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R W Mercer

Publications and source records attributed to R W Mercer.

47 records · Page 3Linked to original sources
PubMed1988

In vitro expression of the alpha and beta subunits of the Na,K-ATPase.

Our analysis of the cloned alpha 3 protein strongly suggests that this c-DNA represents a bona fide Na+,K+ ATPase isoform. Its similarity to alpha + may have made its detection in tissues by gel migration or immunoreactivity difficult. Expression of an enzymatically active alpha 3 beta Na+K+ATPase either in a completely in vitro system or in a heterologous tissue culture system will clearly establish the biochemical properties of this isoform. Development of alpha 3 specific immunochemical probes will allow a proper assessment of its in vivo expression.

Animals↗
PubMed1987

Expression of genes for the alpha and beta subunits of the Na,K-ATPase in normal and drug resistant cells.

The studies described in this report show that the genes for sodium, potassium-ATPase exhibit more complexity than was originally expected on the basis of studies of the Na,K-ATPase proteins or their transport activities. First, there are at least three isoforms of the alpha subunit. Second, expression of the beta subunit, although apparently always leading to the same protein product, involves complex variability in the sizes and predominance of different mRNA classes. The biologic basis for this heterogeneity of mRNA transcripts is not known. Third, ouabain resistance in at least one cell line, the human Hela C+ cell line, involves amplification of two independent genetic units, the alpha and beta subunit genes, as well as high levels of expression of the mRNAs. These results serve as strong evidence for the importance of the beta subunit, although no discrete functions have yet been assignable to this protein. It is otherwise difficult to understand why the alpha and beta subunits are both amplified in these cells, and both decline upon withdrawal of the selective agent. On the basis of evidence gathered in our laboratory and that of other groups, we conclude that the genetic basis for sodium transport in eukaryotic tissues is far more complex than previously anticipated. There appears to be a need for specialization of sodium, potassium-ATPases in different tissues and for complex generation of multiple beta-subunit mRNA species. Our molecular genetic approach has also allowed us to demonstrate that there is probably an important role played by the beta-subunit in Na,K-ATPase function.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Rat-brain Na,K-ATPase beta-chain gene: primary structure, tissue-specific expression, and amplification in ouabain-resistant HeLa C+ cells.

We deduced the complete amino acid sequence of the rat brain Na,K-ATPase beta-subunit from cDNA. The rat brain beta-subunit exhibits a high degree of primary sequence and secondary structural homology with the human and Torpedo beta-subunit polypeptides. Analysis of rat tissue RNA reveals that the beta-subunit gene encodes four separate mRNA species which are expressed in a tissue-specific fashion. In ouabain-resistant HeLa C+ cells, beta-subunit DNA sequences are amplified (approximately 20-fold) and beta-subunit mRNAs are overproduced relative to levels in parental HeLa cells. These results suggest that the beta-subunit plays an important role in Na,K-ATPase structure-function and in the mechanism underlying cellular resistance to the cardiac glycosides.

Amino Acid Sequence↗
PubMed1985

Molecular cloning of rat brain Na,K-ATPase alpha-subunit cDNA.

We have isolated a cDNA clone for the rat brain Na,K-ATPase alpha subunit. A lambda gt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, lambda rb5, containing a 1200-base-pair cDNA insert expressed a beta-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the alpha subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of lambda rb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney greater than brain greater than liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to lambda rb5 cDNA were amplified approximately 40-fold. Analysis of the lambda rb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase alpha-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the lambda rb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase alpha subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase.

Animals↗
PubMed1982

Electrokinetic behavior of inside-out vesicles from human red cell membranes.

The electrokinetic behavior of red cell membrane vesicles of normal (ROV) and inverted (IOV) sidedness has been characterized using the laser Doppler technique of electrophoretic light scattering (ELS). At neutral pH ROV have a (approx. 25%) higher electrophoretic mobility than IOV and the two peaks can be resolved in the ELS spectrum to provide a quantitative estimate of the IOV/ROV ratio which is consistent with the ratio determined by assay of the activity of acetylcholinesterase. The ROV peak coincides with the mobility of fresh red blood cells and of resealed ghosts. Neuraminidase treatment reduces the ROV mobility by a factor of 2.6, while the IOV peak is reduced only slightly (less than 5%). Treatment with trypsin results in a single narrow ELS peak at about 60% of the mobility of ROV. Treatment of IOV with phospholipase C leaves the electrophoretic mobility unaltered, whereas treatment with phospholipase D increases their mode mobility by 22%. The mobility titration curve of IOV from pH 2 to pH 10 reveals three distinct inflection points which may be assigned to chemical groups on the cytoplasmic surface of the red cell membrane.

Acetylcholinesterase↗
PubMed1981

Biphasic effect of orthophosphate on the (Na, K)-pump of human red cells.

Orthophosphate (Pi) can both stimulate and inhibit the (Na, K)-pump in red cells. At concentrations below 0.5 mmol/l cells, Pi stimulated the pump, but at higher concentrations Pi was inhibitory. The stimulation was demonstrated in intact cells by preincubation with inosine (which leads to a reduction in cellular Pi concentration), and then by incubating cells in media with various Pi concentrations (which relieved the inhibition caused by inosine). In inosine-treated cells there was an inverse relationship between the hematocrit during measurement of the fluxes and inhibition of the (Na, K)-pump; this was also a reflection of cellular Pi, which was lower in inosine-treated cels at low hematocrit. The stimulation of the (Na, K)-pump by Pi below 0.5 mmol/l cells was an indirect effect, due to synthesis of ATP by membrane-bound glycolytic enzymes, which required the appropriate substrates (in addition to Pi). This was shown by studies on inside-out vesicles made from red cell membranes. In the absence of the other substrates, Pi was inhibitory to Na transport in the vesicles. Above 0.5 mmol/l cells Pi was inhibitory to Na transport, both in inside-out vesicles and in intact cells. The mechanism of inhibition, probably a direct effect on the (Na, K)-pump, was not determined, though product inhibition seemed likely. The dependence on Pi of abnormal modes of Na transport by the pump (uncoupled Na efflux and Na/Na exchange) at low Pi concentrations was less than the dependence of normal Na/K exchange. This was attributed to a requirement by the abnormal modes of a lower rate of synthesis of ATP or a lower ATP concentration.

Adenosine Triphosphate↗
PubMed1981

Vanadate binding to the (Na + K)-ATPase.

A particulate (Na + K)-ATPase preparation from dog kidney bound [48V]-ortho-vanadate rapidly at 37 degrees C through a divalent cation-dependent process. In the presence of 3 mM MgCl2 the Kd was 96 nM; substituting MnCl2 decreased the Kd to 12 nM but the maximal binding remained the same, 2.8 nmol per mg protein, consistent with 1 mol vanadate per functional enzyme complex. Adding KCl in the presence of MgCl2 increased binding, with a K0.5 for KCl near 0.5 mM; the increased binding was associated with a drop in Kd for vanadate to 11 nM but with no change in maximal binding. Adding NaCl in the presence of MgCl2 decreased binding markedly, with an I50 for NaCl of 7 mM. However, in the presence of MnCl2 neither KCl nor NaCl affected vanadate binding appreciably. Both the nonhydrolyzable, beta, gamma-imido analog of ATP and nitrophenyl phosphate, a substrate for the K-phosphatase reaction that this enzyme also catalyzes, decreased vanadate binding at concentrations consistent with their acting at the low-affinity substrate site of the enzyme, the presence of KCl increased the concentration of each required to decrease vanadate binding. Oligomycin decreased vanadate binding in the presence of MgCl2, whereas dimethyl sulfoxide and ouabain increased it. With inside-out membrane vesicles from red blood cells vanadate inhibited both the K-phosphatase and (Na + K)-ATPase reactions; however, with the K-phosphatase reaction extravesicular K+ (corresponding to intracellular K+) both stimulated catalysis and augmented vanadate inhibition, whereas with the (Na + K)-ATPase reaction intravesicular K+ (corresponding to extracellular K+) both stimulated catalysis and augmented vanadate binding.

Animals↗
PubMed1981

Membrane-bound ATP fuels the Na/K pump. Studies on membrane-bound glycolytic enzymes on inside-out vesicles from human red cell membranes.

ATP stimulates Na transport into inside-out vesicles (IOVs) made from human red cell membranes; strophanthidin inhibits the ATP-stimulated transport. The substrates for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) (glycolytic enzymes bound to the cytoplasmic surface of the red cell membrane) also stimulate Na transport into IOVs without added ATP. The elution of GAPDH from the membranes prevents the stimulation by the substrates, but not by exogenous ATP. Hexokinase plus glucose (agents that promote breakdown of ATP) prevent stimulation of Na transport by exogenous ATP but not by the substrates for GAPDH and PGK. [32P]orthophosphate is incorporated into a membrane-bound organic phosphate compound shown chromatographically to be ATP. The level of membrane-bound ATP is decreased when Na is added, and this decrease is inhibited by strophanthidin. When further synthesis of [32P]ATP is blocked by the addition of unlabeled orthophosphate, all of the membrane-bound [32P]ATP is dissipated by the addition of Na. From these observations it was concluded that membrane-bound glycolytic enzymes synthesize ATP and deposit it in a membrane-associated compartment from which it is used by the Na/K pump.

Adenosine Triphosphate↗
PubMed1977

Cast ejector.

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Acrylic Resins↗