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P L Pedersen

Publications and source records attributed to P L Pedersen.

At least 91 records · Page 5Linked to original sources
PubMed1990

Glucose catabolism in brain. Intracellular localization of hexokinase.

A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer mitochondrial membrane. In this study, the subcellular location of hexokinase in brain has been rigorously investigated. Mitochondrial fractions containing hexokinase (greater than 500 milliunits/mg protein) were prepared by two different procedures, and then subjected to density gradient centrifugation before and after loading with barium phosphate, a technique designed to increase the density of the mitochondria. The gradient distribution patterns of both unloaded and loaded preparations show that brain hexokinase does not distribute exclusively with mitochondrial marker enzymes. This is particularly evident in the loaded preparations where there is a clear distinction between the peak activities of hexokinase and mitochondrial markers. The same observation was made when the mitochondrial fraction of either untreated or barium phosphate-loaded mitochondria was subjected to titration with digitonin. In fact, at concentrations of digitonin, which almost completely solubilize marker enzymes for both the inner and outer mitochondrial membranes, a significant fraction of the total hexokinase remains particulate bound. Electron microscopy confirmed that particulate material is still present under these conditions. Significantly, hexokinase is released from particulate material only at high concentrations of digitonin which solubilize the associated microsomal marker NADPH-cytochrome c reductase. Glucose 6-phosphate, which is known to release hexokinase from the brain "mitochondrial fraction" also releases hexokinase from this unidentified particulate component. These results on brain, a normal glucose utilizing tissue, differ from those obtained previously on highly glycolytic tumor cells where identical subfractionation procedures revealed a strictly outer mitochondrial membrane location for particulate hexokinase (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). It is concluded that in brain, hexokinase has a greater propensity to localize at nonmitochondrial receptor sites than to those known to be associated with the outer mitochondrial membrane.

Animals↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

F0 "proton channel" of rat liver mitochondria. Rapid purification of a functional complex and a study of its interaction with the unique probe diethylstilbestrol.

The F0 portion of the rat liver mitochondrial ATP synthase (F0F1-ATPase) has been purified by a rapid, high yield procedure. F0 is selectively extracted from inner membrane vesicles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) after prior treatment of the vesicles with guanidine HCl to remove F1. The resultant F0 is functional in proton translocation assays and separates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis into four major and three minor Coomassie-stainable bands, all with apparent molecular masses below 30 kDa. This CHAPS-purified F0 preparation was characterized in detail for its capacity to interact with the unique probe diethylstilbestrol (DES) which, depending on conditions, has been shown to interact with rat liver F0F1 to either inhibit or promote ATP hydrolysis (McEnery, M. W., and Pedersen, P.L. (1986) J. Biol. Chem. 261, 1745-1752). DES-inhibitory sensitivity could be conferred on F1-ATPase activity with the same concentration dependence on F0 as conferral of oligomycin sensitivity. DES was shown also to inhibit the magnitude of valinomycin induced proton influx, while initiating proton efflux in asolectin vesicles reconstituted with F0 and loaded with K+. The potency of DES in producing the latter effects was shown to be highly dependent on hydroxyl groups in "para" positions of the two benzene rings within the DES molecule. Finally, in the absence of F0, DES was shown to act as a catalyst of proton influx in K+-loaded asolectin vesicles upon addition of valinomycin. A model based on the structure of DES is presented to account for both the inhibitory and uncoupling properties of this compound.

Animals↗
PubMed1989

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

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

4-Chloro-7-nitrobenzofurazan↗
PubMed1989

Investigation of the substrate structure and metal cofactor requirements of the rat liver mitochondrial ATP synthase/ATPase complex.

The F1 moiety of the rat liver mitochondrial ATP synthase/ATPase complex contains as isolated 2 mol Mg2+/mol F1, 1 mol of which is nonexchangeable and the other which is exchangeable (N. Williams, J. Hullihen, and P.L. Pedersen, (1987) Biochemistry 26, 162-169). In addition, the enzyme binds 1 mol ADP/mol F1 and 3 mol AMP.PNP, the latter of which can bind in complex formation with divalent cation and displace the Mg2+ at the exchangeable site. Thus, in terms of ligand binding sites the fully loaded rat liver F1 complex contains 3 mol MgAMP.PNP, 1 mol ADP, and 1 mol Mg2+. In this study we have used several metal ATP complexes or analogs thereof to gain further insight into the ligand binding domains of rat liver F1 and the mechanism by which it catalyzes ATP hydrolysis in soluble and membrane bound form. Studies with LaATP confirmed that MgATP is the most likely substrate for rat liver F1, and provided evidence that the enzyme may contain additional Mg2+ binding sites, undetected in previous studies of F1-ATPases, that are required for catalytic activity. Thus, F1 containing the thermodynamically stable LaATP complex in place of MgATP requires added Mg2+ to induce ATP hydrolysis. As Mg2+ cannot readily displace La2+ under these conditions there appears to be a catalytically important class of Mg2+ binding sites on rat liver F1, distinct from the nonexchangeable Mg2+ site and the sites involved in binding MgATP. Additional studies carried out with exchange inert metal-nucleotide complexes involving rhodium and the Mg2+ and Cd2+ complexes of ATP beta S and ATP alpha S imply that the rate-limiting step in the ATPase reaction pathway occurs subsequent to the P gamma-O-P beta bond cleavage steps, perhaps at the level of Mg(ADP)(Pi) hydrolysis or MgADP release. Evidence is presented that Mg2+ remains coordinated to the leaving group of the reaction, i.e., the beta phosphoryl group. Finally, in contrast to soluble F1, F1 bound to F0 in the inner mitochondrial membrane failed to discriminate between the Mg2+ complexes of the ATP beta S isomers. This indicates that a fundamental difference may exist between the catalytic or kinetic mechanism of F1 and the more physiologically intact F0F1 complex.

Adenosine Triphosphatases↗
PubMed1988

Functional significance of mitochondrial bound hexokinase in tumor cell metabolism. Evidence for preferential phosphorylation of glucose by intramitochondrially generated ATP.

Previous studies from this laboratory have shown that mitochondrial bound hexokinase is markedly elevated in highly glycolytic hepatoma cells (Parry, D. M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). A pore-forming protein, porin, within the outer membrane appears to comprise at least part of the receptor site (Nakashima, R.A., Mangan, P.S., Colombini, M., and Pedersen, P.L. (1986). Biochemistry 25, 1015-1021). In studies reported here experiments were carried out to assess the functional significance of mitochondrial bound tumor hexokinase. Two approaches were used to determine whether the bound enzyme has preferred access to mitochondrially generated ATP relative to cytosolic ATP. The first approach compared the time course of glucose 6-phosphate formation by AS-30D hepatoma mitochondria under conditions where ATP was regenerated endogenously via oxidative phosphorylation or exogenously by added pyruvate kinase and phosphoenolpyruvate. The second approach involved the measurement of the specific radioactivity of glucose 6-phosphate formed following the addition of [gamma-32P]ATP to either phosphorylating or nonphosphorylating AS-30D mitochondria. Both approaches provided results which show that the source of ATP for bound hexokinase is derived preferentially from the ATP synthase residing within the inner mitochondrial membrane compartment rather than from the medium (i.e. from the cytosolic compartment). These results provide the first direct demonstration that the exceptionally high level of hexokinase bound to mitochondria of highly glycolytic tumor cells has preferred access to mitochondrially generated ATP, a finding that may have rather profound metabolic significance for such tumors.

Adenosine Triphosphate↗
PubMed1988

Mitochondrial ATP synthase. Overexpression in Escherichia coli of a rat liver beta subunit peptide and its interaction with adenine nucleotides.

The C-terminal two-thirds of the rat liver ATP synthase beta subunit has been overexpressed and exported to the Escherichia coli periplasm under the direction of the alkaline phosphatase (phoA) promoter and leader peptide. The processed soluble protein contains the 358 amino acids from glutamate 122 to the rat liver beta C-terminal serine 479, including all the regions that have been predicted by chemical and genetic modification studies to be involved in nucleotide, Pi, and Mg2+ binding. Through a simple sequence of Tris/EDTA/lysozyme treatment, osmotic lysis, and alkaline pH washes, the processed beta subunit fragment can be prepared in greater than 95% purity and at a yield of greater than 20 mg/liter of culture. It interacts with 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) which exhibits a strong enhancement of fluorescence upon binding. A similar enhancement is observed upon interaction with TNP-ADP. Enhancement observed with both TNP-nucleotides is markedly reduced by prior addition of either ATP or ADP and almost completely prevented by the ATP synthase inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. Both TNP-ATP and TNP-ADP bind at a stoichiometry of approximately 1 mol of nucleotide/mol of beta subunit fragment. Under the same conditions, TNP-AMP does not exhibit a fluorescence enhancement. This work demonstrates that, in the absence of interaction with other ATP synthase subunits, the rat liver beta subunit sequence from glutamate 122 to the C terminus exhibits no more than one readily detectable nucleotide binding domain. This success in producing a "functional" beta subunit fragment has significance for the pursuit of genetic and physical studies focused on the structure and function of the rat liver ATP synthase beta subunit.

Adenine Nucleotides↗
PubMed1988

Purification and characterization of a bindable form of mitochondrial bound hexokinase from the highly glycolytic AS-30D rat hepatoma cell line.

Recent studies from this laboratory have demonstrated that a form of hexokinase characteristic of rapidly growing, highly glycolytic tumor cells is bound to an outer mitochondrial membrane receptor complex containing a Mr 35,000 pore protein (D. M. Parry and P. L. Pedersen, J. Biol. Chem., 258: 10904-10912, 1983; R. A. Nakashima, et al., Biochemistry, 25: 1015-1021, 1986). In new studies reported here the specificity of this receptor complex for binding hexokinase is defined, and a purification scheme is described which leads to a homogeneous and bindable form of the tumor hexokinase. In the AS-30D hepatoma, hexokinase activity is elevated more than 100-fold relative to liver tissue. The relative increase in hexokinase activity is 8 times greater than that of any other glycolytic enzyme. Hexokinase is the only glycolytic enzyme of AS-30D cells to exhibit a mitochondrial/cytoplasmic specific activity ratio greater than 1, showing a 3.5-fold elevation in the mitochondrial fraction. Purification of hexokinase is accomplished by preferential solubilization of the mitochondrial bound enzyme with glucose-6-phosphate, followed by high-performance liquid chromatography on gel permeation and anion exchange columns. The final fraction has a specific activity of 144 units per mg of protein, with a Km for glucose of 0.13 mM and for ATP of 1.4 mM. The purified tumor enzyme migrates as a single species upon sodium dodecyl sulfate: polyacrylamide gel electrophoresis with an apparent molecular weight of 98,000. Significantly, the purified tumor enzyme retains its activity for mitochondrial binding. Additional results derived from chromatographic, polyclonal antibody, and amino acid analysis studies indicate that the predominant rat hepatoma hexokinase species is related most closely to isozymic form(s) of the enzyme commonly referred to as type II, and least related to the liver type IV isozyme (glucokinase).

Amino Acids↗
PubMed1988

Beta subunit of rat liver mitochondrial ATP synthase: cDNA cloning, amino acid sequence, expression in Escherichia coli, and structural relationship to adenylate kinase.

The amino acid sequence of all but a few N-terminal residues of the beta subunit of rat liver ATP synthase has been determined from cDNA clones. Rat liver F1-beta is shown to contain 17 amino acid differences from that reported for F1-beta of bovine heart, 2 differences of which involve differences in charge. This may account in part for the observation that bovine heart F1 binds nucleotides with much greater affinity than the rat liver enzyme. Rat liver F1-beta also contains homologous regions with another nucleotide binding protein, adenylate kinase, for which high-resolution structural studies are available. Adjacent to one of these homologous regions is an eight amino acid stretch which bears striking homology to the phosphorylation region of the (Na+,K+)-ATPase. The combination of these two homology regions may constitute at least part of a nucleotide binding domain in F1-beta. Significantly, both rat liver and bovine heart beta contain these regions of homology, whereas the 17 amino acid differences between the two enzymes lie outside this region. The possibility of a second nucleotide binding domain which differs between the two enzymes is discussed. A cDNA clone containing all the regions of homology as well as 11 of the 17 amino acid differences between the bovine heart and rat liver beta subunits has been ligated into the bacterial expression vector pKK223-3. After transformation of a protease-deficient strain of Escherichia coli, this cDNA clone is expressed as a 36-kilodalton protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Kinase↗
PubMed1988

Mitochondrial ATP synthase. Interaction of a synthetic 50-amino acid, beta-subunit peptide with ATP.

A 50-amino acid peptide predicted by chemical modification studies of F1 and by comparison with adenylate kinase to comprise part of an ATP-binding domain within the beta-subunit of mitochondrial ATP synthase has been synthesized and purified. In the numbering system used for bovine heart beta, the peptide consists of amino acid residues from aspartate 141 at the N-terminal end to threonine 190 at the carboxyl end. In Tris-Cl buffer, pH 7.4, the peptide undergoes a dramatic reaction with ATP resulting in precipitate formation. Analysis of the precipitate shows it to contain both peptide and ATP. Similar to the ATPase activity of F1 and the binding of nucleotide to the enzyme, the capacity of ATP to induce precipitation of the peptide is decreased markedly by lowering pH. Interaction of the peptide with the fluorescent ATP analog, TNP-ATP (2'(3')-O-(2,4-6-trinitrophenyl)-adenosine 5'-triphosphate), can be demonstrated in solution at low concentrations. A 7-fold enhancement in fluorescence is observed when 2.5 microM TNP-ATP interacts with 2.5 microM peptide. Divalent cation is neither required for ATP-induced precipitation of the peptide nor for demonstrating interaction between TNP-ATP and peptide, just as Mg2+ is not required for nucleotide binding to F1. These results indicate that the beta-subunit peptide studied here comprises at least part of a nucleotide-binding domain within the mitochondrial ATP synthase complex.

Adenosine Triphosphate↗
PubMed1988

ATP synthases--structure of the F1-moiety and its relationship to function and mechanism.

A great deal of progress has been made in understanding both the structure and the mechanism of F1-ATPase. The primary structure is now fully known for at least five species. Sequence comparison between chloroplast, photobacteria, aerobic bacteria, and mitochondrial representatives allow us to infer more general functional relationships and evolutionary trends. Although the F1 moiety is the most studied segment of the H+-ATPase complex, there is not a full understanding of the mechanism and regulation of its hydrolytic activity. The beta subunit is now known to contain one and probably two nucleotide binding domains, one of which is believed to be a catalytic site. Recently, two similar models have been proposed to attempt to describe the "active" part of the beta subunits. These models are mainly an attempt to use the structure of adenylate kinase to represent a more general working model for nucleotide binding phosphotransferases. Labelling experiments seem to indicate that several critical residues outside the region described by the "adenylate kinase" part of this model are also actively involved in the ATPase activity. New models will have to be introduced to include these regions. Finally, it seems that a consensus has been reached with regard to a broad acceptance of the asymmetric structure of the F1-moiety. In addition, recent experimental evidence points toward the presence of nonequivalent subunits to describe the functional activity of the F1-ATPase. A summary diagram of the conformational and binding states of the enzyme including the nonequivalent beta subunit is presented. Additional research is essential to establish the role of the minor subunits--and of the asymmetry they introduce in F1--on the physiological function of the enzyme.

Amino Acid Sequence↗
PubMed1988

Purification in a single step and kinetic characterization of the pyruvate kinase of Trypanosoma brucei.

The pyruvate kinase of Trypanosoma brucei can be purified to homogeneity in one step by affinity elution from a phosphocellulose column with the substrate phosphoenolpyruvate (PEP) and the allosteric activator fructose-2,6-diphosphate (FDP). The purified enzyme has a specific activity of 175 mumol min-1 (mg protein)-1 and a subunit molecular mass of 59 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinetic studies of the pure enzyme show that an increase in the PEP concentration decreases the apparent Km for adenosine diphosphate (ADP) and that an increase in the ADP concentration decreases the half saturation point (S0.5) for PEP. Likewise, the allosteric activator FDP decreases both the apparent Km for ADP and the S0.5 for PEP. ADP concentrations above 0.2 mM inhibit trypanosomal pyruvate kinase.

Adenosine Diphosphate↗
PubMed1987

Mitochondrial ATP synthase: dramatic Mg2+-induced alterations in the structure and function of the F1-ATPase moiety.

The ATPase activity of the F1 moiety of rat liver ATP synthase is inactivated when incubated prior to assay at 25 degrees C in the presence of MgCl2. The concentration of MgCl2 (130 microM) required to induce half-maximal inactivation is over 30 times higher than the apparent Km (MgCl2) during catalysis. Moreover, the relative efficacy of divalent cations in inducing inactivation during prior incubation follows an order significantly different from that promoting catalysis. Inactivation of F1-ATPase activity by Mg2+ is accompanied by the dramatic dissociation from the F1 complex of alpha subunits and part of the gamma-subunit population. The latter form a precipitate while the beta, delta, and epsilon subunits, and the remaining part of the gamma-subunit population, remain soluble. Dissociation is not a sudden "all or none" event but parallels loss of ATPase activity until alpha subunits have almost completely dissociated together with about 50% of the gamma-subunit population. Mg2+-induced loss of F1-ATPase activity cannot be prevented by including either the hydrolytic substrates ATP, GTP, or ITP in the incubation medium or the product ADP. Ethylenediaminetetraacetic acid, mercaptoethanol, and dithiothreitol are also ineffective in preventing loss of ATPase activity. Significantly, KPi at high concentration (greater than or equal to 200 mM) is effective in partially protecting F1 against inactivation. However, the most effective means of preventing Mg2+-induced inactivation of F1-ATPase activity is to rebind F1 to its F0 moiety in F1-depleted particles. When bound to F0, F1 is protected completely against divalent cation induced inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Mitochondrial ATP synthase complex: interaction of its F1 adenosinetriphosphatase moiety with the heavy atom iodine.

Studies were carried out to determine whether a simple electron-dense "heavy atom" like iodine could be introduced selectively into one or more of the subunits of the mitochondrial ATP synthase complex of rat liver. Surprisingly, very low amounts of iodine are incorporated into the isolated F1 moiety of this complex under conditions which result in a marked loss of catalytic activity. ATPase activity is inactivated in a concentration-dependent manner at pH 7.5 with half-maximal inactivation occurring at about 40 microM iodine. A maximum of only 10 atoms of iodine are incorporated per F1 molecule under conditions where inhibition of ATPase activity is linearly related to iodine incorporation. The molecular size of F1 after iodination is unchanged, indicating that inactivation is due to modification of essential amino acid residues rather than subunit dissociation. Treatment of F1, with 20-50 microM [125I]iodine followed sequentially by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that the beta subunit is preferentially labeled. Significantly, about two atoms of iodine per beta subunit are incorporated. Some iodine amounting to less than 23% of the total radioactivity placed on the gels is recovered in the alpha and gamma subunits whereas no radioactivity is detected in the delta and epsilon subunits. Iodination of F1 appears to modify essential residues other than those involved in substrate or product binding per se. Thus, nucleotide binding to F1 is unaltered by iodine, and neither phosphate, MgADP, nor MgATP protects F1 against inhibition by this agent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗