Endogenous hyperphosphorylation in plasma membrane from an ascites hepatocarcinoma cell line

1988 ◽  
Vol 66 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Jon G. Church ◽  
Shobha Ghosh ◽  
Basil D. Roufogalis ◽  
Antonio Villalobo
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Line ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Membrane Lipids ◽  
Normal Adult ◽  
Plasma Membrane Proteins ◽  
Phosphoprotein Phosphatase ◽  
Membrane Bound

Plasma-membrane-bound kinases of AS-30D ascites from transplantable rat hepatocarcinoma were shown to extensively catalyze the phosphorylation of plasma membrane proteins and membrane lipids, using [γ-32P]ATP or [γ-32P]GTP as a phosphate donor. In contrast, plasma membranes from normal adult rat liver or fast-growing regenerating liver (24 h after partial hepatectomy) produce significantly less activity for protein phosphorylation and little phosphorylation of the lipids. However, neonatal (24 h old) rat liver plasma membrane preparations show levels of phosphorylation of proteins and lipids intermediate between those in the tumor cell line and normal adult plasma membrane preparations. Phosphatidic acid was identified as one of the 32P-labelled lipids in the tumor plasma membrane chloroform–methanol (2:1, v/v) extract. Phosphorylation of protein was not affected by cAMP or cGMP. However, calcium ion (in the presence or absence of calmodulin) significantly modifies the 32P labelling of a series of proteins in normal tissue but has little effect with the neoplastic preparations. Some plasma membrane proteins were capable of nucleotide binding, instead or in addition to being phosphorylated. Finally, the presence of membrane-bound phosphoprotein phosphatase(s) was also demonstrated in all the preparations examined by means of chase experiments with nonlabelled ATP or GTP, and (or) by the use of the phosphoprotein phosphatase inhibitor, orthovanadate.

Myogenesis of normal and dystrophic chick embryonic skeletal muscle cells in vitro: biosynthesis of plasma membrane proteins

1980 ◽  
Vol 58 (10) ◽  
pp. 1156-1164 ◽  
Author(s):  
Paul C. Holland ◽  
George A. Cates ◽  
Byron S. Wenger ◽  
Barbara L. Raney
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Protein Biosynthesis ◽  
Sodium Dodecyl ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Plasma Membrane Proteins ◽  
Dystrophic Muscle

Plasma membranes were prepared from primary cell cultures of normal and genetically dystrophic chick embryonic pectoral muscle. These membranes were analyzed both by one-dimensional sodium dodecyl sulphate – polyacrylamide slab gel electrophoresis and by two-dimensional electrophoresis using isoelectric focusing in the first dimension. No marked and reproducible abnormalities could be detected in the synthesis, or accumulation, of plasma membrane proteins of dystrophic muscle cells maintained in culture for periods of up to 6 days. Analysis of the relative rates of protein turnover, analysis of fucose incorporation into plasma membrane proteins, and comparison of iodinated cell surface proteins also failed to reveal distinct abnormalities in plasma membranes derived from cultured dystrophic muscle cells. Although the results obtained do not rule out an early defect in plasma membrane protein biosynthesis during the development of dystrophic skeletal muscle in vivo, they do demonstrate that the synthesis and assembly of at least the major plasma membrane proteins occur normally during the initial phases of terminal differentiation of isolated dystrophic skeletal muscle cells in tissue culture.

Cell-free reconstitution of the transport of viral glycoproteins from the TGN to the basolateral plasma membrane of MDCK cells

1996 ◽  
Vol 109 (7) ◽  
pp. 1667-1676 ◽  
Author(s):  
A. Mayer ◽  
I.E. Ivanov ◽  
D. Gravotta ◽  
M. Adesnik ◽  
D.D. Sabatini
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Great Promise ◽  
Plasma Membrane Proteins ◽  
In Vitro System ◽  
Viral Glycoproteins ◽  
Basolateral Plasma Membrane

An in vitro system to study the transport of plasma membrane proteins from the TGN to the basolateral plasma membrane of polarized MDCK cells has been developed in which purified cell fractions are combined and transport between them is studied under controlled conditions. In this system, a donor Golgi fraction derived from VSV or influenza virus-infected MDCK cells, in which 35S-labeled viral glycoproteins were allowed to accumulate in the TGN during a low temperature block, is incubated with purified immobilized basolateral plasma membranes that have their cytoplasmic face exposed and are obtained by shearing-lysis of MDCK monolayers grown on cytodex beads. Approximately 15–30% of the labeled glycoprotein molecules are transferred from the Golgi fraction to the acceptor plasma membranes and are recovered with the sedimentable (1 g) beads. Transport is temperature, energy and cytosol dependent, and is abolished by alkylation of SH groups and inhibited by the presence of GTP-gamma-S, which implicates GTP-binding proteins and the requirement for GTP hydrolysis in one or more stages of the transport process. Endo H-resistant glycoprotein molecules that had traversed the medial region of the Golgi apparatus are preferentially transported and their luminal domains become accessible to proteases, indicating that membrane fusion with the plasma membrane takes place in the in vitro system. Mild proteolysis of the donor or acceptor membranes abolishes transport, suggesting that protein molecules exposed on the surface of these membranes are involved in the formation and consumption of transport intermediates, possibly as addressing and docking proteins, respectively. Surprisingly, both VSV-G and influenza HA were transported with equal efficiencies to the basolateral acceptor membranes. However, low concentrations of a microtubular protein fraction preferentially inhibited the transport of HA, although this effect was not abolished by microtubule depolymerizing agents. This system shows great promise for elucidating the mechanisms that effect the proper sorting of plasma membrane proteins in the TGN and their subsequent targeting to the appropriate acceptor membrane.

Incorporation of proteins into (Xenopus) oocytes by proteoliposome microinjection: functional characterization of a novel aquaporin

1996 ◽  
Vol 109 (6) ◽  
pp. 1285-1295
Author(s):  
F. Le Caherec ◽  
P. Bron ◽  
J.M. Verbavatz ◽  
A. Garret ◽  
G. Morel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Digestive Tract ◽  
Xenopus Oocytes ◽  
Plasma Membranes ◽  
Expression System ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes ◽  
Plasma Membrane Proteins ◽  
Cicadella Viridis

Xenopus laevis oocytes are widely used as an expression system for plasma membrane proteins, achieved by cytoplasmic microinjection of messenger RNA. In the present study, we propose an alternative system allowing functional insertion of exogenous proteins into the plasma membrane of Xenopus oocytes. We microinjected proteoliposome suspensions into the cytoplasm and then analyzed membrane protein function. The proteins used in this work were members of the MIP family: the human erythrocyte water channel aquaporin 1 (AQP1), the major intrinsic protein (MIP26) from bovine eye lens and a 25 kDa polypeptide (P25) from a water shunting complex found in the digestive tract of an homopteran sap-sucking insect (Cicadella viridis). Proteoliposomes containing either AQP1, MIP26, or P25 were injected into Xenopus oocytes. The subsequent insertion of these proteins into the plasma membrane of oocytes was demonstrated by immunocytochemistry. Oocytes microinjected with either AQP1 or P25-proteoliposomes exhibited significantly increased osmotic membrane water permeabilities (Pf = 3.16 +/- 026 and 4.03 +/- 0.26 × 10(−3) cm/second, respectively) compared to those measured for oocytes injected with liposomes alone or with MIP26-proteoliposomes (Pf = 1.39 +/- 0.07 and 1.44 +/- 0.10 × 10(−3) cm/second, respectively). These effects were inhibited by HgCl2 in a reversible manner. Arrhenius activation energies of water transfer were low when AQP1 or P25 were present in oocyte plasma membranes (Ea = 2.29 and 3.01 kcal/mol, respectively, versus Ea = 11.75 kcal/mol for liposome injected oocytes). The properties observed here for AQP1 are identical to those widely reported following AQP1 cRNA expression in oocytes. From the present study, we conclude that: (1) exogenous plasma membrane proteins incorporated into liposomes and microinjected into the cytoplasm of Xenopus oocytes are subsequently found in the plasma membrane of the oocytes in a functional state; and (2) in this system, the P25 polypeptide from the MIP family found in the digestive tract of Cicadella viridis exhibits properties similar to those described for the archetype of water channels AQP1, and thus is a new member of the aquaporin family.

scholarly journals Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration

1983 ◽  
Vol 212 (2) ◽  
pp. 445-452 ◽  
Author(s):  
G Deliconstantinos ◽  
G Ramantanis
Keyword(s):  
Plasma Membrane ◽  
Liver Regeneration ◽  
Membrane Fluidity ◽  
Plasma Membranes ◽  
Membrane Lipids ◽  
Biological Significance ◽  
Hill Coefficient ◽  
Membrane Bound ◽  
The Hill ◽  
Membrane Bound Enzymes

A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5′-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5′-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration.

scholarly journals Loss of the transferrin receptor during the maturation of sheep reticulocytes in vitro. An immunological approach

1983 ◽  
Vol 210 (1) ◽  
pp. 37-47 ◽  
Author(s):  
B T Pan ◽  
R Blostein ◽  
R M Johnstone
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Transferrin Receptor ◽  
Plasma Membranes ◽  
Plasma Membrane Proteins ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Cell Plasma ◽  
A Subunit

Sheep reticulocyte-specific antiserum absorbed with mature sheep red cells has been used to isolate and identify reticulocyte-specific plasma-membrane proteins and to monitor their loss during incubation in vitro. Specific precipitation of labelled plasma-membrane proteins is obtained when detergent-solubilized extracts of 125I-labelled reticulocyte plasma membranes are incubated with this antiserum and Staphyloccus aureus, but not when mature-cell plasma membranes are treated similarly. During maturation of reticulocytes in vitro (up to 4 days at 37 degrees C), there is a marked decrease in the immunoprecipitable material. The anti-reticulocyte-specific antibodies have been identified as anti-(transferrin receptor) antibodies. By using these antibodies as a probe, the transferrin receptor has been shown to have a subunit molecular weight of 93 000. The data are consistent with reported molecular weights of this receptor and with the proposal that the receptor may exist as a dimer, since [125I]iodotyrosyl-peptide maps of the 93 000- and 186 000-mol.wt. components isolated are shown to be identical. Evidence is presented for the transmembrane nature of the receptor and for the presence of different binding sites for transferrin and these antibodies on the receptor.

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