scholarly journals Up-regulation of sodium pump activity in Xenopus laevis oocytes by expression of heterologous β1 subunits of the sodium pump

1991 ◽  
Vol 279 (2) ◽  
pp. 329-336 ◽  
Author(s):  
G Schmalzing ◽  
S Gloor ◽  
H Omay ◽  
S Kröner ◽  
H Appelhans ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Binding Sites ◽  
Sodium Pump ◽  
Binding Capacity ◽  
Polyclonal Antiserum ◽  
Scatchard Analysis ◽  
Xenopus Laevis Oocytes ◽  
Ouabain Binding ◽  
Na Pump

Recent evidence suggests that the beta subunit of the Na+ pump is essential for the alpha subunit to express catalytic activity and for assembly of the holoenzyme in the plasma membrane. We report here that injection into Xenopus laevis oocytes of cRNAs specific for beta 1 subunit isoforms of the Na+ pump of four species (Torpedo californica, chicken, mouse and rat) causes a time-dependent increase in the number of ouabain-binding sites, both in the plasma membrane and in internal membranes. Expression of the beta 1 subunit of the Na+ pump of mouse and rat in the oocytes could be substantiated by immunoprecipitation using a polyclonal antiserum against the mouse beta 1 subunit. Scatchard analysis in permeabilized cells disclosed that the affinity for ouabain is unchanged after expression of each of the beta 1 subunits. A proportional increase in ouabain-sensitive 86Rb+ uptake indicates that the additionally expressed ouabain-binding sites on the cell surface represent functional Na+ pumps. The findings support the concept of Geering. Theulaz, Verrey, Häuptle & Rossier [(1989) Am. J. Physiol. 257, C851-C858] that beta 1 subunits expressed in oocytes associate with an excess of endogenous alpha subunits of the Na+ pump to form a hybrid enzyme. In addition, all of the beta 1 isoforms investigated in the present study were also capable of combining with the co-expressed alpha 1 subunit of the Torpedo Na+ pump to produce a functional enzyme. Injection of cRNA encoding for the Torpedo alpha 1 subunit alone had no effect on the ouabain-binding capacity of the surface and intracellular membranes of the oocyte.

scholarly journals Evidence for intracellular sodium pumps in permeabilized Xenopus laevis oocytes

1989 ◽  
Vol 260 (2) ◽  
pp. 395-399 ◽  
Author(s):  
G Schmalzing ◽  
S Kröner ◽  
H Passow
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Binding Capacity ◽  
Intracellular Sodium ◽  
Xenopus Laevis Oocytes ◽  
Ouabain Binding ◽  
Intact Cells ◽  
Sodium Pumps

Ouabain binding was studied in Xenopus laevis oocytes permeabilized by detergents. The behaviour of markers showed that 10 microM-digitonin selectively disrupts the plasma membrane. In the presence of ATP, oocytes permeabilized at 10 microM-digitonin bound no more ouabain molecules than were required to abolish active 86Rb+ uptake in the intact cells. However, the ouabain binding capacity increased approx. 2-fold when inner membranes were disrupted by SDS or excess digitonin, as judged from the accompanying release of the lysosomal marker beta-hexosaminidase. The results suggest that oocytes have a large internal pool of functional sodium pumps.

Regulation of alpha 1-beta 3-NA(+)-K(+)-ATPase isozyme during meiotic maturation of Xenopus laevis oocytes

1992 ◽  
Vol 262 (6) ◽  
pp. C1520-C1530 ◽  
Author(s):  
D. Pralong-Zamofing ◽  
Q. H. Yi ◽  
G. Schmalzing ◽  
P. Good ◽  
K. Geering
Keyword(s):  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Binding Capacity ◽  
Active Form ◽  
Meiotic Maturation ◽  
Xenopus Laevis Oocytes ◽  
Cell Fractionation ◽  
Binding Studies ◽  
Ouabain Binding

During progesterone-induced maturation of Xenopus oocytes, the transport and ouabain binding capacity of Na(+)-K(+)-ATPase at the plasma membrane is completely downregulated. To elucidate the mechanism and the physiological significance of this process, we have followed the fate of oocyte alpha-beta 3-Na(+)-K(+)-ATPase complexes during meiotic maturation and early embryonic development. An immunocytochemical follow-up of the catalytic alpha-subunit, ouabain binding studies, cell surface iodination, and oocyte cell fractionation combined with immunochemical subunit detection provides evidence that following progesterone treatment Na(+)-K(+)-ATPase molecules are retrieved from the oocyte plasma membrane. The enzyme complexes are recovered in an active form in an intracellular compartment in both in vitro and in vivo matured eggs. Exogenous Xenopus alpha 1- and beta 1-complexes expressed in the oocyte from injected cRNAs are regulated by progesterone similar to endogenous Na(+)-K(+)-ATPase complexes. Finally, active Na(+)-K+ pumps internalized during oocyte maturation appear to be redistributed to plasma membrane fractions during blastula formation in Xenopus embryos. In conclusion, our data suggest that endocytosis of alpha 1- and beta 3-complexes during meiotic maturation of Xenopus oocytes is responsible for downregulation of Na(+)-K(+)-ATPase activity and results in an intracellular pool of functional enzymes, which might be reexpressed during early development in response to physiological needs.

Observations on ouabain binding and membrane phosphorylation by the sodium pump

1976 ◽  
Vol 193 (1112) ◽  
pp. 217-234 ◽  
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Kidney Cortex ◽  
Relative Molecular Mass ◽  
Type B ◽  
Ouabain Binding ◽  
Na Pump ◽  
Pump Mechanism ◽  
Morphological Differences ◽  
Equilibrium Dissociation

A study has been made of ouabain binding and the formation of phosphoprotein from ATP and inorganic phosphate (P i ) with plasma membranes from rabbit and guinea-pig kidney cortex. The aim of the work was first to see whether apparently conflicting results in the literature arise from membranes being prepared by different methods and, secondly, to evaluate the results in relation to the Na pump mechanism. Three different methods were used to prepare membranes, types A, Au and B. The preparations differed markedly when ouabain binding was supported by Mg alone both in the amount bound and in the affinity. Mgdependent binding was influenced by 1 mM P i but the extent of stimulation varied according to the preparations. The main effect of P i was to decrease the equilibrium dissociation constant marginally for type A membranes but eightfold for type B membranes. In contrast, the maximum number of binding sites was little affected. The membrane affinity for ouabain in relation to Mg and P i therefore depended on the method of preparation. In the reaction with Mg-ATP, type Au and B membranes were both phosphorylated to about the same extent. On the other hand, they reacted differently with P i , type B membranes being phosphorylated (in the presence of Mg and ouabain) to the same extent as with ATP, whereas under the same conditions, type Au membranes gave only 15 % of the phosphorylation found with ATP. The phosphoprotein, however formed, whether from ATP or P i , or type Au or type B membranes, migrated in the same way on gel electrophoresis to give a relative molecular mass of approximately 90000. With each preparation, over a tenfold range of ATPase activity, there was a constant value of 1.2 in the ratio of the maximum phosphorylation by ATP compared with the maximum number of ouabain-binding sites. These results show that membranes prepared in different ways exhibit some consistent properties of the Na pump but also striking anomalies. In view of likely morphological differences in the preparations, it is concluded that the inconsistent features, notably the responses to Mg and P i , are an unreliable guide to the pump mechanism.

Juvenile hormone increases ouabain-binding capacity of microsomal preparations from vitellogenic follicle cells

1983 ◽  
Vol 61 (7) ◽  
pp. 826-831 ◽  
Author(s):  
T. T. Ilenchuk ◽  
K. G. Davey
Keyword(s):  
Juvenile Hormone ◽  
Binding Capacity ◽  
Follicle Cell ◽  
Follicle Cells ◽  
Follicular Epithelium ◽  
Curvilinear Relationship ◽  
Scatchard Analysis ◽  
Ouabain Binding ◽  
Binding Characteristics ◽  
Brain Microsomes

A comparison has been made of the effects of juvenile hormone (JH) on the binding characteristics for ouabain of microsomes prepared from brain and from cells of the follicular epithelium surrounding previtellogenic or vitellogenic oocytes in Rhodnius. JH has no effect on the binding of ouabain to brain microsomes and decreases the Kd, but does not alter the Bmax for previtellogenic follicle cells. For vitellogenic follicle cells, Scatchard analysis reveals a curvilinear relationship, which is interpreted as indicating that a new population of JH-sensitive ouabain-binding sites develops as the follicle cell enters vitellogenesis. These results are related to earlier data obtained on the effect of JH on ATPase activity, volume changes in isolated follicle cells, and the development of spaces between the cells of the follicular epithelium.

Insulin receptors on Xenopus laevis oocytes: effects of injection of ob/ob mouse liver mRNA

1991 ◽  
Vol 100 (1) ◽  
pp. 167-171
Author(s):  
D.A. Diss ◽  
B.D. Greenstein
Keyword(s):  
Xenopus Laevis ◽  
Binding Sites ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Cell Types ◽  
Follicle Cells ◽  
Vitelline Membrane ◽  
Xenopus Laevis Oocytes ◽  
Endogenous Insulin ◽  
Order Of Magnitude

We describe here conditions for the detection of insulin binding sites on Xenopus laevis oocytes. The binding of 125I-labelled insulin displayed sigmoidal behaviour, which is characteristic of the binding relationship between insulin and its receptor. Resolution of the resulting curvilinear Scatchard plot into two components revealed KD values of 8.86 × 10(−10) +/− 1.9 × 10(−10) and 5.32 × 10(−9) +/− 2.4 × 10(−9) M and n values of 9.7 × 10(7) +/− 0.4 × 10(7) and 3.3 × 10(8) +/− 0.5 × 10(8) binding sites per oocyte, respectively. The possibility cannot be excluded, however, that receptors for IGF-1 were also being detected. Also described are conditions for the rapid and efficient removal of all tissues surrounding the oocyte, including the vitelline membrane. We could not detect any specific 125I-labelled insulin binding to oocytes that had their follicle cells or vitelline membrane removed and this was not due to the enzymic treatment used in the process. Microinjection of oocytes without follicular layers did not result in the appearance of any detectable insulin binding sites, which were, however, observed if oocytes were first stripped of the vitelline membrane. We suggest that oocytes may possess endogenous insulin receptors on their surface in numbers of the same order of magnitude as those present on somatic cells. The removal of tissues surrounding the oocyte should facilitate studies aimed at determining functional interactions of the various cell types during oocyte development and for studying insulin receptors on the oocyte-follicular cell complex.

Potassium-induced relaxation of arteries in hypertension: modulation by extracellular calcium

1989 ◽  
Vol 256 (3) ◽  
pp. H707-H712 ◽  
Author(s):  
G. Rinaldi ◽  
D. F. Bohr
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Salt Solution ◽  
Binding Sites ◽  
Muscle Relaxation ◽  
Smooth Muscle Relaxation ◽  
Physiological Salt Solution ◽  
Spontaneously Hypertensive ◽  
Na Pump ◽  
Wistar Kyoto

Smooth muscle relaxation due to activation of Na+-K+-adenosinetriphosphatase (ATPase) (K+ relaxation) was studied in isolated tail arteries from stroke-prone spontaneously hypertensive (SHRSP) and Wistar-Kyoto (WKY) rats. Exposure to K+ (7 mM) relaxed a norepinephrine (1 microM) contraction induced in K+-free physiological salt solution (PSS) by 46 +/- 3% in WKY and by 81 +/- 3% in SHRSP. Incubation with monensin (10 microM) augmented the K+-relaxation in WKY to 61 +/- 5%. Incubation with amiloride (10 microM) or with low-Na+ (10 mM) PSS attenuated the K+ relaxation in SHRSP to 34 +/- 3 and 5 +/- 2%, respectively. Incubation with high-Ca2+ (6.6 mM) PSS attenuated the K+ relaxation in WKY to 25 +/- 3% but resulted in only a slight decrease in the relaxation in SHRSP (to 73 +/- 2%). We conclude 1) that a greater Na+ leakiness of the plasma membrane in SHRSP than in WKY while the Na+ pump is inactive in K+-free PSS can explain the greater relaxation observed when the Na+ pump is reactivated and 2) that the Na+ leakiness of the membrane is more attenuated by Ca2+ in the WKY than in the SHRSP. We hypothesize that this Ca2+ effect reflects a normally large amount of this ion that can be bound to and thereby stabilize the membrane of the WKY, decreasing its permeability to Na+. The membrane of the SHRSP is deficient in its Ca2+-binding sites. Hence the added Ca2+ does not further stabilize its membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist-induced internalisation of the angiotensin II-binding sites from plasma membranes of isolated rat hepatocytes

1997 ◽  
Vol 152 (3) ◽  
pp. 407-412 ◽  
Author(s):  
M Montiel ◽  
M C Caro ◽  
E Jiménez
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Rat Hepatocytes ◽  
Receptor Complex ◽  
Ang Ii ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

Angiotensin II (Ang II) provokes rapid internalisation of its receptor from plasma membranes in isolated rat hepatocytes. After 10 min stimulation with Ang II, plasma membrane lost about 60% of its 125I-Ang II-binding capacity. Internalisation was blocked by phenylarsine oxide (PhAsO), whereas okadaic acid, which markedly reduced the sustained phase of calcium mobilization, did not have a preventive effect on Ang II–receptor complex sequestration. These data suggest that Ang II receptor internalisation is probably independent of a phosphorylation/dephosphorylation cycle of critical serine/threonine residues in the receptor molecule. To establish a relationship between sequestration of the Ang II receptor and the physical properties of the Ang II-binding sites, 125I-Ang II–receptor complex profiles were analysed by isoelectric focusing. In plasma membrane preparations two predominant Ang II-binding sites, migrating to pI 6·8 and 6·5 were found. After exposure to Ang II, cells lost 125I-Ang II-binding capacity to the Ang II–receptor complex migrating at pI 6·8 which was prevented in PhAsO-treated cells. Pretreatment of hepatocytes with okadaic acid did not modify Ang II–receptor complex profiles, indicating that the binding sites corresponding to pI 6·5 and pI 6·8 do not represent a phosphorylated and/or non-phosphorylated form of the Ang II receptor. The results show that the Ang II–receptor complex isoform at pI 6·8 represents a functional form of the type-1 Ang II receptor. Further studies are necessary to identify the Ang II-related nature of the binding sites corresponding to pI 6·5. Journal of Endocrinology (1997) 152, 407–412

Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules

1981 ◽  
Vol 241 (6) ◽  
pp. F605-F611 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Collecting Tubule ◽  
The Difference ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.

Homeoviscous adaptation and thermal compensation of sodium pump of trout erythrocytes

1991 ◽  
Vol 260 (5) ◽  
pp. R916-R924 ◽  
Author(s):  
R. S. Raynard ◽  
A. R. Cossins
Keyword(s):  
Cold Acclimation ◽  
Scatchard Analysis ◽  
Transport Activity ◽  
Turnover Number ◽  
Ouabain Binding ◽  
Homeoviscous Adaptation ◽  
Membrane Order ◽  
Na Pump ◽  
Pump Activity ◽  
Cholesterol Supplementation

The effects of thermal acclimation of trout on the transport activity and turnover number of the erythrocyte Na+ pump have been determined. Na+ pump activity was estimated by measuring the ouabain-sensitive K+ influx in Na(+)-loaded cells and the number of active pumps determined by Scatchard analysis of [3H]ouabain binding and by correlation of ouabain binding with pump inhibition. Cold acclimation was associated with an increase in pump activity of up to 60%, although the furosemide-sensitive and residual fluxes were unaffected. The number of ouabain binding sites was similar in both acclimation groups at approximately 21,000-23,000 sites/cell. This means that cold acclimation induced an increase in the transport turnover number of pump molecules from approximately 6 to 9 s-1. Cold acclimation was also associated with a decrease in membrane order as indicated by steady-state fluorescence polarization of the membrane probe, 1,3-diphenyl-1,3,5-hexatriene, with a homeoviscous efficacy of 25-41%. That membrane order may influence pump transport activity is supported by experiments on cholesterol supplementation, which caused both an increase in membrane order and a decrease in pump turnover number. The degree of pump compensation was dependent on the season, with greatest responses in the late spring and declining responses through to winter. By contrast, changes in membrane order were observed throughout the year. Expression of pump activity and erythropoiesis may vary throughout the seasonal cycle in complex ways that confuse the direct comparison study of cellular properties in a heterogeneous population of cells.

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