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.

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.

Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes

1990 ◽  
Vol 258 (1) ◽  
pp. C179-C184 ◽  
Author(s):  
G. Schmalzing ◽  
P. Eckard ◽  
S. Kroner ◽  
H. Passow
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Meiotic Maturation ◽  
Xenopus Laevis Oocytes ◽  
Sodium Pumps

During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by [gamma-32P]ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from [3H]ouabain bound to the cell surface before maturation could be phosphorylated with inorganic [32P]phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.

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.

Enhancement of specific [3H]ouabain binding and ouabain sensitive 86rubidium influx in intact human lymphocytes by a dialysable factor in human and fetal calf serum

1987 ◽  
Vol 72 (1) ◽  
pp. 71-79 ◽  
Author(s):  
V. M. S. Oh ◽  
E. A. Taylor ◽  
J. L. Ding ◽  
N. A. Boon ◽  
J. K. Aronson ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Plasma Membrane ◽  
Human Serum ◽  
Major Part ◽  
Fetal Calf Serum ◽  
Human Lymphocytes ◽  
Calf Serum ◽  
Thymidine Uptake ◽  
Ouabain Binding ◽  
Intact Cells

1. We have measured specific [3H]ouabain binding and ouabain sensitive 86rubidium influx in intact human lymphocytes incubated for up to 7 days in media containing different concentrations of fetal calf serum and human serum. 2. Incubation for periods of up to 7 days with fetal calf serum and human serum produced increases in both specific [3H]ouabain binding and ouabain sensitive 86rubidium influx that were dependent on concentration and time. 3. Neither specific [3H]ouabain binding nor ouabain sensitive 86rubidium influx was altered when dialysed serum was used, suggesting that both fetal calf serum and human serum contain a dialysable factor or factors which stimulate specific [3H]ouabain binding and ouabain sensitive 86rubidium influx in intact human lymphocytes. 4. To further elucidate the mechanisms underlying these changes we also measured the activity of two other enzymes of the lymphocyte plasma membrane, 5′-nucleotidase and γ-glutamyltransferase, the uptake of [3H]thymidine by the intact cells, and the effects of cycloheximide, puromycin, and anisomycin, inhibitors of protein synthesis. 5. The activity of 5′-nucleotidase was increased after incubation of the lymphocytes in fetal calf serum for 72 h, but the activity of γ-glutamyltransferase was not changed, suggesting some selectivity of the stimulatory effect. 6. Measurements of [3H]thymidine uptake by the lymphocytes showed that the major part of the observed changes in specific [3H]ouabain binding and ouabain sensitive 86rubidium influx was not attributable to transformation of the lymphocytes to lymphoblasts. 7. All three inhibitors of protein synthesis prevented the increase in specific [3H]ouabain binding due to fetal calf serum.

scholarly journals Functional and Morphological Correlates of Connexin50 Expressed in Xenopus laevis Oocytes

1999 ◽  
Vol 113 (4) ◽  
pp. 507-524 ◽  
Author(s):  
Guido A. Zampighi ◽  
Donald D.F. Loo ◽  
Michael Kreman ◽  
Sepehr Eskandari ◽  
Ernest M. Wright
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Xenopus Laevis ◽  
Coated Vesicles ◽  
Xenopus Laevis Oocytes ◽  
Cross Sectional ◽  
Membrane Area ◽  
Gap Junction Channels ◽  
Vesicle Exocytosis ◽  
Cell Current

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 ± 3 helices, (b) when their density reached 300–400/μm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1–2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in ∼0.1-μm diameter “coated” and in larger 0.2–0.5-μm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5–40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80,000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60–500 μm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in ∼24 h.

Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes

2007 ◽  
Vol 292 (3) ◽  
pp. C1132-C1136 ◽  
Author(s):  
Alexandre Leduc-Nadeau ◽  
Karim Lahjouji ◽  
Pierre Bissonnette ◽  
Jean-Yves Lapointe ◽  
Daniel G. Bichet
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Xenopus Laevis ◽  
Western Blot ◽  
Plasma Membranes ◽  
Transient Receptor Potential ◽  
Expression System ◽  
Vitelline Membrane ◽  
Transient Receptor Potential Vanilloid ◽  
Xenopus Laevis Oocytes

Over the past two decades, Xenopus laevis oocytes have been widely used as an expression system to investigate both physiological and pathological properties of membrane proteins such as channels and transporters. Past studies have clearly shown the key implications of mistargeting in relation to the pathogenesis of these proteins. To unambiguously determine the plasma membrane targeting of a protein, a thorough purification technique becomes essential. Unfortunately, available techniques are either too cumbersome, technically demanding, or require large amounts of material, all of which are not adequate when using oocytes individually injected with cRNA or DNA. In this article, we present a new technique that permits excellent purification of plasma membranes from X. laevis oocytes. This technique is fast, does not require particular skills such as peeling of vitelline membrane, and permits purification of multiple samples from as few as 10 and up to >100 oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations. We have validated this technique essentially with Western blot assays on three plasma membrane proteins [aquaporin (AQP)2, Na+-glucose cotransporter (SGLT)1, and transient receptor potential vanilloid (TRPV)5], using both wild-type and mistargeted forms of the proteins. Purified plasma membrane fractions were easily collected, and samples were found to be adequate for Western blot identification.

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