Changes in protein composition in cellular membranes of various parts of secondary dormant cucumber seeds treated with ethanol

2000 ◽  
Vol 10 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Y. Sreenivasulu ◽  
Dilip Amritphale
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Time Course ◽  
Plasma Membranes ◽  
Protein Composition ◽  
Cellular Membrane ◽  
Embryonic Axis ◽  
Dormancy Breaking ◽  
Cucumis Sativus L ◽  
Microsomal Membranes

AbstractSecondary seed dormancy in cucumber (Cucumis sativus L.) cv.Poinsett 76 could be broken with ethanol. Breakage of dormancy was accompanied by significant changes in the composition of membrane proteins in various seed parts. Compared to the intracellular membranes, a change in the protein composition of plasma membranes from the embryonic axis and cotyledons was evident early in the time-course of ethanol-induced germination of dormant seeds. While few alterations in the membrane protein composition in response to ethanol treatment occurred both in dormant and nondormant seeds, a number of changes occurred exclusively in ethanol-treated dormant seeds. Notably, a 14 kD protein in the plasma membrane from the embryonic axis and a 35 kD protein in the microsomal membranes from the perisperm-endosperm envelope disappeared in the ethanol-treated dormant seeds, but not in the ethanol-treated nondormant seeds. Also marked decrease in the content of a 23 kD protein in the plasma membrane from the cotyledons was observed in ethanol-treated dormant seeds only. It is suggested that the particular changes in cellular membrane proteins, which occurred in ethanol-treated dormant seeds much before the first visible indication of germination, might be related to dormancy breaking rather than to the germination process.

scholarly journals Reconstitution of the l-lactate carrier from rat and rabbit erythrocyte plasma membranes

1988 ◽  
Vol 254 (2) ◽  
pp. 385-390 ◽  
Author(s):  
R C Poole ◽  
A P Halestrap
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Time Course ◽  
Plasma Membranes ◽  
Gel Filtration ◽  
Erythrocyte Membranes ◽  
Rabbit Erythrocyte ◽  
Bovine Erythrocyte ◽  
Freeze Thaw ◽  
Erythrocyte Plasma Membrane

1. Rat and rabbit erythrocyte plasma-membrane proteins were solubilized with decanoyl-N-methylglucamide and reconstituted into liposomes. The procedure includes detergent removal by gel filtration, followed by a freeze-thaw step. 2. The rate of [1-14C]pyruvate uptake into these vesicles was inhibited by approx. 70% by alpha-cyano-4-hydroxycinnamate and p-chloromercuribenzenesulphonate. The extent of uptake at equilibrium was not affected by the presence of these inhibitors, but was dependent on the osmolarity of the suspending medium. 3. Reconstituted bovine erythrocyte membranes, which have no lactate carrier, showed a much slower time course of pyruvate uptake, with no inhibitor-sensitive component. 4. L- but not D-lactate competed for alpha-cyano-4-hydroxycinnamate-sensitive [1-14C]pyruvate uptake.

The γ-subunit of Na-K-ATPase is incorporated into plasma membranes of mouse IMCD3 cells in response to hypertonicity

2005 ◽  
Vol 288 (4) ◽  
pp. F650-F657 ◽  
Author(s):  
Kaarina Pihakaski-Maunsbach ◽  
Shigeki Tokonabe ◽  
Henrik Vorum ◽  
Christopher J. Rivard ◽  
Juan M. Capasso ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Osmotic Shock ◽  
Collecting Duct ◽  
Choline Chloride ◽  
Basolateral Membrane ◽  
Γ Subunit ◽  
Subunit Expression

Hypertonicity mediated by chloride upregulates the expression of the γ-subunit of Na-K-ATPase in cultured cells derived from the murine inner medullary collecting duct (IMCD3; Capasso JM, Rivard CJ, Enomoto LM, and Berl T. Proc Natl Acad Sci USA 100: 6428–6433, 2003). The purpose of this study was to examine the cellular locations and the time course of γ-subunit expression after long-term adaptation and acute hypertonic challenges induced with different salts. Cells were analyzed by confocal immunofluorescence and immunoelectron microscopy with antibodies against the COOH terminus of the Na-K-ATPase γ-subunit or the γb splice variant. Cells grown in 300 mosmol/kgH2O showed no immunoreactivity for the γ-subunit, whereas cells adapted to 600 or 900 mosmol/kgH2O demonstrated distinct reactivity located at the plasma membrane of all cells. IMCD3 cell cultures acutely challenged to 550 mosmol/kgH2O with sodium chloride or choline chloride showed incorporation of γ into plasma membrane 12 h after osmotic challenge and distinct membrane staining in ∼40% of the cells 48 h after osmotic shock. In contrast, challenging the IMCD3 cells to 550 mosmol/kgH2O by addition of sodium acetate did not result in expression of the γ-subunit in the membranes of surviving cells after 48 h. The present results demonstrate that the Na-K-ATPase γ-subunit becomes incorporated into the basolateral membrane of IMCD3 cells after both acute hyperosmotic challenge and hyperosmotic adaptation. We conclude that the γ-subunit has an important role in the function of Na-K-ATPase to sustain the cellular cation balance over the plasma membrane in a hypertonic environment.

scholarly journals The phosphoprotein that regulates platelet Ca2+ transport is located on the plasma membrane, controls membrane-associated Ca2+-ATPase and is not glycoprotein Ib β-subunit

1991 ◽  
Vol 273 (2) ◽  
pp. 429-434 ◽  
Author(s):  
A Darnanville ◽  
R Bredoux ◽  
K J Clemetson ◽  
N Kieffer ◽  
N Bourdeau ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Plasma Membranes ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Glycoprotein Ib ◽  
Dependent Protein ◽  
Dose Dependent ◽  
Fold Stimulation

The localization and identity of the human platelet 24 kDa cyclic AMP (cAMP)-dependent phosphoprotein, previously reported to regulate Ca2+ transport, was investigated. It was found to be located on plasma membranes after isolation of these membranes from microsomes. Thus cAMP-dependent regulation of Ca2+ transport was associated with the plasma membrane fraction. Time course studies showed that the catalytic subunit of cAMP-dependent protein kinase (c-sub) induced a maximal 2-fold stimulation of Ca2+ uptake by the plasma membrane vesicles. This stimulation was dose-dependent up to 15 micrograms of c-sub/ml. The increase in Ca2+ uptake also depended upon the outside Ca2+ concentration, and was maximal at 1 microM. As regards the identity of the phosphoprotein, it was clearly distinct from the beta-subunit of glycoprotein Ib, as after electrophoresis under reduced conditions it appeared as a 24 kDa protein, but under non-reduced conditions it appeared as a 22 kDa and not as a 170 kDa protein. Nevertheless, glycoprotein Ib was certainly present, because it was detected with two polyclonal antibodies raised against its two subunits. Furthermore, the 24 kDa phosphoprotein was also present in membranes isolated from platelets obtained from patients with Bernard Soulier Syndrome; these membranes contain no glycoprotein Ib.

scholarly journals Comparative studies on the soluble and plasma membrane associated nitrate reductase from Cucumis sativus L.

2014 ◽  
Vol 63 (3-4) ◽  
pp. 309-314 ◽  
Author(s):  
Grażyna Kłobus ◽  
Jolanta Marciniak ◽  
Józef Buczek
Keyword(s):  
Plasma Membrane ◽  
Nitrate Reductase ◽  
Plasma Membranes ◽  
Polyclonal Antiserum ◽  
Soluble Form ◽  
Phase System ◽  
Two Phase ◽  
Cucumis Sativus L ◽  
Cucumber Roots ◽  
Biochemical Comparison

The biochemical comparison between two forms of nitrate reductase from cucumber roots: the soluble enzyme and the plasma membrane-associated one was made. Soluble nitrate reductase was purified on the blue-Sepharose 4B. The nitrate reductase bound with plasma membranes was isolated from cucumber roots by partition of microsomes in the 6.5% dextran-PEG two phase system. The molecular weight of native enzyme estimated with HPLC was 240 kDa and 114 kDa for the soluble and membrane bounded enzyme, respectively. Temperature induced phase separation in Triton X-114 indicated a huge difference in hydrophobicity of the plasma membrane associated nitrate reductase and soluble form of enzyme. Small differences were observed in partial activities of plasma membrane nitrate reductase and soluble nitrate reductase. Also experiments with polyclonal antiserum raised against the native nitrate reductase showed some differences in the immunological properties of both forms of the nitrate reductase. The above results indicated that in cucumber roots two different forms of the nitrate reductase are present.

Aberrant Protein Sorting to the Nucleus during Erythroblast Enucleation: Mechanistic Basis for Membrane Protein Loss in Hereditary Elliptocytosis and Spherocytosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1559-1559
Author(s):  
Marcela A. Salomao ◽  
Sarah Short ◽  
Gloria Lee ◽  
Xiuli An ◽  
Mohandas Narla ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Protein Sorting ◽  
Transmembrane Proteins ◽  
Hereditary Elliptocytosis ◽  
Skeletal Protein ◽  
Aberrant Protein ◽  
Protein 4.1R

Abstract During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of membrane components to plasma membranes surrounding expelled nuclei and young reticulocytes. This protein partitioning performs a crucial role in regulating the protein content of reticulocyte plasma membranes. Although it is known that cytoskeletal actin, spectrin and protein 4.1R distribute to reticulocytes, little is known about the sorting patterns of erythroblast transmembrane proteins. In hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), erythrocytes contain well-described deficiencies of various transmembrane proteins, in addition to those encoded by the mutant genes. For example, elliptocytic human and murine erythrocytes resulting from mutations in the 4.1R gene lack not only protein 4.1R but also transmembrane protein glycophorin C (GPC), known to be a 4.1R binding partner with a key role in linking cytoskeleton to bilayer. Similarly, in HS resulting from mutations in the ankyrin gene, deficiencies of band 3, Rh and GPA have been documented. Various molecular mechanisms could explain deficiencies of membrane proteins in HS and HE erythrocytes including: perturbed trafficking to the erythroblast membrane; aberrant protein sorting during erythroblast enucleation; and selective loss during reticulocyte membrane remodeling. We explored whether aberrant protein sorting during enucleation might be responsible for GPC deficiency in HE. First we performed immunochemical analysis of the sorting pattern of GPC using highly purified extruded nuclei and immature reticulocytes derived from terminally differentiated murine erythroblast cultures. Proteins from equivalent numbers of expelled nuclei and reticulocytes were analyzed by Western blotting. Using antibodies specific for GPC we determined that 90% of GPC sorted to reticulocyte plasma membranes. To validate these results we used live cell, three-color immunofluorescent microscopy and analyzed enucleating erythroblasts, reticulocytes and extruded nuclei from freshly harvested murine wild type bone marrow. Independently confirming the Western blot data, we found that GPC sorted almost exclusively to reticulocytes with little or no GPC in association with nuclear plasma membrane. Strikingly, in 4.1R null erythroblasts GPC was distributed exclusively to expelled nuclei. These findings unequivocally establish that skeletal protein 4.1R is critical for normal sorting of GPC to young reticulocytes and provide clear evidence that specific skeletal protein associations can regulate protein sorting during enucleation. Moreover, our data provide a molecular explanation for the underlying basis of GPC deficiency observed in 4.1R-deficient individuals with HE. We speculate that aberrant protein sorting may be a prevalent mechanism for the deficiencies of various membrane proteins in HS and HE and that their differential loss could contribute to the variable phenotypic expression of these hemolytic disorders.

Skeletal muscle plasma membrane glucose transport and glucose transporters after exercise

1990 ◽  
Vol 68 (1) ◽  
pp. 193-198 ◽  
Author(s):  
L. J. Goodyear ◽  
M. F. Hirshman ◽  
P. A. King ◽  
E. D. Horton ◽  
C. M. Thompson ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Time Course ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Male Rats ◽  
Intrinsic Activity ◽  
Plasma Membrane Vesicles

Recent reports have shown that immediately after an acute bout of exercise the glucose transport system of rat skeletal muscle plasma membranes is characterized by an increase in both glucose transporter number and intrinsic activity. To determine the duration of the exercise response we examined the time course of these changes after completion of a single bout of exercise. Male rats were exercised on a treadmill for 1 h (20 m/min, 10% grade) or allowed to remain sedentary. Rats were killed either immediately or 0.5 or 2 h after exercise, and red gastrocnemius muscle was used for the preparation of plasma membranes. Plasma membrane glucose transporter number was elevated 1.8- and 1.6-fold immediately and 30 min after exercise, although facilitated D-glucose transport in plasma membrane vesicles was elevated 4- and 1.8-fold immediately and 30 min after exercise, respectively. By 2 h after exercise both glucose transporter number and transport activity had returned to nonexercised control values. Additional experiments measuring glucose uptake in perfused hindquarter muscle produced similar results. We conclude that the reversal of the increase in glucose uptake by hindquarter skeletal muscle after exercise is correlated with a reversal of the increase in the glucose transporter number and activity in the plasma membrane. The time course of the transport-to-transporter ratio suggests that the intrinsic activity response reverses more rapidly than that involving transporter number.

scholarly journals Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells.

1975 ◽  
Vol 64 (2) ◽  
pp. 438-460 ◽  
Author(s):  
A L Hubbard ◽  
Z A Cohn
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Sodium Dodecylsulfate ◽  
Electron Microscope Autoradiography ◽  
L Cells ◽  
L Cell ◽  
Slab Gels ◽  
Pinocytic Vesicles

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane.

EVIDENCE FOR TWO DIFFERENT Ca2+TRANSPORT SYSTEMS ASSOCIATED WITH PLASMA AND INTRACELLULAR HUMAN PLATELET MEMBRANES

1987 ◽  
Author(s):  
J Enouf ◽  
R Breadux ◽  
N Bourdeau ◽  
S Levy-Toledano
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Plasma Membranes ◽  
Human Platelets ◽  
Transport Systems ◽  
Maximal Response ◽  
Potassium Oxalate ◽  
Intracellular Enzyme ◽  
Intracellular Membranes ◽  
Membrane Enzyme

The regulation of Ca2+ concentration in different cells involves two Ca2+ pumps. The presence of such mechanisms in human platelets is still controverted. We then investigated this question by using plasma and intracellular membranes obtained after simultaneous isolation by centrifugation ca 40% sucrose from a mixed 100,000 g membrane fraction.The Ca2+ uptake by the different membrane vesicles has been studied. Both membrane fractions took up Ca2+ and the Ca2+ transport systems exhibited a high affinity towards Ca 2+.However, the two associated Ca2+ transport systems showed a different time course and exhibited different oxalate sensitivity. The plasma membranes are not permeable to potassium oxalate, while the Ca2+ uptake was stimulated by potassium oxalate in intracellular membranes.Two Ca2+ activated ATPase activities are associated with the isolated membrane fractions and appeared different for the following parameters : 1) a different time course of the two enzyme activities; 2)a similar apparent affinity towards Ca2+ (10−7 M), though inhibition of the Ca2+ ATPase activity was only observed in intracellular membranes at 10−6 M Ca2+ ; 3)a different pH dependence with a maximum at pH 7 for the enzyme of intracellular membranes and pH 8 for the enzyme of plasma membranes; 4)a 10 fold difference in the ATP requirement of the enzymes, thus the maximal response was obtained with 20 uM for the intracellular membrane enzyme and with 200 uM for the plasma membrane enzyme ; 5) a different affinity for various nucleotides as energy donors with a higher specificity of the plasma membrane enzyme towards ATP ; 6) a different vanadate inhibition-dose reponse which did not exceed 60% for the plasma enzyme while it reached 100% for the intracellular enzyme.Taken together, these studies agree with the possible role of both a plasma membrane and a dense tubular system Ca2+ -ATPases in the regulation of Ca2+ concentration in human platelets.

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 Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method.

1987 ◽  
Vol 105 (6) ◽  
pp. 2589-2601 ◽  
Author(s):  
R S Molday ◽  
L L Molday
Keyword(s):  
Plasma Membrane ◽  
Perturbation Method ◽  
Plasma Membranes ◽  
Prolonged Exposure ◽  
Gradient Centrifugation ◽  
Protein Composition ◽  
Major Protein ◽  
Binding Studies ◽  
Western Blots ◽  
Retinal Rod

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes.

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