Biomedical subjects

J B Lingrel

Publications and source records attributed to J B Lingrel.

At least 19 recordsLinked to original sources

PubMed1996

Substitution of glutamic 779 with alanine in the Na,K-ATPase alpha subunit removes voltage dependence of ion transport.

The effects of changing Glu-779, located in the fifth transmembrane segment of the Na,K-ATPase alpha subunit, on the phosphorylation characteristics and ion transport properties of the enzyme were investigated. HeLa cells were transfected with cDNA coding the E779A substitution in an ouabain-resistant sheep alpha1 subunit (RD). Steady state phosphorylation stimulated by Na+ concentrations less than 20 mM or by imidazole were similar for RD and E779A enzymes, an indication that phosphorylation and Na+ occlusion were not altered by this mutation. With E779A enzyme, higher Na+ concentrations reduced the level of phosphoenzyme and stimulated Na-ATPase activity in the absence of K+. These effects were a consequence of Na+ increasing the rate of protein dephosphorylation. In voltage-clamped HeLa cells expressing E779A enzyme, a prominent electrogenic Na+-Na+ exchange was observed in the absence of extracellular K+. Thus, increased Na-ATPase activity and Na+-dependent dephosphorylation result from Na+ acting as a K+ congener with low affinity at extracellular binding sites. These data suggest that E779A does not directly participate in ion binding but does affect the connection between extracellular ion binding and intracellular enzyme dephosphorylation. In cells expressing control RD enzyme, Na,K-pump current was dependent on membrane potential and extracellular K+ concentration. However, Na,K-pump current in cells expressing E779A enzyme was voltage independent at all extracellular K+ tested. These results indicate that Glu-779 may be part of the access channel determining the voltage dependence of ion transport by the Na, K-ATPase.

Alanine↗

PubMed1996

Critical effects on catalytic function produced by amino acid substitutions at Asp804 and Asp808 of the alpha1 isoform of Na,K-ATPase.

At two intramembrane carboxyl-containing amino acids of the sheep alpha1 isoform of Na,K-ATPase (Asp804 and Asp808), both charge-conserving (Asp to Glu) and charge-deleting (Asp to Asn, Leu and Ala) replacements were made and the altered enzymes studied. Nucleotide changes encoding the amino acid substitutions were placed in a cDNA encoding a ouabain-resistant enzyme (sheep alpha1 RD) and the encoded enzymes were expressed in ouabain-sensitive HeLa cells. Transfections with cDNAs carrying all Asp804 substitutions, along with those carrying Asp808Ala, Asp808Asn, and Asp808Leu replacements failed to confer ouabain resistance to the cells, indicating critical roles for Asp804 and Asp808. Only the expression of the Asp808Glu enzyme produced ouabain-resistant HeLa cells, demonstrating that the altered protein was functional. When the inactive proteins Asp804Ala and Asp808Ala were expressed using an alternative selection system (the protein carrying the amino acid substitution was the ouabain-sensitive wild-type sheep alpha1 Na,K-ATPase, which was expressed in ouabain-resistant 3T3 cells), intact cells were able to bind extracellular ouabain with high affinity (Kd = 1-30 nM), indicating that the inactive proteins were synthesized and folded properly in the plasma membrane. The results demonstrate that carboxyl side chains at positions 804 and 808 are critical for enzyme catalytic function.

3T3 Cells↗

PubMed1996

Ouabain interactions with the H5-H6 hairpin of the Na,K-ATPase reveal a possible inhibition mechanism via the cation binding domain.

Cardiac glycosides such as ouabain and digoxin specifically inhibit the Na,K-ATPase. Three new residues in the carboxyl half of the Na, K-ATPase, Phe-786, Leu-793 (PFLIF786IIANIPL793PLGT797), and Phe-863 (FTYF863VIM) have been identified as ouabain sensitivity determinants using random mutagenesis. Polymerase chain reaction was utilized to randomly mutate the DNA sequence encoding the amino acids between Lys-691 and Lys-945 in the alpha subunit of the Na, K-ATPase. This region contains four transmembrane segments (H5, H6, H7, and H8) and the connecting extracellular and cytoplasmic loops. Diverse substitutions of these three residues resulted in proteins displaying 2.8-48-fold increases in the I50 of different cardiac glycosides for inhibition of the Na,K-ATPase activity. By locating these residues, in conjunction with Thr-797 (Feng, J., and Lingrel, J. B (1994) Biochemistry 33, 4218-4224), a new region of the protein containing the H5-H6 hairpin and the H7 transmembrane segment emerges as a major determinant of ouabain inhibition. Thus, a link between the cardiac glycoside binding site and the cation transport sites of the Na,K-ATPase transpires giving a structural base to the cation antagonism of ouabain binding. Furthermore, this link suggests a possible mechanism for cardiac glycoside inhibition of the Na,K-ATPase, such that ouabain binding to the implicated region blocks the movement of the H5 and H6 transmembrane domains which may be required for energy transduction and cation transport.

Amino Acid Sequence↗

PubMed1996

Analysis of developmental switching in transgenic mice with 5' and 3' deletions in the human beta globin gene.

Our interest in the cis-acting elements that promote the up-regulation of the beta globin gene has led to a systematic deletion analysis of portions of the beta globin gene in the context of the HS2 and gamma globin gene using transgenic mice. In constructs that delete the 5' region to only 265 bp, high-level, erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels. A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased beta globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between -122 and -265. In addition, a construct in which the entire beta globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3' region of the beta globin gene was deleted in order to examine the effect of a previously defined 3' enhancer region. With deletion of this region, the expression of the human beta globin gene in transgenic mice is unchanged relative to the parental constructs.

Animals↗

PubMed1995

Substitutions of serine 775 in the alpha subunit of the Na,K-ATPase selectively disrupt K+ high affinity activation without affecting Na+ interaction.

The functional role of serine 775, predicted to be located in the fifth transmembrane segment of the alpha subunit of the Na,K-ATPase (YTLTSNIPE), was studied using site-directed mutagenesis, expression, and kinetic analysis. Substitutions S775A, S775C, and S775Y were introduced into an ouabain-resistant alpha 1 sheep isoform and expressed in HeLa cells. cDNAs carrying substitutions S775C and S775A produced ouabain-resistant colonies only when extracellular K+ was increased from 5.4 mM to 10 or 20 mM, respectively. No ouabain-resistant colonies were obtained for substitutions S775Y at any tested K+ concentration. Kinetic characterization of S775C and S775A substituted enzymes showed expression levels higher than control enzyme, reduced Vmax and turnover, and normal phosphorylation and high affinity ATP binding. Dephosphorylation experiments indicated that S775A substituted enzyme is insensitive to ADP but readily dephosphorylated by K+. The K+ K1/2 values for the activation of the Na,K-ATPase were markedly altered, with S775C displaying a 13-fold increase and S775A exhibiting a 31-fold increase. These large changes in the Na,K-ATPase affinity for K+ are consistent with the participation of this amino acid in binding K+ during the translocation of this cation. Substitutions of Ser775 did not change Na+ affinity, indicating that this residue is likely not involved in Na+ binding and occlusion. These data show that the electronegative oxygen and the small side chain of Ser775 are required for efficient enzyme function. Moreover, these results suggest Ser775 plays a distinct role in K+ transport and not in Na+ interactions, revealing a possible mechanism for the enzymatic differentiation of these cations by the Na,K-ATPase.

Amino Acid Sequence↗

PubMed1995

Amino acid replacement of Asp369 in the sheep alpha 1 isoform eliminates ATP and phosphate stimulation of [3H]ouabain binding to the Na+, K(+)-ATPase without altering the cation binding properties of the enzyme.

Modification of aspartic acid 369 in the sheep alpha 1 Na+,K(+)-ATPase to asparagine results in a membrane-associated form of Na+,K(+)-ATPase that can bind [3H]ouabain with high affinity in the presence of Mg2+ alone (KD = 20.4 +/- 2.6 nM). Ouabain binding to the D369N mutant is not stimulated by inorganic phosphate, confirming that Asp369 is both the catalytic phosphorylation site and the only Pi interaction site which stimulates ouabain binding. Cation inhibition of Mg(2+)-stimulated ouabain binding to the D369N mutant demonstrated that three Na+ and two K+ ions inhibit [3H]ouabain binding and suggests that this inhibition must occur via a cation-sensitive conformational change which does not directly involve dephosphorylation of the enzyme. In the presence of 10 mM Mg2+, ATP stimulates ouabain binding to the wild type protein, (AC50 = 21.4 +/- 2.7 microM) but inhibits the binding to the D369N mutant (IC50 = 2.52 +/- 0.17 microM) indicating that the mutation does not destroy the high affinity site for MgATP but does change the nature of the protein conformation normally induced by a nucleotide-Na+,K(+)-ATPase interaction. Increasing the Mg2+ from 1 to 10 mM did not alter the AC50 or IC50 values for ATP and reveals that the Mg2+ interaction which stimulates ouabain binding in the absence of nucleotide involves a distinct divalent cation site not associated with the binding of the magnesium-nucleotide complex. Thus, altering the catalytic phosphorylation site of Na+,K(+)-ATPase does not affect the expression of the ouabain-sensitive protein in the membrane fraction of NIH 3T3 cells and does not disrupt the binding of Na+, K+, Mg2+, ouabain, or ATP to the enzyme. However, the D369N substitution does inhibit the formation of a nucleotide-protein complex with high affinity for ouabain.

Adenosine Triphosphate↗

PubMed1995

Glutamic acid 327 in the sheep alpha 1 isoform of Na+,K(+)-ATPase is a pivotal residue for cation-induced conformational changes.

The cation binding characteristics of the mutant E327A formed in the sheep alpha 1 isoform of the Na+,K(+)-ATPase were examined using [3H]ouabain binding as a function of monovalent cation concentrations. Equilibrium competition binding assays in the presence of Mg2+, inorganic phosphate and various amounts of unlabelled ouabain indicated that both wild-type sheep alpha 1 protein and the E327A mutant expressed in 3T3 cells had similar affinities for ouabain (KD = 1.53 and 1.31 nM respectively). Sodium inhibition of ouabain binding appeared competitive in both enzymes. However, binding of three Na+ ions was required to explain the steep character of the Na+ inhibition curve for the wild-type Na+,K(+)-ATPase (Ki = 12.8 +/- 1.6 mM), whereas the binding of two Na+ ions was detected for the mutant E327A (Ki = 19.2 +/- 2.5 mM). Potassium binding of [3H]ouabain binding displayed a partially competitive nature with Hill coefficients of 2 for both wild-type sheep alpha 1 (Ki = 0.743 +/- 0.044 mM) and E327A (Ki = 0.875 +/- 0.067 mM). At concentrations of K+ above 10 mM, the sheep alpha 1 competition curve levelled off whereas the inhibition curve for E327A displayed a stimulation in ouabain binding. This stimulation in [3H]ouabain binding also occurred with Rb+, Cs+ and Li+, but was never observed with choline or Na+, suggesting that this effect was not due to ionic strength. From these [3H]ouabain-binding studies, it is obvious that the mutant enzyme E327A in the presence of Mg2+, Pi and ouabain, interacts with monovalent cations in a unique fashion. One interpretation of these data is that the glutamic acid residue at position 327 is involved in a conformational transition induced by the binding of monovalent cations to the Na+,K+-ATPase and that this transition is inhibited by the mutation of E327A.

3T3 Cells↗

PubMed1995

Comparison of the effects of potassium on ouabain binding to native and site-directed mutants of Na,K-ATPase.

We examined the effect of K+ on Mg(2+)- and P(i)-supported [3H]ouabain binding to Na,K-ATPases, including partially purified enzyme from sheep kidney and wild-type and mutant sheep alpha 1 isoforms (C104A, Y108A, E116Q, P118K, Y124F, R880P, R880L, and N122D) expressed in NIH3T3 cells. In the presence of increasing concentrations of K+, [3H]ouabain binding to these enzymes decreases but never reaches nonspecific binding levels, consistent with the concept that ouabain is still able to bind to the K(+)-complexed enzyme but with reduced affinity. A partially competitive model for K+ inhibition of ouabain binding is proposed which satisfactorily fits the binding data. The model is consistent with the sequential binding of two K+ ions to the enzyme. Ki values (approximately 1.0 mM) for K+ obtained from this model are comparable to the apparent K+ affinities of the rat alpha isoforms determined by measuring the K+ dependence of Na,K-ATPase activity [E. A. Jewell and J. B. Lingrel (1991) J. Biol. Chem. 266, 16925-16930]. This is consistent with the concept that K+ inhibition of Mg2+ plus P(i) supported ouabain binding is mediated by K+ binding to the same high-affinity binding sites present in the native enzyme under physiological conditions. While the mutants exhibit binding constants for ouabain which vary more than 30-fold from that of the wild-type enzyme, their affinities for K+ differ less than twofold from that of the native enzyme. Our results suggest that the ouabain and K+ binding sites are not the same and are differentially affected by mutations of the enzyme. The system described here should prove useful in the analysis of cation binding to other mutants of the Na,K-ATPase, especially those carrying amino acid replacements which result in an inactive enzyme.

3T3 Cells↗

PubMed1995

Glutamic acid 327 in the sheep alpha 1 isoform of Na+,K(+)-ATPase stabilizes a K(+)-induced conformational change.

By combining the tools of site-directed mutagenesis and [3H]ouabain binding, the functional role of glutamic acid 327 in the fourth transmembrane domain of the sheep alpha 1 isoform of Na+,K(+)-ATPase was examined with respect to its interactions with ouabain, Na+,K+,Mg2+, and inorganic phosphate. Using site-directed mutagenesis, this glutamic acid was substituted with alanine, aspartic acid, glutamine, and leucine. The mutant proteins were constructed in a sheep alpha 1 protein background such that [3H]ouabain binding could be utilized as a highly specific probe of the exogenous protein expressed in NIH 3T3 cells. Na+ competition of [3H]ouabain binding to the mutant forms of Na+,K(+)-ATPase revealed only slight alterations in their affinities for Na+ and in their abilities to undergo Na(+)-induced conformational changes which inhibit ouabain binding. In contrast, K+ competition of [3H]ouabain binding to all four mutant forms of Na+,K(+)-ATPase displayed severely altered interactions between these proteins and K+. Interestingly, [3H]ouabain binding to the mutant E327Q was not inhibited by the presence of K+. This mutant was previously reported to be functionally able to support cation transport with a 5-fold reduced K0.5 for K(+)-dependent ATPase activity (Jewell-Motz, E. A., and Lingrel, J.B. (1993) Biochemistry 32, 13523-13530; Vilsen, B. (1993) Biochemistry 32, 13340-13349). Thus, it appears that this glutamic acid in the fourth transmembrane domain may be important for stabilizing a K(+)-induced conformation within the catalytic cycle of Na+,K(+)-ATPase that is not rate-limiting in the overall ATPase cycle but that displays a greatly reduced affinity for ouabain.

3T3 Cells↗

PubMed1995

Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Krüppel-like factor: identification of a new multigene family.

We have identified and characterized the gene for a novel zinc finger transcription factor which we have termed lung Krüppel-like factor (LKLF). LKLF was isolated through the use of the zinc finger domain of erythroid Krüppel-like factor (ELKF) as a hybridization probe and is closely related to this erythroid cell-specific gene. LKLF is expressed in a limited number of tissues, with the predominant expression seen in the lungs and spleen. The gene is developmentally controlled, with expression noted in the 7-day embryo followed by a down-regulation at 11 days and subsequent reactivation. A high degree of similarity is noted in the zinc finger regions of LKLF and EKLF. Beyond this domain, the sequences diverge significantly, although the putative transactivation domains for both LKLF and EKLF are proline-rich regions. In the DNA-binding domain, the three zinc finger motifs are so closely conserved that the predicted DNA contact sites are identical, suggesting that both proteins may bind to the same core sequence. This was further suggested by transactivation assays in which mouse fibroblasts were transiently transfected with a human beta-globin reporter gene in the absence and presence of an LKLF cDNA construct. Expression of the LKLF gene activates this human beta-globin promoter containing the CACCC sequence previously shown to be a binding site for EKLF. Mutation of this potential binding site results in a significant reduction in the reporter gene expression. LKLF and EKLF can thus be grouped as members of a unique family of transcription factors which have discrete patterns of expression in different tissues and which appear to recognize the same DNA-binding site.

Animals↗

PubMed1995

In situ localization of sodium-potassium ATPase mRNA in developing mouse lung epithelium.

The ontogeny of Na(+)-K(+)-adenosinetriphosphatase (ATPase) mRNA in the mouse lung was examined, using alpha- and beta-isoform-specific probes in Northern blot assays and for in situ hybridization analysis. Northern blot assays demonstrated an increase in Na(+)-K(+)-ATPase expression in the perinatal period, peaking at birth (D1), with alpha 1- and beta 1-isoform levels reaching six to eight times adult levels. In situ alpha 1-isoform hybridization signals were localized primarily to developing airway epithelium and were most intense on D1. Postnatally, alpha 1-isoform hybridization signals persisted in airway epithelium, although progressively diminishing in intensity relative to perinatal levels. In developing alveolar regions, alpha 1-isoform hybridization signals remained slightly above background during this period. beta 1-Isoform hybridization signals increased dramatically during the perinatal period in both developing airway and alveolar epithelia. Postnatally, beta 1-isoform hybridization signals declined slightly in airway epithelium and developed a punctate pattern in alveolar epithelium. These data indicate that the perinatal increase in Na(+)-K(+)-ATPase expression observed in the developing mouse lung is localized primarily to epithelial structures and is therefore likely to be related to the changes in transepithelial ion and fluid transport known to occur in the perinatal period.

Aging↗

PubMed1995

Functional consequences of substitutions of the carboxyl residue glutamate 779 of the Na,K-ATPase.

Carboxyl-containing amino acids in the transmembrane segments appear to be important for sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) activity. Substitution of Glu779 with Leu in a ouabain-resistant isoform inactivates the overall enzyme activity (Jewell-Motz & Lingrel, 1993). Chemical modification of this residue results in inactivation of Na,K-ATPase in a Na+ and K+ protectable manner (Arguello and Kaplan, 1991, 1994). These experiments suggest that this residue is important in cation binding. To further understand the role of Glu779 in Na,K-ATPase function, we have substituted this with four amino acids (Gln, Asp, Ala, and Leu) using site-directed mutagenesis coupled with expression and characterized the expressed enzyme. The amino acid substitutions were introduced into a modified sheep RD alpha 1 isoform that is relatively resistant to this drug. Enzyme carrying the E779Q and E779A replacements conferred ouabain resistance to the sensitive HeLa cells, while expression of enzyme carrying the E779D and E779L substitutions did not. Further analysis of isolated plasma membranes containing altered enzymes E779Q and E779A confirmed that they retain Na,K-ATPase activity. Analysis of cation stimulation of Na,K-ATPase activity revealed that the E779Q substituted enzyme exhibited a similar apparent affinity for K+ and a 2.6-fold decrease in affinity for Na+ compared with control enzyme. The E779A replacement caused a 6.6-fold and 5-fold decrease in apparent affinity for K+ and Na+, respectively. There is no difference in apparent affinity for ATP at the low affinity site for either E779Q or E779A.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1994

A putative fourth Na+,K(+)-ATPase alpha-subunit gene is expressed in testis.

The Na+,K(+)-ATPase alpha subunit has three known isoforms, alpha 1, alpha 2 and alpha 3, each encoded by a separate gene. This study was undertaken to determine the functional status of a fourth human alpha-like gene, ATP1AL2. Partial genomic sequence analysis revealed regions exhibiting sequence similarity with exons 3-6 of the Na+,K(+)-ATPase alpha isoform genes. ATP1AL2 cDNAs spanning the coding sequence of a novel P-type ATPase alpha subunit were isolated from a rat testis library. The predicted polypeptide is 1028 amino acids long and exhibits 76-78% identity with the rat Na+,K(+)-ATPase alpha 1, alpha 2 and alpha 3 isoforms, indicating that ATP1AL2 may encode a fourth Na+,K(+)-ATPase alpha isoform. A 3.9-kb mRNA is expressed abundantly in human and rat testis.

Amino Acid Sequence↗

PubMed1994

The presence of both negative and positive elements in the 5'-flanking sequence of the rat Na,K-ATPase alpha 3 subunit gene are required for brain expression in transgenic mice.

The Na,K-ATPase is an integral plasma membrane protein consisting of alpha and beta subunits, each of which has discrete isoforms expressed in a tissue-specific manner. Of the three functional alpha isoform genes, the one encoding the alpha 3 isoform is the most tissue-restricted in its expression, being found primarily in the brain. To identify regions of the alpha 3 isoform gene that are involved in directing expression in the brain, a 1.6 kb 5'-flanking sequence was attached to a reporter gene, chloramphenicol acetyltransferase (CAT). The alpha 3-CAT chimeric gene construct was microinjected into fertilized mouse eggs, and transgenic mice were produced. Analysis of adult transgenic mice from different lines revealed that the transgene is expressed primarily in the brain. To further delineate regions that are needed for conferring expression in this tissue, systematic deletions of the 5'-flanking sequence of the alpha 3-CAT fusion constructs were made and analyzed, again using transgenic mice. The results from these analyses indicate that DNA sequences required for mediating brain-specific expression of the alpha 3 isoform gene are present within 210 bp upstream of the transcription initiation site. alpha 3-CAT promoter constructs containing scanning mutations in this region were also assayed in transgenic mice. These studies have identified both a functional neural-restrictive silencer element as well as a positively acting cis element.

Animals↗

PubMed1994

Identification of an amino acid substitution in human alpha 1 Na,K-ATPase which confers differentially reduced affinity for two related cardiac glycosides.

The ouabain-resistant cell line H1C1 displays a 30-fold differential of reduced sensitivity to the structurally related cardiac glycosides digoxin and digitoxin (Baker, R. M. (1976) in Biogenesis and Turnover of Membrane Macromolecules (Cook, J.S., ed) pp. 93-103, Raven Press, New York). Since these ligand congeners differ only by the presence of a hydroxyl group at C-12 of digoxin we predicted that the H1C1 phenotype must reflect a mutation which alters the binding site of the cardiac glycoside receptor (Na,K-ATPase). Complementary DNA encoding the alpha 1 Na,K-ATPase was prepared from H1C1 cell total RNA by reverse transcription-coupled polymerase chain reaction and these cDNAs were cloned. Sequence analysis of the reverse transcriptase-polymerase chain reaction clones revealed several independent isolates containing a G > A transition at nucleotide 332 of the propeptide coding sequence, generating the amino acid substitution C108Y. The ability of this substitution to confer differential sensitivity for digoxin and digitoxin was tested and confirmed by expressing a human alpha 1 C108Y-Na,K-ATPase in wild type HeLa cells and assaying for inhibition of cell growth and inhibition of Na,K-ATPase activity. Phenylalanine or alanine substitutions of this cysteine also confer this pattern of ligand discrimination. Ouabain-resistant Na,K-ATPase substitutions, at positions other than Cys-108 failed to exhibit differential sensitivity indicating that this ligand discrimination is unique to Cys-108 substitutions rather than a general property of cardiac glycoside-resistant mutants. It is proposed that differential resistance of the C108Y receptor for these ligands is a consequence of altering two features of the ligand-receptor interaction; one, a disruption of a common hydrogen bond resulting in general loss of affinity for cardiac glycosides and the other, formation of a new H-bond between the C-12 hydroxyl of digoxin and the receptor, specifically augmenting the stability of this ligand-receptor complex.

Amino Acid Sequence↗

PubMed1994

Na+,K(+)-ATPase.

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Adenosine Triphosphate↗

PubMed1994

Positive transcriptional regulation of the human gamma-globin gene. Gamma PE is a novel nuclear factor with multiple binding sites near the gene.

A novel nuclear factor involved in human gamma-globin gene regulation has been identified. Co-migrating and cross-competing complexes were formed with five individual fragments from the 5'- and 3'-flanking regions of the gene in DNA-protein binding assays. This indicates that a nuclear factor, termed gamma PE, has multiple binding sites near the gamma-globin gene. This characteristic is shared by other important factors in globin gene regulation, such as GATA-1. The five gamma PE binding sites can be placed in two categories based on DNA-protein binding affinity and DNA sequence composition. The consensus sequence for the two higher affinity binding sites is ATTANNNGGAANNCT(N)TNNNTAATGG and for the three lower affinity sites is AAAAN(A/T)A(A/T)TT. Both the ATTA and the TAAT motifs of a high affinity binding site are required for efficient DNA-protein binding. The tissue distribution of gamma PE binding activity is broad, including both erythroid and non-erythroid cell types. Transcription of either a gamma-globin or heterologous promoter is increased in the presence of nearby gamma PE binding sites. Therefore, gamma PE may be involved in activating the gamma-globin gene in fetal erythroid cells. UV cross-linking analysis indicates that the major protein interacting with a high affinity gamma PE binding site has a molecular mass of 108 kDa.

Animals↗

PubMed1994

Tissue- and isoform-specific kinetic behavior of the Na,K-ATPase.

The objective of this study has been to delineate the side-specific effects of Na+ and K+ on the transport kinetics of tissue-specific Na/K pumps. Two experimental systems have been used. In one, Na/K pumps of exogenous microsomal membrane sources (rat axolemma, kidney) were delivered by membrane fusion into dog erythrocytes, and in the other, the three isoforms of the catalytic subunit of the rat enzyme were individually transfected into HeLa cells as in previous studies (Jewell, E.A., and Lingrel, J. B (1991) J. Biol. Chem. 266, 16925-16930), with the alpha 2 and alpha 3 isoforms rendered relatively resistant to ouabain by site-directed mutagenesis. Whereas the kidney microsomes comprise the alpha 1 catalytic isoform, the axolemma microsomes were predominantly alpha 3 (approximately 60%) with lesser amounts of alpha 2 (approximately 25%) and alpha 1 (approximately 15%) as measured by the ouabain-sensitive profile of phosphoenzyme as well as by immunoblotting with isoform-specific antibodies using membranes of known specific activity as standards (alpha 1 of kidney, alpha 1 and alpha 2 of muscle). Both systems were analyzed with respect to the effects of varying concentrations of cytoplasmic Na+ and extracellular K+ on pump-mediated 86Rb+(K+) influx. With the individual isoform-transfected HeLa cells and monensin added to vary and control the intracellular Na+ concentration, differences in apparent affinities of the alpha 3 isoform compared with the alpha 1 and alpha 2 isoforms were observed, i.e. a approximately 3-fold higher affinity for extracellular K+ and approximately 4-fold lower affinity for cytoplasmic Na+. Thus, in the presence of 10 mM extracellular Na+, apparent K0.5 values for extracellular K+ activation of K+(Rb+) influxes were 0.22 +/- 0.02 mM for alpha 1, 0.20 +/- 0.02 mM for alpha 2, and 0.09 +/- 0.01 mM for alpha 3. At high intracellular K+ (> or = 100 mM) and saturating extracellular K+ concentrations, apparent K0.5 values for cytoplasmic Na+ activation were 17.6 +/- 1.1 mM for alpha 1, 19.7 +/- 1.0 mM for alpha 2, and 63.5 +/- 9.1 mM for alpha 3. The functional differences observed with the individual isoform-transfected cells were completely consistent with the kinetic differences observed with the axolemma and kidney pumps fused into erythrocytes. Axolemma pumps had a approximately 3-fold lower K0.5 for extracellular K+ and a approximately 2-fold higher K0.5 for cytoplasmic Na+.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗