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E M Price

Publications and source records attributed to E M Price.

At least 19 recordsLinked to original sources
PubMed1992

Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase.

Drug-resistant tumor cells actively extrude a variety of chemotherapeutic agents by the action of the multi-drug resistance (MDR1) gene product, the plasma membrane P-glycoprotein. In this report we show that the expression of the human MDR1 gene in cultured Sf9 insect cells via a baculovirus vector generates a high activity vanadate-sensitive membrane ATPase. This ATPase is markedly stimulated by drugs known to interact with the P-glycoprotein, such as vinblastine and verapamil, and the ability of the various drugs to stimulate the ATPase corresponds to their previously observed affinity for this transporter. The drug-stimulated ATPase is not present in uninfected or mock-infected Sf9 cells, and its appearance correlates with the appearance of the MDR1 gene product detected with a monoclonal anti-MDR protein antibody and by labeling with 8-azido-ATP. The drug-induced ATPase requires magnesium ions, does not utilize ADP or AMP as substrates, exhibits a half-maximal activation at about 0.5 mM MgATP, and its maximal activity (about 3-5 mumol/mg MDR protein/min) approaches that of the well characterized ion transport ATPases. These results provide the first direct demonstration of a high capacity drug-stimulated ATPase activity of the human multidrug resistance protein and offer a new and simple assay for the investigation of functional interactions of various drugs with this clinically important enzyme.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Biochemical characterization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis epithelial cells.

Affinity-purified polyclonal antibodies, raised against two synthetic peptides corresponding to the R domain and the C terminus of the human cystic fibrosis transmembrane conductance regulator (CFTR), were used to characterize and localize the protein in human epithelial cells. Employing an immunoblotting technique that ensures efficient detection of large hydrophobic proteins, both antibodies recognized and approximately 180-kDa protein in cell lysates and isolated membranes of airway epithelial cells from normal and cystic fibrosis (CF) patients and of T84 colon carcinoma cells. Reactivity with the anti-C terminus antibody, but not with the anti-R domain antibody, was eliminated by limited carboxypeptidase Y digestion. When normal CFTR cDNA was overexpressed via a retroviral vector in CF or normal airway epithelial cells or in mouse fibroblasts, the protein produced had an apparent molecular mass of about 180 kDa. The CFTR expressed in insect (Sf9) cells by a baculovirus vector had a molecular mass of about 140 kDa, probably representing a nonglycosylated form. The CFTR in epithelial cells appears to exist in several forms. N-glycosidase treatment of T84 cell membranes reduces the apparent molecular mass of the major CFTR band from 180 kDa to 140 kDa, but a fraction of the T84 cell CFTR could not be deglycosylated, and the CFTR in airway epithelial cell membranes could not be deglycosylated either. Moreover, wheat germ agglutinin absorbs the majority of the CFTR from detergent-solubilized T84 cell membranes but not from airway cell membranes. The CFTR in all epithelial cell types was found to be an integral membrane protein not solubilized by high salt or lithium diiodosalicylate treatment. Sucrose density gradient fractionation of crude membranes prepared from the airway epithelial cells, previously surface-labeled by enzymatic galactosidation, showed a plasma membrane localization for both the normal CFTR and the CFTR carrying the Phe508 deletion (delta F 508). The CFTR in all cases co-localized with the Na+, K(+)-ATPase and the plasma membrane calcium ATPase, while the endoplasmic reticulum calcium ATPase and mitochondrial membrane markers were enriched at higher sucrose densities. Thus, the CFTR appears to be localized in the plasma membrane both in normal and delta F 508 CF epithelial cells.

3T3 Cells↗
PubMed1992

Small linear chloride channels are endogenous to nonepithelial cells.

We used both single-channel and whole cell patch-clamp techniques to characterize chloride channels and currents endogenous to Sf9 cells, 3T3 fibroblasts, and Chinese hamster ovary cells. In cell-attached patches from these cell types, anion channels were observed with low ohmic conductance (4-11 ps), linear current-voltage relationships, and little time- or voltage-dependent behavior. These channels are very similar to the Cl- channels reported to appear concomitant with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in these cell lines. The presence of such endogenous channels suggests either that low levels of CFTR are present in all of these cell lines prior to transfection or that an endogenous non-CFTR channel is present in these cell types. Our results suggest that at least some of the channel behaviors attributed to expressed, recombinant CFTR in previous studies may have been due to these endogenous Cl- channels.

3T3 Cells↗
PubMed1990

Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit.

It has recently been shown that replacement of the border residues (Gln-111 and Asn-122) of the H1-H2 extracellular domain of the sheep Na,K-ATPase alpha subunit with the charged amino acids Arg and Asp generates a ouabain-resistant enzyme (Price, E. M. and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to further study structure-function relationships in Na,K-ATPase, six additional mutations have been made at these border positions. Two of these mutants were single amino acid substitutions (Gln-111 to Arg or Asn-122 to Asp). These mutations change one or the other H1-H2 border residue to a charged amino acid. The remaining substitutions were double mutants in which both of the H1-H2 border residues were simultaneously changed to charged amino acids. Changes were made which introduced either positively charged amino acids (Lys at positions 111 and 122), negatively charged amino acids (Glu at positions 111 and 122) or oppositely charged amino acids (Lys at position 111 and Glu at 122; Asp at position 111 and Arg at 122) at the borders of the H1-H2 extracellular domain. HeLa cells transfected with any of these sheep Na,K-ATPase alpha subunit mutants were able to grow in concentrations of ouabain that were toxic to untransfected cells or cells transfected with the wild type sheep alpha subunit. Crude membranes isolated from the transfectants were analyzed for ouabain inhibitable Na,K-ATPase activity. All of the transfectants contained a relatively ouabain-resistant component of enzyme activity, with the ouabain I50 values ranging from 4 x 10(-3) M to 1 x 10(-6) M. The most resistant enzyme was the double mutant that contained Asp at position 111 and Arg at 122, whereas the least resistant were the enzymes containing the single amino acid substitutions. There was no correlation between the type of charged amino acid present at the border position and the degree of ouabain resistance. These data demonstrate the functional importance, in terms of ouabain binding, of the border positions of the H1-H2 extracellular domain of the Na,K-ATPase alpha subunit.

Animals↗
PubMed1990

Molecular genetics of Na,K-ATPase.

Researchers in the past few years have successfully used molecular-genetic approaches to determine the primary structures of several P-type ATPases. The amino-acid sequences of distinct members of this class of ion-transport ATPases (Na,K-, H,K-, and Ca-ATPases) have been deduced by cDNA cloning and sequencing. The Na,K-ATPase belongs to a multiple gene family, the principal diversity apparently resulting from distinct catalytic alpha isoforms. Computer analyses of the hydrophobicity and potential secondary structure of the alpha subunits and primary sequence comparisons with homologs from various species as well as other P-type ATPases have identified common structural features. This has provided the molecular foundation for the design of models and hypotheses aimed at understanding the relationship between structure and function. Development of a hypothetical transmembrane organization for the alpha subunit and application of site-specific mutagenesis techniques have allowed significant progress to be made toward identifying amino acids involved in cardiac glycoside resistance and possibly binding. However, the complex structural and functional features of this protein indicate that extensive research is necessary before a clear understanding of the molecular basis of active cation transport is achieved. This is complicated further by the paucity of information regarding the structural and functional contributions of the beta subunit. Until such information is obtained, the proposed model and functional hypotheses should be considered judiciously. Considerable progress also has been made in characterizing the regulatory complexity involved in expression of multiple alpha-isoform and beta-subunit genes in various tissues and cells during development and in response to hormones and cations. The regulatory mechanisms appear to function at several molecular levels, involving transcriptional, posttranscriptional, translational, and posttranslational processes in a tissue- or cell-specific manner. However, much research is needed to precisely define the contributions of each of these mechanisms. Recent isolation of the genes for these subunits provides the framework for future advances in this area. Continued application of biochemical, biophysical, and molecular genetic techniques is required to provide a detailed understanding of the mechanisms involved in cation transport of this biologically and pharmacologically important enzyme.

Amino Acid Sequence↗
PubMed1989

Site-directed mutagenesis of a conserved, extracellular aspartic acid residue affects the ouabain sensitivity of sheep Na,K-ATPase.

Site-specific mutagenesis was used to study the function of a conserved, extracellular aspartic acid residue from the sheep Na,K-ATPase alpha subunit. This amino acid, Asp-121, is the penultimate residue of the first extracellular domain of the alpha subunit. The border residues of this particular extracellular loop of the alpha subunit have been shown to be determinants of ouabain sensitivity (Price, E. M., and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to determine if Asp-121 is involved in ouabain binding, five different amino acid substitutions at this position were generated. Four of the five mutant alpha subunits, containing either Asn, Ala, Glu, or Ser in place of Asp-121, conferred ouabain resistance to HeLa cells when expressed in those cells. Cloned sublines of cells selected in ouabain were characterized in terms of ouabain-inhibitable cell growth and Na,K-ATPase activity. The cells expressing the mutant Na,K-ATPase alpha subunit containing either Asn, Ala, Glu, or Ser in place of Asp-121 contained a component of Na,K-ATPase activity that was nearly 100-times more resistant to ouabain than the endogenous HeLa (human) or sheep enzyme. Apparently, conservative (Glu for Asp), isosteric (Asn for Asp), and nonconservative (Ala or Ser for Asp) substitutions all significantly decreased ouabain sensitivity. These data suggest that Asp-121 of the sheep Na,K-ATPase alpha subunit participates in the binding interaction between the enzyme and ouabain.

Amino Acid Sequence↗
PubMed1989

Folate coenzyme and antifolate transport proteins in normal and neoplastic cells.

The transport systems for folate coenzymes and antifolate compounds into various types of normal and neoplastic cells display considerable diversity in their pharmacokinetics and also in terms of the apparent molecular weights of the proteins involved. Further, several uptake routes may exist in a given cell type. A variety of neoplastic tissues have been reported to rely upon a single major transport system that has a relatively high affinity for the reduced form of folate compounds and for antifolates such as methotrexate. Using a photoaffinity analogue of methotrexate, we have identified the involvement of a 48 kDa membrane protein and a 38 kDa cytosolic or peripheral membrane protein in the transport of this compound into murine L1210 leukemia cells. Such an uptake is absent in mutant L1210 cells that are defective in methotrexate transport. We propose a model for the uptake of reduced folate coenzymes in L1210 cells in which the compound is initially transported across the cell membrane by the 48 kDa protein and delivered on the cytoplasmic surface to the 38 kDa protein; the 38 kDa protein then carries the folate compound to a specific enzyme of folate metabolism. Antibodies to the membrane folate binding protein from human placenta cross-react with the 48 kDa protein in L1210 cell membranes indicating an immunological relationship between these two proteins. Comparison of the amino acid sequences of peptides of the placental receptor obtained by digestion with S. aureus V8 protease indicate the presence of two homologous forms of the folate binding protein in placenta; one of these forms appears to have an identical sequence to the soluble and membrane associated folate binding proteins in human epidermoid carcinoma (KB) cells, which in turn share the primary structure of the soluble and membrane associated folate binders in human milk in the regions that have been sequenced. These results indicate that the folate coenzyme transport proteins in various tissues may be structurally related and in several instances may even be identical. In the latter cases the observed differences in apparent molecular weights may be due to differences in glycosylation and/or proteolysis. In support of this view is our observation that the deglycosylated and/or partially proteolyzed placental receptor retains the ability to bind folate.

Affinity Labels↗
PubMed1988

Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme.

Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the alpha 1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat alpha 1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase alpha subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep alpha 1 subunit, Gln-Ala-Ala-Thr-Glu-Glu-Glu-Pro-Gln-Asn-Asp-Asn, was changed to that of the rat, Arg-Ser-Ala-Thr-Glu-Glu-Glu-Pro-Pro-Asn-Asp-Asp. When expressed in HeLa cells, this mutated sheep alpha 1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep alpha 1 cDNA containing only two amino acid substitutions. This double mutation was a Gln-111----Arg and Asn-122----Asp change at the amino terminus and carboxyl terminus, respectively, of the H1-H2 extracellular region. The resistant cells, whether transfected with the rat alpha 1 cDNA, the rat/sheep chimera, or the mutant sheep alpha 1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86Rb+ uptake, and Na,K-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: involvement of a membrane receptor and a cytosolic protein.

A radioiodinated photoaffinity analogue of methotrexate, N alpha-(4-amino-4-deoxy-10-methyl-pteroyl)-N epsilon-(4-azidosalicylyl)-L- lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells [Price, E. M., & Freisheim, J. H. (1987) Biochemistry 26, 4757-4763]. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degrees C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degrees C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Photoaffinity analogues of methotrexate as folate antagonist binding probes.

A photoaffinity analogue of methotrexate, APA-[125I]ASA-Lys, specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation. An excess of methotrexate blocks incorporation of the photoprobe. Following cyanogen bromide digestion of the radiolabeled enzyme and high-pressure liquid chromatographic separation of the generated peptides, a majority of the label was centered around residues 63-65 (Lys-Asn-Arg), part of the inhibitor binding domain. This photoprobe is also transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a Vmax similar to that for methotrexate. Ultraviolet irradiation at 4 degrees C of a cell suspension that had been incubated with the radiolabeled photoprobe resulted in the covalent modification of a 46-48 Kd protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the Kt for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. In addition, no labeling occurs when a cell line that has a defective methotrexate transport system is similarly treated. Evidence that, in the absence of irradiation and at 37 degrees C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (Mr = 38 Kd and 21 Kd) derived from the cell homogenate supernatant.

Animals↗
PubMed1987

Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region.

N alpha-(4-Amino-4-deoxy-10-methylpteroyl)-N epsilon-(4-azido-5- [125I]iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the 125I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme.

Affinity Labels↗
PubMed1987

Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells.

A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a Kt of 506 +/- 79 nM and a Vmax of 17.9 +/- 4.2 pmol min-1 (mg of total cellular protein)-1. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate (I50 = 1.0 microM). The parent compounds of the iodinated photoprobe inhibit [3H]methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a Ki of 66 +/- 21 nM. UV irradiation, at 4 degrees C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the Kt for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. In addition, no labeling occurs when a cell line that has a defective methotrexate transport system is similarly treated. Evidence that, in the absence of irradiation and at 37 degrees C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (Mr 38K and 21K) derived from the cell homogenate supernatant.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed1975

Scope and limitations of propionylthiocholinesterase in the characterisation of cholinesterase variants.

1. A manual procedure, using propionylthiocholine, for the determination of serum cholinesterase and its genetic variants has been assessed in respect of its precision and of its ability to classify the genetic variants of the enzyme. 2. It is shown that the propionylthiocholinesterase activity alone is a useful guide in identifying individuals who are likely to be sensitive to succinylcholine. 3. In phenotyping with dibucaine and fluoride, sera of phenotypes U and UF could not be distinguished from each other with confidence, but were distinguishable from sera of phenotype UA.

Choline↗