Biomedical subjects

J B Lingrel

Publications and source records attributed to J B Lingrel.

At least 55 records · Page 3Linked to original sources

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.

Explore the source record for details and available documents.

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↗

PubMed1994

Analysis of amino acid residues in the H5-H6 transmembrane and extracellular domains of Na,K-ATPase alpha subunit identifies threonine 797 as a determinant of ouabain sensitivity.

Several amino acid residues of the alpha subunit of the Na,K-ATPase have been identified which alter ouabain sensitivity. These residues are located in the N-terminal half of the alpha 1 subunit suggesting that this portion of the molecule may represent the binding site for cardiac glycosides. However, not all extracellular and transmembrane regions have been investigated, including the H5-H6 membrane-spanning region. To determine if this region of the alpha subunit contributes to ouabain sensitivity, amino acids which have the potential to form hydrogen bonds were substituted with alanine, a non-hydrogen-bonding amino acid. cDNAs encoding enzyme containing these individual amino acid replacements were expressed in ouabain-sensitive HeLa cells, and the ability of the altered enzymes to confer ouabain resistance was examined. Nineteen amino acid substitutions were investigated. T797A (Thr 797 to Ala) was the only substitution which conferred ouabain resistance to sensitive HeLa cells. Three additional substitutions at this position (T797V, T797S, and T797D) were generated in order to examine the effects of the replacements of Thr 797 on ouabain inhibition of Na,K-ATPase activity. The T797V substitution conferred ouabain resistance, but T797S and T797D substitutions did not. The ouabain-resistant cell lines expressing the T797A and T797V substitutions exhibited Na,K-ATPase activity that was 60 and 70 times more resistant to ouabain than the endogenous HeLa or sheep enzymes. The absence of a hydroxyl group at amino acid 797 may be responsible for the reduced sensitivity of the enzyme with substitutions at this position.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Ouabain binding kinetics of the rat alpha two and alpha three isoforms of the sodium-potassium adenosine triphosphate.

The Na,K-ATPase has three alpha isoforms which differ in cardiac glycoside sensitivity and tissue distribution. The rodent alpha 1 isoform is relatively resistant to cardiac glycosides, while the alpha 2 and alpha 3 isoforms are quite sensitive. Because both the alpha 2 and alpha 3 isoforms are generally expressed in the same tissue, it has been difficult to differentiate and accurately determine the kinetics of ouabain binding to these isoforms. To more fully understand the interactions of the alpha 2 and alpha 3 isoforms with cardiac glycosides, the association and dissociation rates of ouabain binding were measured in transfected cell lines. cDNA's coding for the rat alpha 2 and alpha 3 isoforms were transfected into NIH 3T3 cells and characterized by Na,K-ATPase activity and [3H]ouabain binding. By individually expressing the alpha 2 and alpha 3 isoforms in ouabain-insensitive NIH 3T3 cells, the ouabain-binding characteristics of each isoform could be accurately determined. The association rate constants of the alpha 2 and alpha 3 isoforms were similar while the dissociation rate constant was 33 times slower for the alpha 3 isoform than the alpha 2 isoform. Calculation of the dissociation constant (Kd) from these rate constants yielded values of 115 and 1.6 nM for rat alpha 2 and alpha 3 isoforms, respectively. Scatchard analysis of the rat alpha 2 isoform produced a similar value for Kd of 37 +/- 9 nM. Inhibition of Na,K-ATPase activity indicates the rodent alpha 1 isoform has an IC50 1000-fold higher than the alpha 2 or alpha 3 isoform at 4.8 x 10(-5) M. The results are consistent with the hypothesis that the order of ouabain affinity between the rat alpha isoforms of the Na,K-ATPase is alpha 3 > alpha 2 >> alpha 1.

3T3 Cells↗

PubMed1994

Structure-function studies of the Na,K-ATPase.

Na,K-ATPase catalyzes the movement of sodium and potassium ions across the cell membrane utilizing ATP as an energy source. This enzyme is present in almost all tissues of higher organisms but is most abundant in the kidney where it is responsible for reabsorbing sodium ions from the glomerular filtrate. The enzyme is composed of two subunits and serves as the receptor for cardiac glycosides. Utilizing an expression/selection system it has been possible to identify several amino acid residues that affect sensitivity to the cardiac glycoside, ouabain. Those identified to date are located in the first transmembrane region and first extracellular region. The fact that amino acid residues within a transmembrane region affect ouabain sensitivity suggests that the drug is partially internalized in the lipid bilayer. In an effort to determine whether any of the amino acid residues which affect ouabain sensitivity interact with the sugar part of cardiac glycosides, ouabain and ouabagenin were tested in terms of their ability to inhibit enzyme containing substitutions at these positions. The two compounds differ in that ouabagenin lacks a sugar moiety. Interestingly, the ratio of I50's for the substituted enzymes remains the same as the wild type, which suggests that the amino acids identified as determinants of ouabain sensitivity to date are not likely to interact with the sugar. Another set of studies focused on cation binding. It has been proposed that cation transport involves negatively charged residues in one or more transmembrane regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Site-directed mutagenesis of the Na,K-ATPase: consequences of substitutions of negatively-charged amino acids localized in the transmembrane domains.

Site-directed mutagenesis was used to examine the importance of five carboxyl-containing amino acids localized in the putative membrane-spanning regions of the Na,K-ATPase (i.e., E327, E778, D803, D807, and D925 of the rat alpha 2 isoform). The substitutions were introduced into a cDNA encoding a ouabain-resistant isoform (i.e., rat alpha 2* which was mutated to encode a ouabain-resistant isoform), and the effect of these substitutions on Na,K-ATPase function was assessed by screening the altered enzymes for their ability to confer ouabain resistance when expressed in otherwise ouabain-sensitive cells. The expression of the alpha isoform containing certain substitutions at positions 327 and 925 was able to confer ouabain resistance to HeLa cells while the expression of rat alpha 2* containing substitutions at positions 778, 803, and 807 was not. In particular, amino acids in each of these positions were substituted with leucine to evaluate the importance of the carboxyl-containing side chain. The ability of rat alpha 2* containing E327L and D925L to confer ouabain resistance to HeLa cells indicates that neither the negative charge nor the oxygen-containing side chain is absolutely essential for overall function in this position. In contrast, the inability of rat alpha 2* carrying E778L, D803L, and D807L to confer ouabain resistance suggests that the naturally occurring amino acid may be more critical structurally and/or functionally for the Na,K-ATPase. Other more conservative substitutions introduced to further characterize the role of particular amino acid side chains include E327D, E327Q, D803N, D803E, and D925N.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Kinetic analysis of ouabain binding to native and mutated forms of Na,K-ATPase and identification of a new region involved in cardiac glycoside interactions.

Cardiac glycosides inhibit the Na,K-ATPase by binding to the catalytic alpha subunit of the enzyme. Site-directed mutagenesis of the H1-H2 domain has demonstrated the importance of this region in determining cardiac glycoside affinity. In this study, random mutagenesis was used to identify an amino acid, arginine 880, in the COOH-terminal portion of the alpha subunit which influences the sensitivity of the enzyme to ouabain. This residue is predicted to reside in the H7-H8 extracellular loop. Conversion of arginine 880 to a proline causes a 10-fold increase in the dissociation rate constant and a 2-fold increase in the association rate constant for [3H]ouabain binding. This results in an enzyme with a KD for ouabain 5-fold higher than the wild-type sheep alpha 1 isoform. These data are compatible with arginine 880 comprising a portion of the ouabain binding site. Furthermore, if arginine 880 is at the physical binding site, then this finding lends support to models that place this amino acid extracellularly since cardiac glycosides interact with the extracellular surface of the Na,K-ATPase. The ouabain binding characteristics of substitution R880P were compared with those of several different Na,K-ATPases, each of which contains a single amino acid substitution in the H1-H2 region of the alpha subunit. The substituted enzymes, C104A, Y108A, E116Q, P118K, and Y124F, vary considerably in their rates of dissociation (1-4-fold increase in the dissociation rate constant). In addition, the rate of association of [3H]ouabain binding to substitution P118K is 2-fold slower than that of the wild-type enzyme. These results suggest that the H1-H2 domain may participate directly in ouabain binding as well as be involved in conformational changes, both of which could affect the sensitivity of the enzyme to ouabain.

3T3 Cells↗

PubMed1993

A helix-loop-helix transcription factor-like gene is located at the mi locus.

The mouse microphthalmia phenotype is complex and consists of one or more of the following phenotypic alterations: a lack of pigmentation, small eyes, a mast cell defect, and bone abnormalities. The locus for this allele has been assigned to chromosome 6. A single gene defect that produces such a pleiotropic effect has suggested some involvement at a control point in development. Recently a mutant line of mice carrying a transgene insertion, which represents a new allelic form of mi, was described. The integration site of the transgene from these mi(tg) mice was cloned and analyzed. An exon sequence was discovered adjacent to the insertion. Computer analysis of this nucleotide sequence revealed the presence of a motif indicative of the helix-loop-helix class of transcription factors. The gene was expressed in a number of tissues from wild type animals but was absent in the tissue RNA from mi(tg) mice. Southern blot analysis demonstrated a deletion of some of the genetic material for this gene in the mi(tg) mice. This is consistent with the lack of expression in the mi(tg) mice. Interestingly, when DNA from other mi allelic variants was subjected to a similar analysis, a deletion was also observed in this gene in two other mi lines. Taken together, these data suggest that the gene encoding this new helix-loop-helix DNA-binding protein, and residing in the mi locus, is a strong candidate for the mi gene.

Amino Acid Sequence↗