Overexpression, purification, and function of first nucleotide-binding fold of cystic fibrosis transmembrane conductance regulator.
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ATP-Binding Cassette Transporters↗
Biomedical subjects
P L Pedersen
Publications and source records attributed to P L Pedersen.
Glucose catabolism in cancer cells. The type II hexokinase promoter contains functionally active response elements for the tumor suppressor p53.
The p53 tumor suppressor is found to be mutated and abundant in a wide variety of tumors. Within tumors showing rapid growth, the Type II isoform of hexokinase is also highly expressed to facilitate high rates of glucose catabolism, which in turn promote their rapid proliferation. We previously reported isolation of the proximal promoter of the Type II hexokinase gene from the highly glycolytic hepatoma AS-30D (Mathupala, S. P., Rempel, A., and Pedersen, P. L. (1995) J. Biol. Chem. 270, 16918-16925). Here, we show that a p53 protein, exhibiting two point mutations in its cDNA, is abundantly expressed in the AS-30D hepatoma. Co-expression studies showed that p53 overexpression significantly and reproducibly activated the Type II hexokinase promoter. Two functional p53 motifs were identified within this promoter by footprint and gel retardation analyses. Presence of functional p53 response elements on the Type II hexokinase promoter and the positive regulatory effect on the promoter by the mutant p53 indicates that in rapidly growing liver tumors, and perhaps in many other tumors as well, this highly abundant p53 protein plays a role in maintaining a high glycolytic rate. This is the first report of a possible link between loss of cell cycle control in rapidly growing cancer cells and their high glycolytic phenotype.
Amino Acid Sequence↗
Novel insights into the chemical mechanism of ATP synthase. Evidence that in the transition state the gamma-phosphate of ATP is near the conserved alanine within the P-loop of the beta-subunit.
The chemical mechanism by which the F1 moiety of ATP synthase hydrolyzes and synthesizes ATP remains unknown. For this reason, we have carried out studies with orthovanadate (Vi), a phosphate analog which has the potential of "locking" an ATPase, in its transition state by forming a MgADP.Vi complex, and also the potential, in a photochemical reaction resulting in peptide bond cleavage, of identifying an amino acid very near the gamma-phosphate of ATP. Upon incubating purified rat liver F1 with MgADP and Vi for 2 h to promote formation of a MgADP.Vi-F1 complex, the ATPase activity of the enzyme was markedly inhibited in a reversible manner. When the resultant complex was formed in the presence of ultraviolet light inhibition could not be reversed, and SDS-polyacrylamide gel electrophoresis revealed, in addition to the five known subunit bands characteristic of F1 (i.e. alpha, beta, gamma, delta, and epsilon), two new electrophoretic species of 17 and 34 kDa. Western blot and N-terminal sequencing analyses identified both bands as arising from the beta subunit with the site of peptide bond cleavage occurring at alanine 158, a conserved residue within F1-ATPases and the third residue within the nucleotide binding consensus GX4GK(T/S) (P-loop). Quantification of the amount of ADP bound within the MgADP. Vi-F1 complex revealed about 1.0 mol/mol F1, while quantification of the peptide cleavage products revealed that no more than one beta subunit had been cleaved. Consistent with the cleavage reaction involving oxidation of the methyl group of alanine was the finding that [3H] from NaB[3H]4 incorporates into MgADP.Vi-F1 complex following treatment with ultraviolet light. These novel findings provide information about the transition state involved in the hydrolysis of ATP by a single beta subunit within F1-ATPases and implicate alanine 158 as residing very near the gamma-phosphate of ATP during catalysis. When considered with earlier studies on myosin and adenylate kinase, these studies also implicate a special role for the third residue within the GX4GK(T/S) sequence of many other nucleotide-binding proteins.
Adenosine Diphosphate↗
Cystic fibrosis transmembrane conductance regulator: the first nucleotide binding fold targets the membrane with retention of its ATP binding function.
Most cases of cystic fibrosis are caused by a single deletion mutation (deltaF508) within the first nucleotide binding fold (NBF1) of the CFTR protein (cystic fibrosis transmembrane conductance regulator). NBF1 is classically defined as amino acid residues phenylalanine 433 through serine 589, encoded by exons 10-12, and only part of exon 9, of the CFTR gene. This assignment is based on sequence homology of this region of the CFTR protein with that of other nucleotide binding proteins. Here, we report that when the complete modular unit encoded precisely by exons 9-12 is expressed in Escherichia coli as glycine 404 through serine 589, i.e., as [G404-N432]NBF1 or as deltaF508[G404-N432]NBF1, the resultant proteins target the cytoplasmic membrane. Significantly, [G404-N432]NBF1 is readily labeled from the outside of intact E. coli spheroplasts with the water soluble, membrane impermeable probe Biotin-X-NHS, sulfosuccinimidyl-6-(biotinamido)-hexanoate. Similar findings were observed with the disease causing mutant deltaF508[G404-N432]NBF1. Three different control experiments which involved (1) assays for known cytosolic E. coli enzymes, (2) immuno-gold electron microscopy with antibody having an epitope for the biotin moiety, and (3) tests for biotinylation of the cytosolic component, Enzyme 1 of the glucose phosphotransferase system, demonstrated that the spheroplasts used in this study are neither leaky nor permeable to Biotin-X-NHS. In addition, membrane-associated [G404-N432]NBF1, upon solubilization with Triton X-100, was found to bind to an ATP-agarose column and be released therefrom by elution with ATP, emphasizing retention of a native-like structure. In sharp contrast, NBF1 localizes to the cytosol when the [G404-N432]-N-terminal region is replaced with the maltose binding protein. The novel findings reported here implicate a role of the N-terminal region of NBF1 in its subcellular localization and are directly relevant to our understanding of the membrane structure, function, and trafficking of CFTR.
ATP-Binding Cassette Transporters↗
Cystic fibrosis, lung infections, and a human tracheal antimicrobial peptide (hTAP).
In order to understand how lungs of healthy people, unlike those of cystic fibrosis (CF) patients, are protected against bacterial infections such as Pseudomonas aeruginosa, the following three key findings were made. First, P. aeruginosa do not multiply when planted onto tracheal epithelial cells from healthy humans but do so profusely on cells from deltaF508 CF patients. Second, some bacteria bind, and gain entrance into CF cells, even at a physiological salt concentration (104 mM). Third, human tracheal epithelial cells express an approximately 4 kDa peptide (hTAP), which is known in its bovine form to exhibit bactericidal action against P. aeruginosa. A model is proposed depicting both how normal epithelial cells, in a first-line self defense mechanism, may be protected against bacterial infection and how this mechanism may fail during the initial stages of CF.
Amino Acid Sequence↗
Frontiers in research on cystic fibrosis: understanding its molecular and chemical basis and relationship to the pathogenesis of the disease.
In recent years a new family of transport proteins called ABC transporters has emerged. One member of this novel family, called CFTR (cystic fibrosis transmembrane conductance regulator), has received special attention because of its association with the disease cystic fibrosis (CF). This is an inherited disorder affecting about 1 in 2000 Caucasians by impairing epithelial ion transport, particularly that of chloride. Death may occur in severe cases because of chronic lung infections, especially by Pseudomonas aeruginosa, which cause a slow decline in pulmonary function. The prospects of ameliorating the symptoms of CF and even curing the disease were greatly heightened in 1989 following the cloning of the CFTR gene and the discovery that the mutation (deltaF508), which causes most cases of CF, is localized within a putative ATP binding/ATP hydrolysis domain. The purpose of this introductory review in this minireview series is to summarize what we and others have learned during the past eight years about the structure and function of the first nucleotide binding domain (NBF1 or NBD1) of the CFTR protein and the effect thereon of disease-causing mutations. The relationship of these new findings to the pathogenesis of CF is also discussed.
Cystic Fibrosis↗
Modeling of nucleotide binding domains of ABC transporter proteins based on a F1-ATPase/recA topology: structural model of the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR).
Members of the ABC transporter superfamily contain two nucleotide binding domains. To date, the three dimensional structure of no member of this super-family has been elucidated. To gain structural insight, the known structures of several other nucleotides binding proteins can be used as a framework for modeling these domains. We have modeled both nucleotide binding domains of the protein CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) using the two similar domains of mitochondrial F1-ATPase. The models obtained, provide useful insights into the putative functions of these domains and their possible interaction as well as a rationale for the basis of Cystic Fibrosis causing mutations. First, the two nucleotide binding domains (folds) of CFTR are each predicted to span a 240-250 amino acid sequence rather than the 150-160 amino acid sequence originally proposed. Second, the first nucleotide binding fold, is predicted to catalyze significant rates of ATP hydrolysis as a catalytic base (E504) resides near the y phosphate of ATP. This prediction has been verified experimentally [Ko, Y.H., and Pedersen, P.L. (1995) J. Biol. Chem. 268, 24330-24338], providing support for the model. In contrast, the second nucleotide binding fold is predicted at best to be a weak ATPase as the glutamic acid residue is replaced with a glutamine. Third, F508, which when deleted causes approximately 70% of all cases of cystic fibrosis, is predicted to lie in a cleft near the nucleotide binding pocket. All other disease causing mutations within the two nucleotide binding domains of CFTR either reside near the Walker A and Walker B consensus motifs in the heart of the nucleotide binding pocket, or in the C motif which lies outside but near the nucleotide binding pocket. Finally, the two nucleotide binding domains of CFTR are predicted to interact, and in one of the two predicted orientations, F508 resides near the interface. This is the first report where both nucleotide binding domains of an ABC transporter and their putative domain-domain interactions have been modeled in three dimensions. The methods and the template used in this work can be used to analyze the structures and function of the nucleotide binding domains of all other members of the ABC transporter super-family.
ATP-Binding Cassette Transporters↗
Aberrant glycolytic metabolism of cancer cells: a remarkable coordination of genetic, transcriptional, post-translational, and mutational events that lead to a critical role for type II hexokinase.
For more than two-thirds of this century we have known that one of the most common and profound phenotypes of cancer cells is their propensity to utilize and catabolize glucose at high rates. This common biochemical signature of many cancers, particularly those that are poorly differentiated and proliferate rapidly, has remained until recently a "metabolic enigma." However, with many advances in the biological sciences having been applied to this problem, cancer cells have begun to reveal their molecular strategies in maintaining an aberrant metabolic behavior. Specifically, studies performed over the past two decades in our laboratory demonstrate that hexokinase, particularly the Type II isoform, plays a critical role in initiating and maintaining the high glucose catabolic rates of rapidly growing tumors. This enzyme converts the incoming glucose to glucose-6-phosphate, the initial phosphorylated intermediate of the glycolytic pathway and an important precursor of many cellular "building blocks." At the genetic level the tumor cell adapts metabolically by first increasing the gene copy number of Type II hexokinase. The enzyme's gene promoter, in turn, shows a wide promiscuity toward the signal transduction cascades active within tumor cells. It is activated by glucose, insulin, low oxygen "hypoxic" conditions, and phorbol esters, all of which enhance the rate of transcription. Also, the tumor cell uses the tumor suppressor p53, which is usually modified by mutations to debilitate cell cycle controls, to further activate hexokinase gene transcription. This results in both enhanced levels of the enzyme, which binds to mitochondrial porins thus gaining preferential access to mitochondrially generated ATP, and in a decreased susceptibility to product inhibition and proteolytic degradation. Significantly, these multiple strategies all work together to enable tumor cells to develop a metabolic strategy compatible with rapid proliferation and prolonged survival.
Animals↗
ATP synthase. Conditions under which all catalytic sites of the F1 moiety are kinetically equivalent in hydrolyzing ATP.
Conditions have been reported under which the F1 moiety of bovine heart ATP synthase catalyzes the hydrolysis of ATP by an apparently cooperative mechanism in which the slow rate of hydrolysis at a single catalytic site (unisite catalysis) is enhanced more than 10(6)-fold when ATP is added in excess to occupy one or both of the other two catalytic sites (multisite catalysis) (Cross, R. L., Grubmeyer, C., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105). In the novel studies reported here, and in contrast to the earlier report, we have (a) monitored the kinetics of ATP hydrolysis of F1 by using nucleotide-depleted preparations and a highly sensitive chemiluminescent assay; (b) followed the reaction immediately upon addition of F1 to ATP, rather than after prior incubation with ATP; and (c) used a reaction medium with Pi as the only buffer. The following observations were noted. First, regardless of the source of enzyme, bovine or rat, and catalytic conditions (unisite or multisite), the rates of hydrolysis depend on ATP concentration to the first power. Second, the first order rate constant for ATP hydrolysis remains relatively constant under both unisite and multisite conditions declining only slightly at high ATP concentration. Third, the initial rates of ATP hydrolysis exhibit Michaelis-Menten kinetic behavior with a single Vmax exceeding 100 micromol of ATP hydrolyzed per min/mg of F1 (turnover number = 635 s-1) and a single Km for ATP of about 57 microM. Finally, the reaction is inhibited markedly by low concentrations of ADP. It is concluded that, under the conditions described here, all catalytic sites that participate in the hydrolysis of ATP within the F1 moiety of mitochondrial ATP synthase function in a kinetically equivalent manner.
Adenosine Diphosphate↗
Protein inhibitor of mitochondrial ATP synthase: relationship of inhibitor structure to pH-dependent regulation.
In the absence of an electrochemical proton gradient, the F1 moiety of the mitochondrial ATP synthase catalyzes the hydrolysis of ATP. This reaction is inhibited by a natural protein inhibitor, in a process characterized by an increase in ATPase inhibition as pH is decreased from 8.0 to 6.0. In order to gain greater insight into the molecular and chemical events underlying this regulatory process, the relationships among pH, helicity of the inhibitor protein, and its capacity to inhibit F1-ATPase activity were examined. First, peptides corresponding to four regions of the 82-amino-acid inhibitor protein were chemically synthesized and assessed for both retention of secondary structure, and capacity to inhibit F1-ATPase activity. These studies showed that a region of only 24-amino-acid residues, from Phe 22 through Len 45, accounts for the inhibitory capacity of the inhibitor protein, and that retention of native helical structure in this region is not essential for inhibition. Second, three mutants (33P34, 39P40, and 43P44) of the intact inhibitor protein were prepared in which a proline residue was inserted within the inhibitory region to disrupt native helical structure. The secondary structures and inhibitory capacities of these mutants were analyzed as a function of pH. These studies revealed that, despite the initial loss of helical structure within the inhibitory region due to proline insertion, a further loss of helical structure is required to modulate inhibitory activity. These results suggest that a loss of helical structure outside the inhibitory region correlates with an increase in inhibitory capacity. Finally, two separate mutants (H48A and H55A) were prepared in which a conserved histidine residue in the wild-type inhibitor protein was replaced with an alanine. The secondary structures and inhibitory capacities of these mutants were also investigated as a function of pH. Results indicated that, although histidine residues do not directly affect the inhibitory capacity of the protein, they are important for maintaining the inhibitor protein in an inactive form at high pH. Furthermore, these results show that loss in helical structure, although correlated with an increase in inhibitory capacity, is not essential for this function. These novel experiments are consistent with a model in which the inhibitor protein is envisioned as consisting of two regions, an inhibitory region and a regulatory region. It is suggested that reduction of pH allows for the protonation of a histidine residue blocking the interaction between the two regions, thus activating the inhibitory response. The pH reduction also correlates with a partial unfolding of the protein that may either cause or result from the loss of interaction between the two helices. This unfolding may be necessary for further optimization of inhibitor function.
Amino Acid Sequence↗
Glucose catabolism in cancer cells: amplification of the gene encoding type II hexokinase.
Hexokinase type II is highly overexpressed in many cancer cells, where it plays a pivotal role in the high glycolytic phenotype. Here we demonstrate by Southern blot analysis and fluorescence in situ hybridization (FISH) that in the rapidly growing rat AS-30D hepatoma cell line, enhanced hexokinase activity is associated with at least a 5-fold amplification of the type II gene relative to normal hepatocytes. This amplification is located chromosomally, extends to the whole gene, and most likely occurs at the site of the resident gene. No rearrangement of the gene could be detected. Therefore, overexpression of hexokinase type II in AS-30D hepatoma cells may be based, at least in part, on a stable gene amplification. This is the first report describing the amplification of a hexokinase gene in a tumor cell line expressing the high glycolytic phenotype.
Animals↗
Frontiers in ATP synthase research: understanding the relationship between subunit movements and ATP synthesis.
How biological systems make ATP has intrigued many scientists for well over half the 20th century, and because of the importance and complexity of the problem it seems likely to continue to be a source of fascination to both senior and younger investigators well into the 21st century. Scientific battles fought to unravel the vast secrets by which ATP synthases work have been fierce, and great victories have been short-lived, tempered with the realization that more structures are needed, additional subunits remain to be conquered, and that during ATP synthesis, not one, but several subunits may undergo either significant conformational changes, repositioning, or perhaps even physical "rotation" similar to bacterial flagella (1,2). In this introductory article, the author briefly summarizes our current knowledge about the complex substructure of ATP synthases, what we have learned from X-ray crystallography of the F1 unit, and current evidence for subunit movements.
Adenosine Triphosphate↗
The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase.
Cystic fibrosis is caused by mutations in the cell membrane protein called CFTR (cystic fibrosis transmembrane conductance regulator) which functions as a regulated Cl- channel. Although it is known that CFTR contains two nucleotide domains, both of which exhibit the capacity to bind ATP, it has not been demonstrated directly whether one or both domains can function as an active ATPase. To address this question, we have studied the first CFTR nucleotide binding fold (NBF1) in fusion with the maltose-binding protein (MBP), which both stabilizes NBF1 and enhances its solubility. Three different ATPase assays conducted on MBP-NBF1 clearly demonstrate its capacity to catalyze the hydrolysis of ATP. Significantly, the mutations K464H and K464L in the Walker A consensus motif of NBF1 markedly impair its catalytic capacity. MBP alone exhibits no ATPase activity and MBP-NBF1 fails to catalyze the release of phosphate from AMP or ADP. The Vmax of ATP hydrolysis (approximately 30 nmol/min/mg of protein) is significant and is markedly inhibited by azide and by the ATP analogs 2'-(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate and adenosine 5'-(beta, gamma-imido)triphosphate. As inherited mutations within NBF1 account for most cases of cystic fibrosis, results reported here are fundamental to our understanding of the molecular basis of the disease.
Adenosine Triphosphatases↗
Glucose catabolism in cancer cells. Isolation, sequence, and activity of the promoter for type II hexokinase.
One of the most characteristic phenotypes of rapidly growing cancer cells is their propensity to catabolize glucose at high rates. Type II hexokinase, which is expressed at high levels in such cells and bound to the outer mitochondrial membrane, has been implicated as a major player in this aberrant metabolism. Here we report the isolation and sequence of a 4.3-kilobase pair proximal promoter region of the Type II hexokinase gene from a rapidly growing, highly glycolytic hepatoma cell line (AS-30D). Analysis of the sequence enabled the identification of putative promoter elements, including a TATA box, a CAAT element, several Sp-1 sites, and response elements for glucose, insulin, cAMP, Ap-1, and a number of other factors. Transfection experiments with AS-30D cells showed that promoter activity was enhanced 3.4-, 3.3-, 2.4-, 2.1-, and 1.3-fold, respectively, by glucose, phorbol 12-myristate 13-acetate (a phorbol ester), insulin, cAMP, and glucagon. In transfected hepatocytes, these same agents produced little or no effect. The results emphasize normal versus tumor cell differences in the regulation of Type II hexokinase and indicate that transcription of the Type II tumor gene may occur independent of metabolic state, thus, providing the cancer cell with a selective advantage over its cell of origin.
Amino Acid Sequence↗
Glucokinase of Escherichia coli: induction in response to the stress of overexpressing foreign proteins.
A variety of stressful conditions, such as heat shock, ethanol, osmotic shock, glucose deprivation, and oxidative stress, are known to induce the synthesis of specific proteins. Here, we report the induction in Escherichia coli of a protein elicited in response to a hitherto unidentified stress condition, i.e., the overexpression of foreign proteins. The induced protein identified as glucokinase (EC 2.7.1.2) is produced at a level > or = 20-fold higher than the level in wild-type E. coli when foreign proteins are expressed under the control of the alkaline phosphatase (phoA) promoter. The bacterial glucokinase is shown to have a mass of approximately 47 kDa determined by a "renaturation activity stain assay" in situ following sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and exhibits a high specificity for the phosphorylation of glucose. The apparent Km values for glucose and ATP for the enzyme are 0.15 and 0.50 mM, respectively, indicating that the E. coli enzyme is a low Km glucose hexokinase. The enzyme cross-reacts with rabbit antisera raised against hexokinase from higher eukaryotes, implicating some sequence similarity with mammalian hexokinases. Under normal conditions, E. coli glucokinase plays a minor role in glucose metabolism. However, under anabolic stress conditions, this glycolytic enzyme may be required to supplement levels of glucose 6-phosphate. Alternatively, glucokinase, which is predicted in analogy to other hexose-utilizing kinases to have structural folds characteristic of hsp 70, may itself, or in combination with other E. coli proteins, function in the stabilization of newly synthesized proteins.
DNA, Complementary↗
Solution structure and function in trifluoroethanol of PP-50, an ATP-binding peptide from F1ATPase.
PP-50, a synthetic peptide, based on residues 141-190 of the beta-subunit of mitochondrial F1ATPase, containing the GX4GKT consensus sequence for nucleoside triphosphate binding, binds ATP tightly (Kd = 17.5 microM) as found by fluorescence titration at pH 4.0. CD and 2D proton NMR studies at pH 4.0 revealed two beta-turns, regions of extended secondary structure, transient tertiary structure, and flexibility in the GX4GKT region (W.J. Chuang, C. Abeygunawardana, P. L. Pedersen, and A. S. Mildvan, 1992, Biochemistry 31, 7915-7921). CD titration of PP-50 with trifluoroethanol (TFE) reveals a decrease in ellipticity at 208 and 222 nm, saturating at 25% TFE. Computer analysis indicates that 25% TFE increases the helix content from 5.8 to 28.6%, decreases the beta-structure from 30.2 to 20.2% and decreases the coil content from 64 to 51.2%. Fluorescence titrations of H2ATP2- with PP-50 in 25% TFE yields a Kd of 7.3 microM, 2.4-fold tighter than in H2O, probably due to TFE increasing the activity of H2ATP2- . PP-50 completely quenches the fluorescence of H2ATP2- in 25% TFE, while in H2O the fluorescence quenching is only 62%. In H2O the binding of H2ATP2- increases the structure of PP-50 as detected by CD, but in 25% TFE no significant change in CD is found on binding either H2ATP2- or Mg2+ HATP (Kd = 14 microM). The complete proton NMR spectrum of PP-50 in 25% TFE has been assigned. The solution structure, determined by distance geometry, molecular dynamics with simulated annealing, and energy minimization, consists of a coil (residues 1-8), a strand (residues 9-12), a loop (residues 13-22) containing the GX4GKT consensus sequence (residues 16-23), an alpha-helix (residues 23-36), a turn (residues 38-41), and a coil (residues 42-50), similar to that of the corresponding region of the X-ray structure of F1ATPase (J.P. Abrahams, A.G.W. Leslie, R. Lutter, and J. E. Walker, 1994 Nature 370, 621-628) and to the structure of a homologous peptide from the ATP-binding site of adenylate kinase (D. C. Fry, D. M. Byler, H. Susi, E. M. Brown, S. A. Kuby, and A. S. Mildvan, 1988 Biochemistry 27, 3588-3598), beta, gamma-Bidentate Cr3+ ATP binds to PP-50 with the Cr3+ pyrophosphate moiety approaching the epsilon-methylene group of K22 in the GX4GKT consensus sequence, in agreement with the X-ray structure of the Mg2+ AMPPNP complex of F1ATPase.
Adenosine Triphosphate↗
Rat liver ATP synthase. Relationship of the unique substructure of the F1 moiety to its nucleotide binding properties, enzymatic states, and crystalline form.
The F1 moiety of rat liver ATP synthase has a molecular mass of 370,000, exhibits the unique substructure alpha 3 beta 3 gamma delta epsilon, and fully restores ATP synthesis to F1-depleted membranes. Here we provide new information about rat liver F1 as it relates to the relationship of its unique substructure to its nucleotide binding properties, enzymatic states, and crystalline form. Seven types of experiments were performed in a comprehensive study. First, the capacity of F1 to bind [3H]ADP, the substrate for ATP synthesis and [32P]AMP-PNP (5'-adenylyl-beta,gamma-imidodiphosphate), a nonhydrolyzable ATP analog, was quantified. Second, double-label experiments were performed to establish whether ADP and AMP-PNP bind to the same or different sites. Third, total nucleotide binding was assessed by the luciferin-luciferase assay. Fourth, F1 was subfractionated into an alpha gamma and a beta delta epsilon fraction, both of which were subjected to nucleotide binding assays. Fifth, the nucleotide binding capacity of F1 was quantified after undergoing ATP hydrolysis. Sixth, the intensity of the fluorescence probe pyrene maleimide bound at alpha subunits was monitored before and after F1 experienced ATP hydrolysis. Finally, the catalytic activity and nucleotide content of F1 obtained from crystals being used in x-ray crystallographic studies was determined. The picture of rat liver F1 that emerges is one of an enzyme molecule that 1) loads nucleotide readily at five sites; 2) requires for catalysis both the alpha gamma and the beta delta epsilon fractions; 3) directs the reversible binding of ATP and ADP to different regions of the enzyme's substructure; 4) induces inhibition of ATP hydrolysis only after ADP fills at least five sites; and 5) exists in several distinct forms, one an active, symmetrical form, obtained in the presence of ATP and high P(i) and on which an x-ray map at 3.6 A has been reported (Bianchet, M., Ysern, X., Hullihen, J., Pedersen, P. L., and Amzel, L. M. (1991) J. Biol. Chem. 266, 21197-21201). These results are discussed within the context of a multistate model for rat liver F1 and also discussed relative to those reported for bovine heart F1, which has been crystallized with inhibitors in an asymmetrical form and has a propensity for binding nucleotides more tightly.
Adenosine Diphosphate↗
Multidrug resistance--a fascinating, clinically relevant problem in bioenergetics.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗