Permeability and the Mechanism of Transport of Boric Acid Across the Plasma Membrane of Xenopus laevis Oocytes

2001 ◽  
Vol 81 (2) ◽  
pp. 127-139 ◽  
Author(s):  
Christos Dordas ◽  
Patrick H Brown
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Boric Acid ◽  
Xenopus Laevis Oocytes

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.

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.

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.

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.

Protooncogene product, c-mos kinase, is involved in upregulating Na+/H+ antiporter in Xenopus oocytes

1994 ◽  
Vol 267 (6) ◽  
pp. C1717-C1722 ◽  
Author(s):  
K. Rezai ◽  
A. Kulisz ◽  
W. J. Wasserman
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Intracellular Ph ◽  
Xenopus Oocytes ◽  
Stage Iv ◽  
Meiotic Maturation ◽  
Xenopus Laevis Oocytes ◽  
Cell Systems ◽  
Oocyte Meiotic Maturation

Progesterone-stimulated Xenopus laevis oocytes undergo an increase in their intracellular pH from 7.3 to 7.7 because of the activation of Na+/H+ antiporters in their plasma membrane. Activation of Na+/H+ exchangers (NHE) in other cell systems appears to be regulated by phosphorylation of the NHE protein. In the current study we demonstrated that cytoplasm taken from steroid-stimulated oocytes rapidly induced an increase in intracellular pH when microinjected into full-grown stage VI recipient oocytes. The protein within the cytoplasm that appears to be responsible for this activity is c-mos kinase. Microinjected pure mosxe kinase protein rapidly activated the Na+/H+ exchangers in full-grown recipient oocytes. Furthermore, injected mosxe protein rapidly activated the Na+/H+ exchangers in smaller progesterone-insensitive stage IV oocytes. Therefore, it appears that the protooncogene product, p39 c-mos kinase, which is normally synthesized in full-grown stage VI oocytes in response to progesterone stimulation, is involved in the upregulation of the Na+/H+ antiporters during oocyte meiotic maturation.

Sign in / Sign up

Export Citation Format

Share Document