Mitochondrial Phosphate Research Articles

Specific and reversible activation and inactivation of the mitochondrial phosphate carrier by cardiolipin and nonionic detergents, respectively

The phosphate carrier of pig heart mitochondria was solubilized with Triton X-100 and purified by chromatography on hydroxylapatite. Incubation of the phosphate carrier fraction with cardiolipin stimulated the reconstituted [ 32P]phosphate exchange activity in liposomes, whereas increased Triton X-100 concentrations inhibited it. The effects of cardiolipin and Triton X-100 are reversible. The activation by cardiolipin is highly specific and could not be obtained with any other applied phospholipid.

Characterization of sulphydryl groups of the mitochondrial phosphate translocator by a maleimide spin label

A maleimide spin label strongly inhibits the phosphate/H + symporter of rat liver mitochondria. While inducing half-maximal inhibition of transport, the spin label reacts preferentially with the SH groups of the carrier, which are at least of two types. One type of SH group is localized close to the surface of the membrane and its environment does not significantly influence the mobility of the probe. The second type of SH group is buried in the membrane, is not accessible to ascorbate or chromium oxalate and its environment greatly restricts the motion of the probe.

Phosphate-binding proteolipid from brush border.

A proteolipid that binds inorganic phosphate with high affinity and specificity has been extracted from rabbit kidney brush-border membranes. This proteolipid has been partially purified by chromatography on LH-20. The molecular weight of the proteolipid is approximately 3000 as determined by urea-sodium dodecyl sulfate gel electrophoresis. This proteolipid can bind and transport phosphate into an organic phase. The K0.5 for phosphate binding is 8 microM with a Hill coefficient of 1.92. Arsenate inhibits phosphate binding in a competitive manner with a KI of 27.5 microM. The aminoreactive reagent 2,4-dinitrofluorobenzene inhibits phosphate binding to the proteolipid. Similarly, 2,4-dinitrofluorobenzene inhibited Na+-driven Pi uptake in renal brush-border membrane vesicles. In contrast to the mitochondrial phosphate binder, this proteolipid is not inhibited by sulfhydryl reagents. We suggest that this molecular species is a likely candidate for involvement in phosphate uptake in the renal tubule.

Purification of the active mitochondrial phosphate carrier by affinity chromatography with an organomercurial agarose column

The general procedure for the isolation of the phosphate carrier from mitochondria involves solubilization with non-ionic detergents and chromatography on hydroxylapatite [l-3]. With high resolution SDS-gel electrophoresis this preparation can be separated into 4-5 protein bands [2]. Further purification, in particular removal of the ADP/ATP-carrier, was obtained by chromatography on Celite [2], on Mersalyl-Ultrogel [3,4] and by using Triton X-l 14 instead of Triton X-100 [5]. However, after all these procedures the purified phosphate carrier fraction still contained 4-5 protein bands in the &.-region of 30 000-35 000 [5]. Affi-Gel 501 (an organomercurial agarose gel), Dowex AG l-X8 and hydroxylapatite (Bio-Gel HTP) were obtained from Bio-Rad; [32P]phosphate (carrier free) from Amersham; [3H]NEM from New England Nuclear; acrylamide, N,N’methylenebisacrylamide, SDS, Triton X-100 and NEM from Serva; scintillation liquid (Maxifluor) from J. Baker; L-cY-phosphatidylcholine (from egg yolk, Type X-E) from Sigma and cardiolipin from Serdary.

Mitochondrial carbamyl phosphate and citrulline synthesis at high matrix acetylglutamate.

Matrix acetylglutamate of uncoupled rat liver mitochondria increased about 10-fold, to 4.3 nmol/microliters, upon incubation with 5 mM concentrations of that compound. Uncoupled mitochondria incubated with the reagents needed for carbamyl phosphate and citrulline synthesis and 5 mM acetylglutamate synthesized citrulline at velocities which reached 99 nmol/min/mg of protein; simultaneously, as much as 47 nmol/min/mg of carbamyl phosphate accumulated and was distributed between matrix and medium. Maximal total carbamyl phosphate synthesis was, therefore, 146 nmol/min/mg, similar to the activity measured in liver homogenates. Without added acetylglutamate, some carbamyl phosphate accumulated when citrulline synthesis was about 40 nmol/min/mg. The finding that ornithine transcarbamylase can be limiting for citrulline synthesis shows that the activity of this enzyme is greatly restricted in mitochondria. The stimulation by ornithine of mitochondrial carbamyl phosphate synthesis was prevented when ornithine transcarbamylase was inhibited more than 96% by 5 mM delta-N-phosphonacetyl-L-ornithine, suggesting that the normal stimulatory effect of ornithine on carbamyl phosphate synthetase occurs via ornithine transcarbamylase. Lower concentrations of delta-N-phosphonacetyl-L-ornithine were required to achieve a given inhibition of citrulline synthesis from added carbamyl phosphate from endogenously synthesized carbamyl phosphate. The results reported suggest the existence of interactions between carbamyl phosphate synthetase and ornithine transcarbamylase in the matrix.

The mitochondrial phosphate carrier has an essential requirement for cardiolipin

The mitochondrial phosphate carrier has an essential requirement for cardiolipin

Human mitochondrial glycerol phosphate dehydrogenase (GPDm) isozymes.

1. A hydrophobic/phospholipid electrophoretic system has been devised which makes possible the analysis of human mitochondrial glycerol phosphate dehydrogenase (E.C. 1.1.99.5. GPDM). 2. GPDM has a wide tissue distribution in both adult and foetal life and is active in cultured lymphoblastoid cells and fibroblasts but is absent from red cells. 3. The solubilization procedure does not significantly alter the kinetic properties of the enzyme (Km alpha-glycerophosphate = 0.04-0.07 M, Km PMS = 0.19-0.35 mM) but the soluble form is less thermostable. 4. Comparisons of physicochemical characteristics, tissue distribution and coenzyme requirement point to a separate genetic determination and low level of evolutionary relatedness between GPDM and its cytosolic counterpart GPDS.

Synthesis and intracellular transport of mitochondrial carbamyl phosphate synthetase I and ornithine transcarbamylase.

Carbamyl phosphate synthetase I (CPS) and ornithine transcarbamylase (OTC), the first two enzymes of urea synthesis, are localized in the liver mitochondrial matrix of ureotelic animals.1 The two enzymes are coded by nuclear genes, synthesized on cytoplasmic 80 S ribosomes, and subsequently transported across the two mitochondrial membranes to the matrix space. Studies in our2,3 and other laboratories4,5 have shown that both enzymes are synthesized in larger precursor forms (pCPS and pOTC) in cell-free protein-synthesizing systems. These precursors form large aggregates and have conformations different from those of the mature enzymes.6 We further showed that pOTC was transported into isolated rat liver mitochondria in association with the processing of pOTC to the mature form of the enzyme.3,7,8 It has been shown that both pCPS9 and pOTC10 are synthesized on membrane-free polysomes, released into the cytosol and then transported rapidly into mitochondria. The present paper describes detailed kinetic studies of the synthesis, processing and intracellular transport of pCPS and pOTC in isolated rat hepatocytes. The paper also deals with the chemical nature of pOTC and its transport into isolated mitochondria in vi vitro.

PH gradient-dependent phosphate transport catalyzed by the purified mitochondrial phosphate transport protein.

The mitochondrial phosphate transport protein (Wohlrab, H. (1980) J. Biol. Chem. 255, 8170-8173), which co-purifies with the adenine nucleotide translocase, has been isolated with a modified procedure resulting in an increased yield and in a preparation that is stable to storage in liquid nitrogen. The phosphate transport protein was incorporated into liposomes (phosphatidylethanolamine/phosphatidylcholine/phosphatidic acid, 2.75:1:1), and phosphate/phosphate exchange rates were determined. With pHe (extraliposomal) = pHi (intraliposomal) = 7.2, we found a Km of 2.5 mM independent of [(Pi)i] and a Vmax of 12 mumol/min . mg of protein. Parallel phosphate transport experiments were carried out with liposomes containing phosphate transport protein isolated from mitochondria inhibited by N-ethylmaleimide. Phosphate transported unidirectionally [pHe = pHi = 6.8; = 1.0 mM, (Pi)i = 0 mM] reaches a maximum at 30 s and is 0.13 and 0.05 mumol/mg for active and inhibited protein, respectively. At pHe = 6.8 and pHi = 8.0, the respective amounts are 0.45 and 0.05. At pHe = 8.0, the uptake becomes the same with active and inhibited protein (pHi = 6.8, 0.15 and 0.14; pHi = 8.0, 0.25 and 0.20). At all the above pH values, about the same uptake is observed with liposomes prepared without protein as those prepared with inhibited protein. The initial rate of protein catalyzed unidirectional flux [(Pi)e = 1.0 mM, pHe = 6.8; (Pi)i = 0 mM, pHi = 8.0) is 2 mumol/min . mg of protein or 8 mumol/min . mg of phosphate transport protein estimated without the adenine nucleotide translocase.

Growth hormone and rat liver mitochondria effects on urea cycle enzymes

Effects of hypophysectomy and subsequent growth hormone administration on mitochondrial enzymes of the urea cycle were investigated in rat liver. Hypophysectomy increased the activities of the two mitochondrial enzymes, carbamyl phosphate synthetase and ornithine transcarbamylase but not of the cytosolic enzyme, argininosuccinate synthetase. The activity of mitochondrial phosphate dependent glutaminase was not affected. Administration of bovine growth hormone (100 μg/100 g body weight) for two weeks decreased the activities of carbamyl phosphate synthetase and ornithine transcarbamylase almost to the normal level. These results suggest a specific effect of growth hormone on mitochondrial enzymes of the urea cycle and serve to explain the increased urea formation in hypopituitarism.

Purification and reconstitution of the mitochondrial phosphate transporter.

Purification and reconstitution of the mitochondrial phosphate transporter.

Purification of a reconstitutively active mitochondrial phosphate transport protein.

Purification of a reconstitutively active mitochondrial phosphate transport protein.

Computer simulation of metabolism in pyruvate-perfused rat heart. V. Physiological implications

The results of a simulation of metabolism in the pyruvate-perfused rat heart subjected to a sudden increase in work load are interpreted to provide a coherent explanation for the observed physiology. Respiration is most closely correlated with the mitochondrial phosphate potential, calculated from the MgATP and MgADP levels. No correlation between respiration and the pH gradient across the mitochondrial membrane was found. The transient falls in pH in the cytosol and perhaps the mitochondria are due largely to carbonic and lactic acidosis and appear to be only weakly coupled. The heart maintains a high ATP level during the transition to increased work by utilizing its energy reserves in order of decreasing availability in response to physiological signals mediated by Mg2+, Ca2+, and cAMP.

Identification of the N-ethylmaleimide reactive protein of the mitochondrial phosphate transporter

The mitochondrial phosphate carrier is inhibited by the SH reagents p-(hydroxymercuri)benzoate and N-ethylmaleimide. Based on an analysis utilizing dodecyl sulfate-polyacrylamide gels, an SH-containing 32 000-dalton protein has been identified as a component of the phosphate carrier system. Two other N-[3H]ethylmaleimide-labeled proteins of the inner mitochondrial membrane have been eliminated from this role [Wholrab, H., & Greaney, J., Jr. (1978) Biochim. Biophys. Acta 503, 425] on the basis that band IV (45,000 daltons) is absent from heart sonic submitochondrial particles and band VII (6 500 daltons) does not react with p-(hydroxymercuri)benzoate. The mobility of the 32 000-dalton protein (0.43) is lower than that of the gamma subunit of the mitochondrial ATPase (0.46) and the carboxyatractyloside binding protein (0.48) on 12.5% dodecyl sulfate-polyacrylamide gels. In these flight muscle mitochondria, 0.87 nmol of N-[3H]ethylmaleimide per nmol of cytochrome a is bound to the 32,000-dalton protein.

Kinetics of mitochondrial phosphate transport and rates of respiration and phosphorylation during greening of etiolated Avena leaves

Using the technique of silicone oil filtration of organelles and the inhibitor stop method, the kinetics of transport of inorganic phosphate across the inner mitochondrial membrane were tested in relation to different stages of greening (0 to 24 h) of etiolated laminae of Avena sativa L., and compared to the rates of oxygen consumption and ATP formation. The results demonstrate that there is a pronounced increase in phosphate transport after 3 h of greening, reaching values for Vmax (about 17 μmol mg protein(-1) h(-1)) that are three times as high as those measured with mitochondria from etiolated tissue. This is also mirrored by the rates of respiration and oxidative phosphorylation. After 24 h of light treatment (4 Klx), respiration and ATP formation, as well as V decreased again to levels below those of the etiolated stage. In contrast to V, there was no change in the affinity between inorganic phosphate and the binding sites of the transporting systems involved, as indicated by a rather constant Km (0.23 mM) for phosphate transport. Of the inhibitors of phosphate transport tested, mersalyl and methyl mercuric iodide were most efficient with identical characteristics of inhibition; but compared to animal mitochondria, the concentrations needed to result in similar amounts of inhibition, were more than ten times higher. The results are discussed with respect to plastid development.

Inhibitors of mitochondrial phosphate transport

Inhibitors of mitochondrial phosphate transport

Mitochondrial phosphate transport and the N-ethylmaleimide binding proteins of the inner membrane

Mitochondria have been prepared from the flight muscles of mature blowflies ( Sarcophaga bullata). Phosphate transport by these mitochondria, determined by rates of passive swelling in ammonium phosphate, is sensitive to inhibition by N-ethylmaleimide. 20 nmol of N-ethylmaleimide/nmol cytochrome a inhibit the swelling by 90%. When the mitochondria are inhibited by N-[ 3 H] ethylmaleimide , then solubilized in dodecyl sulfate/mercaptoethanol at 100°C and then electrophoresed on dodecyl sulfate-polyacrylamide gels, many labeled protein bands can be detected, including a large labeled peak that has the same mobility as the tracking dye, bromophenol blue. Sonic submitochondrial particles that are prepared from the N-[ 3 H] ethylmaleimidelabeled mitochondria, solubilized, and electrophoresed on dodecyl sulfatepolyacrylamide gels, possess only seven major labeled protein bands with no radioactive peak at the tracking dye. These labeled proteins have molecular weights of 71, 68, 64, 45, 32, 30, and approx. 10 · 10 3. The nmol N-[ 3 H]- ethylmaleimide bound to each of these proteins per nmol cytochrome a are 0.15, 0.19, 0.35, 0.45, 0.87, 0.10, and 0.17, respectively, when the mitochondria are inhibited with 21.5 mol N-[ 3 H] ethylmaleimide/mol cytochrome a at 10 μM cytochrome a. Coty and Pedersen ((1975) J. Biol. Chem. 250, 3515–3521) sensitized rat liver mitochondria to N-[ 3 H] ethylmaleimide and identified five labeled proteins. Only the labeled 32 · 10 3 dalton and the 45 · 10 3 dalton proteins are common to both systems

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.

Effects of hormones and N6O2′-dibutyryl-adenosine 3′ :5′-cyclic monophosphate, administered in vivo, on phosphate transport and metabolism in isolated rat liver mitochondria

1. The administration of glucagon or N6O2'-dibutyryl cyclic AMP to fed rats by intraperitoneal injection was associated with a 2-fold increase in the amounts of endogenous Pi and ATP, and an increase in the rate and extent of transport of exogenous Pi (measured in either the presence or the absence of Ca2+) in mitochondria subsequently isolated from the liver. No change was observed in either the maximum rate of transport of exogenous Pi or in the rate of 32Pi exchange. 2. The changes induced by glucagon and dibutyryl cyclic AMP were markedly decreased by the co-administration of cycloheximide. 3. The administration of insulin to rats resulted in an increase of about 1.3-fold in the concentration of endogenous mitochondrial Pi 4. The amounts of endogenous Pi in mitochondrial isolated from the livers of starved rats were 3 times those in mitochondria isolated from fed animals. 5. It is concluded that the liver mitochondrial phosphatetransport system may be an important site of hormone action. 6. In the course of these experiments, it was shown that Ca2+ markedly stimulates mitochondrial phosphate transports.

On the mechanism of phosphate and dicarboxylate transport in mitochondria

Studies on the swelling of mitochondria in isoosmotic solutions of the ammonium salts of phosphoric acid and of various dicarboxylic and tricarboxylic acids, led to the postulation of 3 functionallyrelated but separate anion transporters in the inner mitochondrial membrane [ 1,2] : a phosphate/OH-, a phosphate/dicarboxylate and a dicarboxylate/tricarboxylate antiporter. In further studies [3-61, using the swelling technique [7-91, or more direct methods to study the transport of anions [lo-l 51, the main conclusions were further substantiated (reviewed [17-211). Although the 2 transport systems for phosphate could be distinguished by the discovery of the specific inhibitors N-ethylmaleimide for phosphate/OHexchange [5,1 l] and butylmalonate for phosphate/ dicarboxylate exchange [3 ,l 1 ] , experiments on the effect of the 2 inhibitors on mitochondrial phosphate and dicarboxylate exchanges, led to the proposition that the inner mitochondrial membrane contains only 1 carrier for phosphate and dicarboxylates [22,23] . Kinetic analysis by a modified Hill-plot of the effect of N-ethylmaleimide and butylmalonate on 32P uptake in m’ o rt chondria clearly demonstrated a mutual interdependence of both inhibitor binding sites [24]. in an investigation on the induction of mitochondrial swelling in NH4 -malate by the phosphate analogues thiophosphate, monoand di-fluorophosphate, we concluded that the uptake of malate into mitochondria does not occur via an exchange with phosphate, but by activation of the transporter by phosphate [ 251.