Pi Carrier Research Articles

Dietary phosphorus-responsive genes in the intestine, pyloric ceca, and kidney of rainbow trout.

Identification of phosphorus (P)-responsive genes is important in diagnosing the adequacy of dietary P intake well before clinical symptoms arise. The mRNA abundance of selected genes was determined in the intestine, pyloric ceca, and kidney of rainbow trout fed low-P (LP) or sufficient-P (SP) diet for 2, 5, and 20 days. The LP-to-SP ratio (LP/SP) of mRNA abundance was used to evaluate the difference in gene expression between LP and SP fish, and to compare the response with bone and serum P, which are conventional indicators of P status. The LP/SP of intestinal, cecal, and renal type II sodium-phosphate cotransporter (NaPi-II) mRNA abundance changed from approximately 1-2 (day 2) to approximately 1.4-4 (day 5) and to approximately 2-10 (day 20). The LP/SP of renal NaPi-II, vitamin D 24-hydroxylase, and vitamin D receptor mRNA abundance correlated inversely with serum P on day 5 but not on day 2 and day 20. In another study, differentially expressed genes between LP and SP fish were examined by subtractive hybridization, confirmed by Northern blot, and evaluated by t-test and correlation with serum and bone P concentrations. About 30 genes were identified as dietary P responsive at day 20, including intestinal meprin and cysteinesulfinic acid decarboxylase, renal S100 calcium-binding protein and mitochondrial P(i) carrier, and cecal apolipoprotein E, somatomedin B-related protein, and NaPi-II. The LP/SP of mRNA abundance of renal mitochondrial P(i) carrier and intestinal cysteinesulfinic acid decarboxylase changed significantly by day 2, and intestinal meprin by day 5. Hence, these genes and NaPi-II are among the earliest steady-response genes capable of predicting P deficiency well before the onset of clinical deficiency.

Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thaliana.

Most cellular ATP is produced within the mitochondria from ADP and Pi which are delivered across the inner-membrane by specific nuclearly encoded polytopic carriers. In Saccharomyces cerevisiae, some of these carriers and in particular the ADP/ATP carrier, are represented by several related isoforms that are distinct in their pattern of expression. Until now, only one mitochondrial Pi carrier (mPic) form, encoded by the MIR1 gene in S. cerevisiae, has been described. Here we show that the gene product encoded by the YER053C ORF also participates in the delivery of phosphate to the mitochondria. We have called this gene PIC2 for Pi carrier isoform 2. Overexpression of PIC2 compensates for the mitochondrial defect of the double mutant Deltamir1 Deltapic2 and restores phosphate transport activity in mitochondria swelling experiments. The existence of two isoforms of mPic does not seem to be restricted to S. cerevisiae as two Arabidopsis thaliana cDNAs encoding two different mPic-like proteins are also able to complement the double mutant Deltamir1 Deltapic2. Finally, we demonstrate that Pic2p is a mitochondrial protein and that its steady state level increases at high temperature. We propose that Pic2p is a minor form of mPic which plays a role under specific stress conditions.

A novel in vitro culture system for analysis of functional role of phosphate transport in endochondral ossification

In vivo expression of the type III sodium-dependent phosphate transporter (NaPiT) Glvr-1 during endochondral ossification, suggests a functional role for inorganic phosphate (Pi) transport in cartilage calcification. For further analysis of this relationship, an in vitro model of endochondral ossification is required. In this context, we investigated the characteristics of Pi transport in the new chondrogenic cell line ATDC5 in relation to extracellular matrix (ECM) formation and mineralization. Pi uptake in ATDC-5 cells and in isolated matrix vesicles (MVs) is mediated by an Na-dependent Pi transporter with a pH dependency characteristic of a type III Pi carrier (lower activity at alkaline pH). Northern blot analysis indicated that ATDC-5 cells express Glvr-1 transcripts during the various stages of their maturation with a maximal level during the proliferating stage. In isolated MVs, Pi transport activity was maximal at day 21, concomitant with the beginning of type X collagen messenger RNA expression. These events preceded the initiation of matrix mineralization, which was apparent at day 25, and then gradually increased until day 47. This temporal relationship between maximal Pi transport activity in MVs and the expression of a marker of mineralizing chondrocytes is compatible with the possible involvement of Pi transport in the ECM calcification observed in ATDC-5 cell cultures. In conclusion, these observations suggest that ATCD-5 cells in culture represent a promising model for the analysis of a functional role of Pi transport in the initial events of endochondral ossification.

Increased risk of chronic liver failure in adults with heterozygous alpha1-antitrypsin deficiency.

Controversy exists whether patients who are genetically heterozygous for 1-antitrypsin deficiency (1ATD), carrying a single PI*Z allele, are at increased risk of developing chronic liver disease. In these investigations, we determined the prevalence of heterozygous 1AT phenotypes (PI MZ, PI SZ) in a well-characterized cohort of patients presenting with chronic liver failure before orthotopic liver transplantation (OLT). We analyzed data collected from all adult patients (n = 641) who underwent OLT at our tertiary referral center between March 1985 and December 1996. Study patients entered a prospective protocol designed to test for all known etiologies of liver disease. Complete testing including 1AT phenotyping was successfully performed in 599 adults. We compared the overall number of heterozygous PI*Z carriers in our OLT cohort with established prevalence figures for general and regional American populations, and examined their distribution among various liver disease subgroups. Fifty-one patients were found to be heterozygous carriers of a single PI*Z allele for 1AT. The predominant phenotype in our transplantation cohort was PI MZ, identified in 49 patients (8.2%), which is a significantly higher prevalence than that reported from previous American population studies (2%-4%). Additionally, a significantly greater number of PI MZ carriers existed in patients with cryptogenic cirrhosis compared with other liver disease categories (26.9%; P < .001). These data suggest that individuals carrying a single PI*Z allele for 1AT may be at increased risk of developing cirrhosis and liver failure, even in the absence of an identifiable coexisting liver disease.

Mitochondrial Energy Metabolism in the Left Ventricular Tissue of Spontaneously Hypertensive Rats: Abnormalities in both Adeninenucleotide and Phosphate Translocators and Enzyme Adenylate-Kinase and Creatine-Phosphokinase Activities

The aim of this study was to investigate the oxidative phosphorylation and additional adenosinetriphosphate (ATP) production mechanisms in mitochondria isolated from hypertrophied left ventricles of spontaneously hypertensive rats (SHR). Measurements of adenosinediphosphate (ADP)/ ATP and inorganic phosphate (Pi) carrier activities showed a significant reduction of Vmax values thus suggesting a general decrease of ATP supply in the hypertrophied ventricles. Investigation of mitochondrial enzyme activities showed 45% and 90% increases of adenylate-kinase and 80% and 110% increases of creatine-phosphokinase in 5- and 24-week-old SHR, before and after the development of the hypertensive state, respectively. The abnormalities found in SHR at the mitochondrial level suggest a profound rearrangement of energy production mechanisms in this model of left ventricular hypertrophy; whether the defects are determined genetically, and then worsen with the hypertensive state, remains to be determined.

Exposure to bone particles stimulates phosphate transport in chicken osteoclasts: Implication of the integrin receptor

The characteristics of the transport system for inorganic phosphate (Pi) have been investigated in chicken osteoclasts. Pi transport in these cells was mainly Nadependent and also energy-dependent, suggesting the existence of Na-Pi cotransport system in these cells. While the time course of Pi transport in chicken osteoclasts was comparable to that in chicken mononuclear cells, the precursors of the osteoclasts, Pi transport was stimulated in a time- and dose-dependent fashion, when the osteoclasts were exposed to bone particles. The stimulation was not due tode novo synthesis of Pi carrier, since the stimulatory effect was very rapid with the maximal effect observed at 1 h and could not be blocked by 50 µM cycloheximide. On the other hand, inhibition of osteoclast attachment to bone particles by adding an excess of Arg-Gly-Asp-Ser peptide blocked the stimulation. In summary, stimulation of Pi transport in chicken osteoclasts was induced by bone particles. Attachment of the osteoclasts to bone particles with integrin receptors appeared to be crucial to this stimulation.

Substrate specificity of the two phosphate transport systems of Acinetobacter johnsonii 210A in relation to phosphate speciation in its aquatic environment.

In natural waters and domestic waste waters in which divalent metal ions are present in excess of Pi, H2PO4-, HPO4(2-), and MeHPO4 prevail at pH values physiological for Acinetobacter johnsonii 210A (pH 5.5-8.0). In view of the ability of this organism to extensively accumulate Pi and divalent cations in cytoplasmic polyphosphate granules, the substrate specificity of its two Pi transport systems was studied. The constitutive, proton motive force-driven Pi carrier, previously shown to be dependent on divalent cations, plays a major role in the divalent cation and Pi flux by translocating MeHPO4 rather than Pi. This notion is confirmed by the observation that divalent cations are cotransported with Pi in a 1:1 stoichiometry in proteoliposomes containing reconstituted Pi carrier protein. In contrast, the Pi repressible, periplasmic binding protein-dependent Pi transport system mediates the uptake of H2PO4- and HPO4(2-). Pi uptake, but not MeHPO4 uptake, was stimulated in cells under Pi limitation, and the periplasmic Pi-binding protein has affinity for H2PO4- and HPO4(2-), but not for MeHPO4. When operating in concert, both systems enable A. johnsonii 210A to efficiently acquire Pi from its habitat through uptake of the predominant Pi species.

Mechanisms of ATP conservation during ischemia in slow and fast heart rate hearts

In the present study we compared the quantitatively most important, Pi-activated mechanisms for conserving ATP during ischemia in dog and rat cardiac muscle. Earlier studies by ourselves showed that dog heart, like all slow heart rate mammalian hearts examined, possesses the ability to inhibit its mitochondrial ATPase by binding IF1, the ATPase inhibitor protein, during ischemia. Rat heart, like other fast heart rate mammalian hearts studied, does not. The present study demonstrated that this IF1-mediated ATPase inhibition in ischemic dog heart, as in other slow heart rate hearts, appears to depend on matrix space acidification mediated largely by Pi-H+ symport via the mitochondrial Pi carrier. The present study further confirmed that maximal glycolytic flux rates are five- to sixfold greater in ischemic rat than in ischemic dog heart. Both of these systems are activated by increasing Pi concentration ([Pi]) during ischemia, and both appear to be regulated somewhat differently in dog than in rat heart. Thus intact dog heart mitochondria exhibited a [Pi]-dependent ATPase inhibition at low external pH, whereas rat heart mitochondria did not. The [Pi] required for maximal ATPase inhibition in dog heart mitochondria was approximately 6 mM. Although both dog and rat heart phosphofructokinase were stimulated by Pi, the enzyme in dog heart was maximally activated by approximately 6 mM Pi, whereas the rat heart enzyme required only approximately 3 mM Pi for its maximal stimulation under otherwise identical conditions. The most active nonmitochondrial ATPase in ischemic dog and rat cardiac muscle, the Ca(2+)-activated actomyosin ATPase, accounted for approximately one-half of the total nonmitochondrial ATPase activity in each species.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of control of oxidative phosphorylation in mitochondria oxidizing NAD-linked substrates

The flux control distribution of the net rate of state 3 respiration was determined in heart and kidney mitochondria incubated with low concentrations of pyruvate (0.5 mM) or 2-oxoglutarate (1 mM), and in conditions that led to activation of NAD-linked dehydrogenases, i.e., high substrate or Ca2+ concentrations. Control of flux was exerted by the ATP/ADP carrier (flux control coefficient, ci = 0.37) and Site 1 of the respiratory chain (ci = 0.28) when dehydrogenase activity was low. Control of the process shifted to the ATP synthase (ci = 0.32) and the Pi carrier (Ci = 0.27) when dehydrogenases were activated by high pyruvate and high Ca2+. The changes in the control exerted by the ATP/ADP carrier and the ATP synthase were not due to changes in the transmembrane potential, nor to a modification of intramitochondrial ATP/ADP ratios. Applying the summation theorem of the control analysis, it was found that at low Ca2+ and pyruvate concentrations the dehydrogenases shared the control of state 3 respiration with other steps. The NAD-linked dehydrogenases did not exert any significant control at high Ca2+ or high pyruvate concentrations.

Characterization of a Pi transport system in cartilage matrix vesicles. Potential role in the calcification process.

The mechanisms by which calcium (Ca2+) and inorganic phosphate (Pi) accumulate into matrix vesicles (MV) have not been elucidated. In the present study the characteristics of Pi uptake into MV isolated from mildly rachitic chicken growth plate cartilage have been investigated. The results indicate that Pi accumulates into MV mainly via a Na(+)-dependent Pi transport system. In the absence of NaCl in the extravesicular medium, Pi uptake was a nonsaturable process. In the presence of 150 mM NaCl, the initial rate of Pi uptake was 4.38 +/- 1.02-fold higher than with 150 mM choline chloride (mean +/- S.E., n = 8, p less than 0.005). Other cations showed partial activity to drive Pi into MV as compared to Na+:Li+ (64.4%) greater than K+ (39.8%) greater than choline (39.0%) greater than tetramethylammonium (30.0%) greater than N-methylglucamine (26.3%). Na(+)-dependent Pi transport activity displayed saturability towards increasing extra-vesicular concentrations of Na+ and Pi. The apparent Km for Pi was 0.68 +/- 0.16 mM. The Na+ concentration producing half-maximum Pi transport activity was 106.2 +/- 11.0 mM. Kinetic analysis suggests that Na+ interacts with the Pi carrier with a stoichiometry of more than one Na+ ion with one Pi molecule. In MV isolated from normal chicken growth plate cartilage, this Na(+)-dependent Pi transport system was barely expressed. In contrast to the effect on Pi uptake by MV, the activity of alkaline phosphatase was not changed when NaCl was substituted for choline chloride in the assay medium. In addition to this observation which suggests that this enzyme is not related to the Pi transport activity described in this study, levamisole, which inhibited alkaline phosphatase activity did not affect the Na(+)-dependent uptake of Pi. Both arsenate and phosphonoformic acid, two inhibitors of the epithelial Na(+)-dependent Pi transport systems, were active inhibitors of the Na(+)-dependent Pi uptake by MV with a higher potency for phosphonoformic acid. Associated with the expression of a facilitated Na(+)-coupled Pi transport in MV, in vitro calcification assessed by 45Ca2+ uptake also showed a marked dependence on extravesicular sodium. This relationship was markedly attenuated in MV isolated from normal chicken growth plate cartilage expressing a weak Na(+)-facilitated Pi transport activity. In conclusion, a saturable Na(+)-dependent Pi carrier has been characterized which facilitates Pi transport in MV. Its potential role for Ca-Pi accumulation into MV and subsequent development of vesicular calcification followed by mineralization of the osteogenic matrix is proposed and remains to be further investigated.

Effects of anthralin on mitochondrial bioenergetics

Isolated mitochondria were used to determine what causes anthralin inhibition of oxidative phosphorylation. In good agreement with other results, the rate of oxygen consumption was not modified by anthralin when mitochondria were first uncoupled with FCCP, suggesting that only the last steps of the process leading to ATP phosphorylation are implicated. No effects were found at the level of the ATPase and the Pi carrier in contrast with a competitive inhibition of the ADP/ATP translocator. These experiments suggest an atractyloside-like effect to explain the action of anthralin on mitochondria.

Mitochondrial phosphate transport. Import of the H+/Pi symporter and role of the presequence.

The rat liver mitochondrial phosphate transporter contains a 44-amino acid presequence. The role of this presequence is not clear since the ADP/ATP carrier and the brown fat uncoupling protein, related members of a family of inner membrane anion transporters, lack a presequence and contain targeting information within the mature protein. Here, we present evidence that the rat liver mitochondrial phosphate transporter can be synthesized in vitro, imported into mitochondria, and processed to a protein of Mr 33,000. Import requires the membrane potential and external nucleotide triphosphate. The presequence inserts into the outer mitochondrial membrane, and import proceeds via a process similar to other proteins destined for the inner membrane or matrix. A mutant phosphate transporter lacking 35 amino acids at the NH2 terminus of the presequence has little capacity for mitochondrial import. The rat liver phosphate transporter is also imported and processed by rat kidney mitochondria and by mitochondria from the yeast Saccharomyces cerevisiae. A site-directed mutation of the N-ethyl-maleimide reactive cysteine 41 does not affect import or processing. The results presented show that optimal import of the mitochondrial phosphate transporter, unlike the ADP/ATP carrier and the brown fat uncoupling protein, is dependent on a presequence. As these carriers are believed to have evolved from a single gene, it seems likely that the H+/Pi carrier, known to be present in prokaryotes, appeared first and that subsequent evolutionary events leading to the other anion carriers eliminated the presequence.

Ornithine/phosphate antiport in rat kidney mitochondria

[14C]Ornithine uptake by rat kidney mitochondria has been investigated according to the stop inhibitor method by using praseodimium chloride as an inhibitor. The existence of an ornithine/Pi exchange was found occurring with 1:1 stoichiometry. Both uptake and efflux follow first-order kinetics with a k of 2.4 min-1. Uptake increases with increasing pH. The activation energy for the process is 58.6 kJ/mol and Q10 is 2.6. Ornithine/Pi exchange is electrical and energy-dependent, as suggested by the sensitivity of the process to the ionophores valinomycin and nigericin. Measurements both of proton movement across the mitochondrial membrane and of membrane potential strongly suggest that ornithine uptake into mitochondria is driven by the electrochemical proton gradient via the dependent ornithine/Pi translocator and delta pH-dependent Pi carrier.

Energy-linked anion transport: Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter

A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe. The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively. The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein. The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e. approximately 10%). It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase. Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology. Analysis of genomic DNA from both rat and S. cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins. This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport.

Energy-transducing complexes in bacterial respiratory chains.

Oxidative phosphorylation in mitochondria and photophosphorylation in chloroplasts are highly organized systems that yield living energy for eukaryotes. By introducing the concept of an electrochemical H+ gradient across the membrane (\(\Delta \tilde \mu \rm H^ + \))—that is, a proton motive force (pmf) composed of ΔΨ and ΔpH as \(\Delta \tilde \mu \rm H^ + _{(mV)} = \Delta \psi - Z\Delta pH\,(Z \simeq 60\,mV)\)—the chemiosmotic hypothesis (Mitchell, 1966) has played a central role in solving the problem of how organelles synthesize ATP using oxidative or photo energy. According to this hypothesis, the components of the electron transfer chain translocate protons across the membrane unidirectionally. The pmf thus established drives protons through an anisotropic reversible proton-translocating ATPase (ATP synthase) to synthesize ATP from ADP and Pi. There are other translocators (H+ symporters such as Pi carrier and electrogenic translocators such as ATP/ADP translocator) in energy-transducing biomembranes that transport substrates using a pmf. These energy-transducing membranes include the cytoplasmic membranes of bacteria. Eubacteria at least (Archaebacteria may have a slightly different ATP synthase) contain the anisotropic F0·F1 type H+-translocating ATPase (ATP synthase) that has very similar molecular architecture and characteristics to those of the ATP synthases of mitochondria and chloroplasts (Amzel and Pedersen, 1983), as well as having several translocators that utilize pmf. In contrast, the bacterial electron transfer chains that produce a pmf show great variety, even if among those in aerobic respiratory systems (Jones, 1977).KeywordsCytochrome OxidaseNADH DehydrogenaseElectron Transfer ChainQuinone OxidoreductaseTerminal OxidaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Relation between intestinal alkaline phosphatase activity and brush border membrane transport of inorganic phosphate, D-glucose, and D-glucose-6-phosphate.

In order to evaluate the role of the alkaline phosphatase in intestinal transport processes, we studied the influence of known modulators of the alkaline phosphatase (polyclonal anti-calf AP antibodies, theophylline and zinc ions) on the absorption rate of glucose, inorganic phosphate and glucose liberated from glucose-6-phosphate into calf duodenal brush border membrane vesicles. Our results allow the following conclusions: First a direct involvement of the AP in the Na+-dependent glucose absorption is unlikely. Indeed, theophylline inhibits strongly the AP activity but rather stimulates the glucose uptake; second the AP is indirectly involved in glucose absorption from glucose-6-phosphate, if its enzymatic hydrolysis is the only source of glucose. In that case the Na+-dependent uptake of glucose was completely suppressed either by phosphatase specific antibodies or by theophylline; third the positive correlation found with calf intestinal BBMV between the inhibition of AP by AP antibodies or by theophylline and the decrease of rate of Na+-dependent Pi uptake rate suggests that the enzyme plays some role in the Pi absorption. It appears from the present study that the AP is probably not a carrier protein itself, but its hydrolytic activity might nevertheless be important for intestinal absorption. After hydrolysis of phosphoric esters the alcohol residues and Pi can be supplied to their specific carriers. Furthermore, the high Pi affinity of the enzyme at physiological pH values, could even favour a transient sequestration of phosphate, which then could be transferred to the Pi carrier.

Phosphate transport by rat intestinal basolateral-membrane vesicles.

The characteristics of phosphate transport across intestinal basolateral membranes of the rat were determined by using enriched preparations in which uphill Na+-dependent D-glucose transport could not be demonstrated, but ATP-dependent Ca2+ transport was present. Phosphate transport was saturable, Na+-dependent and exhibited Michaelis-Menten kinetics. Vmax. was 51.1 +/- 4.2 pmol/10 s per mg of protein and Km was 14 +/- 3.9 microM. The transport process was electroneutral. Tracer-exchange experiments and counter-transport studies confirmed the presence of a Na+-Pi carrier at the basolateral membrane. The presence of inside-positive membrane potential did not enhance phosphate uptake, indicating that the Na+ effect is secondary to the presence of the Na+-Pi carrier rather than an induction of positive membrane potential. The stoichiometry of this carrier at pH 7.4 was 2 Na+:1 phosphate, as shown by direct studies utilizing the static-head method. These studies are the first to determine the presence of a phosphate carrier at the basolateral membrane.

62] Rapid filtration technique for metabolite fluxes across cell organelles

This chapter describes rapid filtration technique. The advantage is that it allows temporal resolution of noncovalent binding and of transport processes without implying the use of a specific carder inhibitor. It is, however, restricted to the cases where the enzyme or membrane fragments can be immobilized or adsorbed within a filter. The rapid filtration technique prevents any buffer washing after the filtration. The experiments––ADP/ATP transport in potato mitochondria, Pi-Pi exchange in intact mitochondria and calcium-induced calcium release in sarcoplasmic reticulum––described to illustrate some applications of the rapid filtration technique. The kinetics of phosphate-phosphate exchange mediated by the Pi carrier of the mitochondrial membrane is studied. Calcium release from the sarcoplasmic reticulum membrane of skeletal muscle triggers the contraction of the muscle fiber. The rapid filtration technique is clearly very well adapted to the study of ion or metabolite transport.

Effect of acute acidemia on phosphate uptake by renal proximal tubular brush-border membranes

Prolonged metabolic acidosis is associated with depressed phosphate (Pi) uptake by the brush-border membrane (BBM) of the proximal tubule. To examine if changes in systemic pH underlie this inhibition, we measured Pi transport by renal cortical BBM from thyroparathyroidectomized rats with respiratory or metabolic acidosis of 1 or 3 h, respectively, and in appropriate controls. Also, Pi transport was measured in BBM prepared using tissue slices from nonacidotic rats that were preincubated for 20 or 45 min at either pH 6.9 (HCO3 = 10 mM, CO2 = 10%) or 7.4 (HCO3 = 10 mM, CO2 = 2.5%). Despite comparable acidemia (pH 7.06 +/- 0.05 with respiratory acidosis and 7.10 +/- 0.03 with metabolic acidosis), Na-dependent Pi uptake at 5 s incubation was reduced by 15.2 +/- 3.5% with respiratory acidosis compared with paired controls. It was not altered with metabolic acidosis. Vmax in respiratory acidosis (1.2 nmol X mg protein-1 X 5 s-1) was less than in controls (1.6); Kt was similar in both groups. 22Na transport and Na-dependent glucose transport were unchanged. Plasma phosphorus (P) increased from 8.75 +/- 0.35 mg/dl to 12.42 +/- 1.9 with respiratory acidosis. Therefore BBM vesicles transport was measured in controls after plasma P was raised. Under these conditions, Pi transport was similar to that with respiratory acidosis. Also Pi transport by BBM was unchanged when tissue slices were preincubated in vitro at high CO2 concentrations for 20 or 45 min. Thus respiratory acidosis specifically inhibits Na-dependent Pi transport by decreasing the number or rate of the BBM Pi carrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of the reconstitutively active phosphate transporter from rat liver mitochondria.

Procedures have been developed for the purification of a nearly homogeneous, highly active phosphate transport system from rat liver mitochondria in either a two-subunit (alpha, beta) or a single subunit (beta) form. Significantly, both forms display a similar high magnitude N-ethylmaleimide (NEM)-sensitive Pi/Pi exchange activity upon incorporation into phospholipid vesicles. The transport system is extracted from hypotonically shocked mitoplasts with Triton X-114 and purified in the presence of cardiolipin by sequential chromatography on hydroxylapatite, DEAE-Sepharose CL-6B, and Affi-Gel 501. Depending on the conditions used to elute the transporter from Affi-Gel 501, preparations are obtained which, when analyzed by high resolution sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis, consist of either a single 33-kDa protein (beta) or a 33-kDa (beta) plus a 35-kDa (alpha) component. In preparations yielding the latter result, both bands display a nearly equivalent Coomassie staining intensity. Furthermore, after alkylation with NEM, the two protein bands co-migrate. Fluorography indicates that the coalesced band contains [3H]NEM. Upon reconstitution of the purified Pi carrier into liposomes, direct measurement of both the initial transport rate and the amount of protein that actually incorporates into the phospholipid vesicles yields a specific transport activity of 22.6 mumol/min/mg of protein. The exchange is characterized by a first order rate constant of 0.85 min-1, a t1/2 of 49 s, and is inhibited by sulfhydryl reagents (i.e., NEM, p-chloromercuribenzoate, and mersalyl). It is also substantially inhibited by diethyl pyrocarbonate, N-acetylimidazole, phenylglyoxal, and 5-dimethylaminoaphthalene-1-sulfonyl chloride. In addition to providing a simple, rapid method for preparing the NEM-sensitive phosphate carrier in nearly homogeneous form, these studies provide new information about the catalytically active species of the carrier, its kinetic properties, and its inhibitor sensitivities.