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I S Edelman

Publications and source records attributed to I S Edelman.

At least 19 recordsLinked to original sources
PubMed1994

Assembly of ordered contigs of cosmids selected with YACs of human chromosome 13.

We have developed an efficient method for assembling ordered cosmid contigs aligned to mega-YACs and midi-YACs (average insert sizes of 1.0 and 0.35 Mb, respectively) and used this general method to initiate high-resolution physical mapping of human chromosome 13 (Chr 13). Chr 13-enriched midi-YAC (mYAC) and mega-YAC (MYAC) sublibraries were obtained from corresponding CEPH total human YAC libraries by selecting colonies with inter-Alu PCR probes derived from Chr 13 monochromosomal cell hybrid DNA. These sublibraries were arrayed on filters at high density. In our approach, the MYAC 13 sublibrary is screened by hybridization with cytogenetically assigned Chr 13 DNA probes to select one or a small subset of MYACs. Inter-Alu PCR products from each MYAC are then hybridized to the MYAC and mYAC sublibraries to identify overlapping YACs and to an arrayed Chr 13-specific cosmid library to select corresponding cosmids. The set of selected cosmids, gridded on filters at high density, is hybridized with inter-Alu PCR products from each of the overlapping YACs to identify subsets of cosmids and also with riboprobes from each cosmid of the arrayed set ("cosmid matrix cross-hybridization"). From these data, cosmid contigs are assembled by a specifically designed computer program. Application of this method generates cosmid contigs spanning the length of a MYAC with few gaps. To provide a high-resolution map, ends of cosmids are sequenced at preselected sites to position densely spaced sequence-tagged sites.

Base Sequence↗
PubMed1992

Serum independence of low K+ induction of Na,K-ATPase: possible role of c-fos.

Cultured ARL15 cells respond to abnormally low extracellular K+ concentrations by increasing the abundance of Na,K-ATPase (the Na/K pump). This response is preceded by significant increases in the mRNAs of the alpha 1 and beta 1 subunits of this enzyme, implying transcriptional or post-transcriptional regulation in the response. The present study concerned the possible participation of serum factors in low K+ induction of Na,K-ATPase. In normal K+ (4.5 mM) or low K+ (0.68 mM) the presence of 10% calf serum had no effect on Na,K-ATPase activity. The serum independence of the response to low K+ raised the possibility that low K+ may itself elicit a "growth" response. Accordingly, the effect of low K+ on mRNA abundances of four proto-oncogenes (c-fos, c-myc, c-jun and c-ski) was evaluated in the early phase of the response by quantitative Northern blot analysis. The mRNA for c-fos was transiently elevated by low K+, with a peak at 30 min. In contrast, low K+ had no measurable effect on the abundances of c-myc, c-jun and c-ski, for up to 2 hr of exposure. The early elevation of c-fos mRNA makes it a candidate mediator in this signal-transduction pathway. Induction of c-fos mRNA by the phorbol ester, PMA, or by dioctanoyl glycerol, however, had no effect on Na,K-ATPase activity. These results indicate that an increase in c-fos mRNA alone is not sufficient to induce Na,K-ATPase. Whether induction of c-fos is necessary for the response to low K+ remains to be determined in future studies.

Animals↗
PubMed1991

Anomalous mobilities of Na,K-ATPase alpha subunit isoforms in SDS-PAGE: identification by N-terminal sequencing.

Three isoforms of the alpha subunit of Na,K-ATPase, alpha 1, alpha 2, and alpha 3 have been characterized at the DNA, mRNA and protein levels. In admixtures, isoforms migrate as doublets (i.e. alpha 1 and another band originally designated alpha +, comprising alpha 2 + alpha 3) when analyzed by SDS-PAGE. As deduced from cDNA sequences their masses range from 111.7 to 112.6 kDa. With conventional protein standards, however, SDS-PAGE yields nominal masses of 85-105 kDa. In this system, the presence of a doublet that reacted with a polyclonal anti-Na,K-ATPase antibody in the kidney was interpreted as indicating two molecular or conformational species of the kidney alpha sub-unit (Siegel, G.J. and Desmond, T.J. (1989) J. Biol. Chem. 264, 4751-4754). We report that Na,K-ATPase purified from dog, guinea pig and rat kidney medulla or from rat brain, can yield two distinct bands when analyzed by SDS-PAGE or STS-PAGE, migrating between 85 and 105 kDa. An additional band migrating at 117 and 120 kDa appears often in enzyme purified from rat and guinea pig kidney medulla. The apparent molecular weights and relative intensities of these bands vary with temperature and duration of incubation during sample preparation. N-terminal sequencing and monospecific antibody probes revealed that the two distinct bands obtained from the kidney enzyme consist only of the alpha 1 isoform. The band appearing at 117-120 kDa also contains only the alpha 1 N-terminal sequence. In contrast, as reported earlier (Sweadner, K.J. (1979) J. Biol. Chem. 254, 6060-6067), the doublet seen in brain preparations consists of alpha 1 and alpha 2 or (alpha 2 + alpha 3). We conclude that monospecific antibody probes or N-terminal sequencing must be used to identify Na,K-ATPase isoforms by SDS- or STS-PAGE. In addition, gel conditions that may affect the mobilities of the isoforms are discussed.

Amino Acid Sequence↗
PubMed1990

Cell-free transcription and translation of Na,K-ATPase alpha and beta subunit cDNAs.

Synthetic mRNAs (i.e. cRNA alpha and cRNA beta) were obtained by cell-free transcription of M13 KS(+) (Bluescript) expression vectors which contained the entire coding region of the alpha or beta subunits of lamb kidney Na,K-ATPase. Translation in reticulocyte lysates of cRNA alpha yielded full length alpha polypeptide, as well as a limited array of immunoprecipitable lower molecular weight products. cRNA beta yielded a single immunoprecipitable full length polypeptide. Association of the alpha polypeptide with the microsomal membranes was obtained only co-translationally. Fifteen to 50% of the membrane-associated alpha subunit was resistant to extraction with alkali. The resistance of a 29-kDa fragment to trypsinolysis indicated that the alpha subunit was inserted into microsomal membranes. In the presence of dog pancreatic microsomes, the beta polypeptide was glycosylated as indicated by the appearance of three higher molecular weight polypeptides that were sensitive to endoglycosidase H and bound to Concanavalin A. The beta subunit was predominantly translocated into the lumen of the endoplasmic reticulum since 90% of the mass of the membrane-associated beta polypeptide was resistant to trypsin (i.e. reduced in size from 40 kDa to 37.5 kDa), and 95% of all of the beta chains were resistant to extraction with alkali. Neither the alpha nor the beta subunits have NH2-terminal leader signal sequences, but both may require the signal recognition receptor for membrane insertion, as evidenced by inhibition of incorporation of both subunits into microsomes pretreated with N-ethylmaleimide. Simultaneous translation of cRNA alpha and cRNA beta did not enhance membrane insertion of either the alpha or beta polypeptide.

Acetylglucosaminidase↗
PubMed1990

Na(+)-K(+)-ATPase in adipocyte differentiation in culture.

Differentiation of 3T3-L1 cells from a fibroblast to an adipocyte phenotype results in an approximately 50% decline in Na(+)-K(+)-ATPase activity and ouabain-sensitive 86Rb uptake. Kinetic analysis revealed a K 1/2 for Na+ of approximately 14 mM, a Km for ATP of approximately 0.4 mM, and maximal activation by sodium dodecyl sulfate at a 0.05 (wt/wt) detergent/protein ratio in both mature fibroblasts and adipocytes. Both fibroblasts and adipocytes exhibited Na(+)-K(+)-ATPase activity with an inhibition constant (Ki) for ouabain of approximately 10(-4) M. In addition, adipocytes exhibited a second component representing 30% of total activity with a Ki of approximately 5 x 10(-7) M. The emergence of biphasic ouabain inhibition kinetics in adipocytes raised the possibility of a change in alpha-subunit isoform composition with cytodifferentiation. This inference was evaluated by isoform-specific mRNA analysis (Northern blots) and by alpha-isoform-specific immunoassays (Western blots). Northern blots revealed a modest decrease in mRNA alpha 1, a striking increase in mRNA alpha 2, and a significant loss of mRNA beta content with differentiation of fibroblasts to adipocytes. By immunoassay, fibroblasts exhibited the alpha 1-isoform. Adipocytes exhibited an admixture of alpha 1- and alpha 2-isoforms, with alpha 2 being the more abundant isoform. There was no one-to-one correspondence either between the mRNA isoform and alpha-subunit abundances or between alpha-subunit abundances and enzymatic activity, suggesting that regulation occurs at multiple levels in this system. Findings indicate, however, that a shift in alpha-isoform composition accompanied by a change in ouabain inhibition kinetics occurs with cytodifferentiation.

Adipose Tissue↗
PubMed1990

Effect of high extracellular K+ on Na-K-ATPase in cultured canine kidney cells.

The Madin-Darby canine kidney (MDCK) cell line was used to evaluate the influence of high extracellular K+, independent of hormonal effects, on renal Na-K-adenosinetriphosphatase (ATPase) activity and abundance. Confluent cell monolayers were incubated in control (5 mM) or high K+ (7.5 mM) medium for 24 h. Exposure to high K+ elicited a 46% rise in Na-K-ATPase activity and a 55% increase in ouabain-sensitive 86Rb uptake. Na-K-ATPase abundance, estimated from the number of ouabain-binding sites, also increased 63% over control in cells exposed to 7.5 mM K+, and as a consequence there was no statistically significant change in the catalytic turnover number. Northern blot analysis using rat cDNA probes for the alpha 1- and beta-subunits showed no corresponding changes in subunit-specific mRNA abundances at 24 h. We conclude that chronic exposure to high extracellular K+ produces a rise in renal epithelial Na-K-ATPase activity and active K+ transport, independent of changes in aldosterone, renal blood flow, or extracellular Na+ concentration. This effect is due to an increase in enzyme abundance rather than a change in catalytic turnover rate. The results of Northern analysis suggest that regulation of Na-K-ATPase activity and abundance by high K+ may involve translational or posttranslational mechanisms, but further study with cDNA probes of canine origin is needed to resolve this issue.

Animals↗
PubMed1989

Internal image properties of a monoclonal auto-anti-idiotypic antibody and its binding to aldosterone receptors.

A monoclonal anti-idiotypic antibody (H10E4C9F) that interacts with the aldosterone receptors was generated using an auto-anti-idiotypic approach by immunizing a mouse with a 3-O-carboxymethyloxime of aldosterone coupled to bovine serum albumin. This antibody, an IgG1, displayed internal image properties of aldosterone and was considered as an Ab2 beta according to the following criteria. (i) H10E bound to Fab fragments of affinity-purified rabbit anti-aldosterone antibody that had high affinity for aldosterone (Kd = 5 x 10(-10) M). Binding was inhibited by aldosterone but not by estradiol. (ii) H10E inhibited [3H]aldosterone binding to rabbit polyclonal antibodies and also to murine monoclonal antibodies raised during the same fusion. Inhibition was concentration-dependent. These results are consistent with the antibody recognizing an interspecies cross-reacting epitope involved in the aldosterone combining site. (iii) The antibody could be affinity-purified on an immobilized monoclonal anti-aldosterone antibody. (iv) It inhibited [3H]aldosterone binding to rabbit kidney cytosolic aldosterone receptors but had no effect on glucocorticoid receptors. Additional evidence for the interaction of H10E with aldosterone receptors was provided by glycerol gradients analyses: the anti-idiotypic antibody displaced [3H]aldosterone and [3H]corticosterone from the native untransformed 9 S aldosterone receptor in the presence of RU 26988, a specific marker of glucocorticoid receptors. All of the above are consistent with the first successful production of a monoclonal antibody that mimics aldosterone and interacts specifically with the steroid binding domain of aldosterone receptors.

Adrenalectomy↗
PubMed1989

Isoforms of Na,K-ATPase in Artemia saline: I. Detection by FITC binding and time course.

Partially purified Na,K-ATPase from whole nauplii at various stages of development, analyzed by SDS-PAGE, reveals a polydisperse beta and two alpha subunits (denoted alpha 1 and alpha 2). In the absence of Ca2+, ATP-inhibitable fluorescein isothiocyanate (FITC) labeling is restricted to the alpha subunit of this enzyme, even in crude naupliar homogenates. The intensity of the alpha-specific fluorescent signal (i.e., the sum of the yield from both alpha isoforms) is proportional to Na,K-ATPase activity during development. FITC-labeled subunits were detected at 8 hr of development prior to the detection of measurable Na,K-ATPase activity. The alpha 2/alpha 1 ratio changed from an initial value of 1.25 to a peak of 1.75 at 32 hr of development, then reverted to a ratio of 1.25 by 42 hr, and remained constant thereafter. Pulse chase studies with 35S-methionine indicated that the developmental increase in enzyme activity is coincident with amino acid incorporation into the alpha subunits, implying that enzyme synthesis is active during enzyme accumulation.

Animals↗
PubMed1989

Isoforms of Na,K-ATPase in Artemia salina: II. Tissue distribution and kinetic characterization.

To characterize the molecular properties conveyed by the isoforms of the alpha subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The alpha isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the alpha 1 isoform, whereas the intestinal enzyme exhibits both the alpha 1 and the alpha 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mumol Pi/mg protein/hr (1 mu)] in whole homogenates was 32 +/- 6 in the salt glands and 12 +/- 3 in the intestinal preparations (mean +/- SEM). The apparent half-maximal activation constants (K1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7 +/- 0.6 mM vs. 23.5 +/- 4 mM (P less than 0.01) for Na+, 16.6 +/- 2.2 mM vs. 8.29 +/- 1.5 mM for K+ (P less than 0.01), and 0.87 +/- 0.8 mM vs. 0.79 +/- 1.1 mM for ATP (NS). The apparent Ki's for ouabain inhibition were 1.1 x 10(-4) M vs. 2 x 10(-5) M, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1989

Identification of an epitope shared by the DNA-binding domain of glucocorticoid receptor and the B chain of insulin.

A monoclonal antibody (8G11-C6) generated by an auto-anti-idiotypic route and directed to a site near the ligand-binding site of the glucocorticoid receptor also binds to native insulin and the B chain of insulin but not to the A chain of insulin. The glucocorticoid receptor and the B chain of insulin, therefore, share a cross-reacting epitope. Examination of the primary sequences of the two proteins revealed a limited number of regions of identity or close homology. Several peptides representative of those regions were synthesized. A heptapeptide sequence of the B chain of insulin with homology to a sequence in the first "zinc finger" of the DNA-binding domain of the glucocorticoid receptor was identified as the cross-reactive epitope. This heptapeptide sequence is restricted to and highly conserved among insulins of various species. Homologous sequences are found in the DNA-binding domains of most steroid receptors and related DNA-binding proteins. Consistent with this is the finding that 8G11-C6 inhibits the binding of glucocorticoid receptor to DNA-cellulose.

Adrenalectomy↗
PubMed1988

Thyroidal regulation of rat renal and hepatic Na,K-ATPase gene expression.

Na,K-ATPase activity, Na,K-ATPase alpha- and beta-subunit mRNA abundance (mRNA alpha and mRNA beta), and gene transcription rates were determined in kidney cortex and liver of hypothyroid and triiodothyronine (T3)-treated rats. In hypothyroid rats, Na,K-ATPase activity (expressed per unit of DNA) was 3.6-fold greater in kidney cortex than liver, and the abundance of mRNA alpha and mRNA beta in kidney cortex exceeded that of liver by 2.8- and 5.2-fold, respectively. In vitro nuclear run-on analysis revealed similar rates of Na,K-ATPase alpha and beta gene transcription in nuclei isolated from either kidney cortex or liver. Administration of T3 for 72 h elicited a 2.3-fold stimulation of renal Na,K-ATPase activity that was associated with a 3.1- and 2.6-fold increase of mRNA alpha and mRNA beta content, respectively. In contrast, T3 induced a 1.3-fold stimulation of liver Na,K-ATPase activity accompanied by a 7.3-fold increase in mRNA alpha and no change in mRNA beta abundance. Transcription rates of alpha and beta genes (assayed by nuclear run-on) in renal cortex were both stimulated 1.8-fold in response to T3 injection. Similarly in liver nuclei, T3 treatment produced a 1.4- and 1.3-fold stimulation in the rate of alpha and beta gene transcription, respectively. These results indicate that significant discrepancies exist in the quantitative relationships between control and T3-induced changes in renal and hepatic enzyme activity, mRNA abundance and rate of gene transcription, and imply that the T3-induced increase in Na,K-ATPase abundance is mediated at both transcriptional and post-transcriptional steps.

Actins↗
PubMed1988

Kinetic analysis of Na,K-activated adenosine triphosphatase induced by low external K+ in a rat liver cell line.

Exposure of ARL 15 cells to medium containing reduced concentrations of K+ (0.65 mM) elicited a 50-100% increase in Na,K-ATPase activity. The inhibition by ouabain of both the basal and the induced enzyme conformed to a single-site model (KI = 1 x 10(-4) M). The low K+-induced increment in Na,K-ATPase activity was accompanied by an equivalent increase in the abundance of Na,K-pump sites estimated by ouabain-stabilized ("back-door") phosphorylation, such that the calculated catalytic turnover number of approximately 8000/min was minimally changed. Comparison of the dependence of ouabain-inhibitable K+ uptake on intracellular Na+ and on extracellular K+ concentrations in control and low K+-treated cells revealed no change in the respective half-maximal stimulatory concentrations for these cations, whereas the maximal rate of active K+ uptake in cells exposed to low external K+ increased by nearly 100%. The derived Hill coefficients for active K+ transport rate were also unchanged by the low K+ treatment (i.e. approximately 1.4 for extracellular K+ and 2.6 for intracellular Na+). Na,K-ATPase activity of basal and low K+-induced cells calculated from the measured maximal Na,K transport rate closely approximated the Na,K-ATPase activity measured enzymatically in unfractionated cell lysates under Vmax conditions, suggesting that all or most of the Na,K-ATPase enzymatic units present in both basal and stimulated states are functionally active. Northern blot analysis of RNA isolated from control cells indicated the presence of the Na,K-ATPase alpha-I isoform of the enzyme which increased by nearly 200% following incubation of the cells in low-K+ medium. By contrast, the alpha-II and alpha-III mRNAs were undetectable in either the basal or low K+-stimulated state. These results indicate that the Na,K-ATPase induced by incubation of ARL 15 cells in low-K+ medium is kinetically and functionally indistinguishable from the basal enzyme, and that only the alpha-I isoform is expressed under control and low-K+ conditions.

Animals↗
PubMed1988

Increased abundance of Na+-K+-ATPase mRNAs in response to low external K+.

Exposure of ARL 15 cells, an established line from adult rat liver, to external K+ concentrations less than 1 mM for 24 h increases Na+-K+ pump abundance (Na+-K+-ATPase) (J. Gen. Physiol. 87:591-606, 1986). We found that treatment of confluent monolayers of ARL 15 cells with low-K+ medium (0.65 mM) caused a 100% increase in total RNA content per plate after 24 h, as well as a 25% increase in DNA and protein content per plate. Concomitant with this growth effect, low-K+ exposure for 6 h elicited 60% increases in mRNA alpha and mRNA beta, the mRNAs that encode the constituent subunits of the Na+-K+-ATPase, in a polyadenylated RNA fraction. At 24 h, however, the abundance of mRNA alpha increased by 290%, whereas mRNA beta increased by only 70%. Moreover, in both control and low-K+-treated cells, mRNA alpha was 30-fold or more greater in abundance than mRNA beta. This discrepancy in abundance was also present in rat liver, but not in cultured MDCK cells. The differences in abundance of mRNA alpha and mRNA beta suggest that the liver may have an unusual subunit composition or biosynthetic mechanism. Nevertheless, the increases in the abundance of mRNA alpha and mRNA beta are sufficient to account for the observed 70-100% increase in Na+-K+-ATPase activity in response to low external K+.

Animals↗
PubMed1987

Low-angle neutron scattering analysis of Na/K-ATPase in detergent solution.

Purified Na/K-ATPase from guinea pig renal outer medulla has been delipidated and solubilized in Brij 58 (polyoxyethylene ether; C-16, E-20). At a concentration of 2 mg of Brij 58/mg of protein, about one-half the enzyme complement was solubilized and almost 50% of Na/K-ATPase activity was retained by the enzyme-micelle complex. Guinier plots of the neutron scattering profiles yielded no evidence of heterogeneity with respect to subunit composition or the state of aggregation in the solubilized oligomers. Contrast matching with D2O used to obtain estimates of the molecular weight of the micellar form of Na/K-ATPase gave a mean value of 310,000 +/- 42,700, which corresponds to an alpha 2 beta 2 tetramer. A Stuhrmann plot of the neutron scattering data yielded an estimated radius of gyration of 67 A. The Stuhrmann plot also indicated an asymmetrical distribution of neutron scattering density. On the basis of the Stuhrmann plot parameters, the estimated molecular weight, and the radius of gyration, a low-resolution model was formulated of the oligomeric unit of Na/K-ATPase.

Animals↗
PubMed1987

Effect of thyroid hormone on the abundance of Na,K-adenosine triphosphatase alpha-subunit messenger ribonucleic acid.

The effects of thyroid hormone on Na,K-ATPase alpha-subunit mRNA (mRNA alpha) content and Na,K-ATPase activity were measured in renal cortex, heart, and cerebrum of hypothyroid rats 24 and 72 h after injection of diluent or T3. Use of a cDNA probe complementary to rat brain mRNA alpha in Northern blot analysis revealed a single 26-27 S band in RNA isolated from these three tissues regardless of thyroid status. Tissue mRNA alpha content was estimated by dot blot analysis of whole cell extracts and isolated total RNA. Injection of T3 augmented mRNA alpha content by 2.1- to 2.5-fold in kidney cortex and myocardium at 24 h. After three daily injections of T3, the increases in mRNA alpha were evident despite a global increase in RNA content associated with hypertrophy of these target tissues. Furthermore, the increases in abundance of mRNA alpha after 72 h of T3 treatment correlated with enhancement of Na,K-ATPase activity. In contrast, both mRNA alpha and enzyme activity were invariant in the cerebrum. These data suggest that T3-induced augmentation of Na,K-ATPase activity is mediated, at least in part, by increased mRNA alpha content in target tissues.

Animals↗