation by the H+-ATPase of mitochondria. Effect of modification by monofunctional reagents of thiol residues in F0 polypeptides

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.     

Reconstitution into liposomes of the B°-like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na⁺ dependent [³H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K⁺. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [³H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na⁺and internal K⁺. The transporter showed low if there is any tolerance towards the substitution of Na⁺ or K⁺ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na⁺ concentration cooperativity index close to 1 was derived, indicating that 1 Na⁺ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na⁺ transport. Optimal rate of 0.1 mM [³H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [³H]glutamine (and [³H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl₂, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue α-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na⁺ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B°-like.     

The uptake of oxalate by rat liver and kidney mitochondria

Oxalate, a metabolic end product, forms calcium oxalate deposits in the tissues under a variety of pathological conditions. In order to determine whether oxalate is able to penetrate the mitochondrial matrix, the uptake of oxalate by rat liver and kidney cortical mitochondria was characterized. Mitochondria did not swell in an iso-osmotic medium of ammonium oxalate unless a small amount of phosphate was provided. This phosphate-induced swelling was prevented by N-ethylmaleimide. The uptake of [14C]oxalate by liver and kidney mitochondria followed first order kinetics and was inhibited by mersalyl an inhibitor of the phosphate and dicarboxylate carriers. Accumulation of [14C]oxalate at equilibrium was significantly higher by mitochondria energized with succinate than by rotenone-inhibited mitochondria due to higher matrix pH as determined by the [14C]5,5'-dimethyloxazolidine-2, 4-dione distribution ratio. The velocity of oxalate accumulation by mitochondria was temperature dependent. The activation energy was 81.5 and 86.5 J/mol for liver and kidney mitochondria, respectively. In both types of mitochondria, the rate of oxalate uptake was hyperbolic with respect to the concentration of oxalate. The apparent Km was 28.8 +/- 0.6 and 13.4 +/- 1.2 mM and the Vmax 87.1 +/- 1.1 and 66.1 +/- 3.1 nmol X mg-1 X min-1 at 12 degrees C for liver and kidney mitochondria, respectively. Phenylsuccinate exhibited mixed inhibition of the rate of oxalate uptake. Oxalate exhibited also a mixed inhibition of the uptake and oxidation of malate by mitochondria. The data obtained provide evidence that oxalate is transported across the mitochondrial membrane by a phosphate-linked, carrier-mediated system similar to or identical to the dicarboxylate transporter.     

Identification of cell-surface heparin/heparan sulfate-binding proteins of a human uterine epithelial cell line (RL95).

The interaction of heparin (HP) with the cell-surface components of a human uterine epithelial carcinoma cell line (RL95) was studied. Binding of [3H]HP to cell surfaces was saturable in a dose- and time-dependent manner. HP and certain forms of heparan sulfate (HS) efficiently compete for [3H]HP binding. In contrast, other glycosaminoglycans, such as chondroitin sulfate, keratan sulfate, hyaluronic acid, and dermatan sulfate, do not compete for binding to these sites. Scatchard analysis revealed that [3H]HP bound to these sites with an apparent KD of 0.7-0.9 microM and a binding capacity of 9 x 10(6) sites/cell to attached cells. EDTA-detached cells displayed a similar apparent KD, but an approximately 2-fold increase in binding capacity. Protease digestion of cells on ice markedly reduced [3H]HP binding, indicating that these binding sites were associated with proteins. In contrast, heparinase treatment of cells stimulated binding by approximately 2-fold, indicating that a large fraction of these binding sites were occupied with endogenous ligand. We examined the structural features of HP/HS required for HP/HS binding. O-Sulfation, substitution of amino groups, and, to a lesser extent, the presence of carboxyl groups were important recognition features of HP/HS by cell-surface HP/HS-binding sites. N-Sulfation was not required. Photoaffinity labeling with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-ethyl-1, 3-dithiopropionate-HP was used to identify HP/HS-binding proteins on RL95 cell surfaces. Proteins with M(r) values of 14,000-18,500 and 31,000 were photolabeled at the surfaces of attached cells. Photolabeling was blocked by the addition of excess HP, but not chondroitin sulfate. Additional proteins with M(r) values greater than 31,000 were photolabeled specifically on EDTA-detached cells. Moreover, the M(r) 14,000-18,500 and 31,000 proteins were retained on the EDTA-detached cells. These observations indicated that certain cell-surface HP/HS-binding proteins were not exposed when cells were attached to substrata. Proteins of similar M(r) values as the photolabeled components as well as many additional proteins were identified by heparin-agarose chromatographic selection of extracts of cells labeled metabolically with [35S]methionine or vectorially with Na125I at the cell surface. Fragments of cell-surface HP/HS-binding proteins were released from intact RL95 and mouse uterine epithelial cells by mild trypsinization and isolated by heparin-agarose affinity chromatography. Three peptides with M(r) values between 6000 and 14,000 required greater than 0.5 M salt for elution from heparin-agarose, retained HP binding activity in a 125I-HP gel overlay assay, and selectively bound [3H]HP in a solid-phase binding assay.(ABSTRACT TRUNCATED AT 400 WORDS)