scholarly journals Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells.

1975 ◽  
Vol 64 (2) ◽  
pp. 461-479 ◽  
Author(s):  
A L Hubbard ◽  
Z A Cohn
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Sodium Dodecyl ◽  
Relative Distribution ◽  
Plasma Membrane Proteins ◽  
Electron Microscope Autoradiography ◽  
L Cells ◽  
Cell Plasma ◽  
Latex Beads ◽  
Discontinuous Sucrose Gradient

The fate of the L-cell plasma membrane proteins labeled by enzymatic iodination was studied. The disappearance of label from growing cells exhibits a biphasic behavior, with 5-20% lost rapidly (t1/2 similar to 2 h) and 80-90% lost relatively slowly (t1/2 similar to 25-33 h). The loss is temperature dependent and serum independent, and is accompanied by the appearance of 51% (125-I)monoiodotyrosine (MIT) in the medium by 47 h. A variable amount (1-14%) of acid-insoluble label can be recovered in the medium over 47 h. Sodium dodecyl sulfate (SDS)-polyacrylamide gel labeling patterns from cells cultured up to 48 h after iodination reveal no change in the relative distribution of radioactivity, indicating similar rates of degradation for most of the labeled membrane proteins. The fate of the labeled membrane proteins was studied at various times after phagocytosis of nondigestible polystyrene particles. Iodinated L cells phagocytose sufficient 1.1 mum latex beads in 60 min to interiorize 15-30% of the total cell surface area. Electron microscope autoradiography confirmed that labeled membrane is internalized during phagocytosis. The latex-containing phagocytic vacuoles are isolated by flotation in a discontinuous sucrose gradient. 15-30% of the total incorporated label and a comparable percentage of alkaline phosphodiesterase I activity (PDase, a plasma membrane enzyme marker) are recovered in the phagocytic vacuole fraction. Lysosomal enzyme activities are found in the latex vacuole fraction, indicating formation of phagolysosomes. SDS gel analyses reveal that all of the radioactive proteins initially present on the intact cell's surface are interiorized to the same relative extent. Incorporated label and PDase activity disappear much more rapidly from the phagolysosomes than from the whole cell. In the phagolysosomal compartment, greater than 70% of the TCA-precipitable labeled proteins and all of the PDase activity are lost rapidly (t1/2 equals 1-2 h) but similar 30% of the labeled proteins in this compartment are degraded with a 17-20 h half-life. The slowly degraded label is due to specific long-lived polypeptides, of 85,000 and 8,000-15,000 daltons, which remain in the phagolysosomal membrane up to 40 h after phagocytosis.

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.

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.

scholarly journals Degradation of proteins in rat liver mitochondrial outer membrane transplanted into different cell types. Evidence for alternative processing

1984 ◽  
Vol 220 (2) ◽  
pp. 489-498 ◽  
Author(s):  
S M Russell ◽  
J S Amenta ◽  
R J Mayer
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Average Rate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
A549 Cells ◽  
Rat Hepatocytes ◽  
Mitochondrial Outer Membrane ◽  
Plasma Membrane Proteins

The degradation of proteins in reductively [3H]methylated mitochondrial outer membrane (MOM) transplanted into cells by a poly(ethylene glycol)-mediated process has been studied. The average rate of degradation (t1/2 24-28 h) of MOM proteins transplanted into HTC cells was not the same as for endogenous MOM proteins (t1/2 56 h), mitoplast proteins (t1/2 120 h), plasma membrane proteins (t1/2 approx. 90 h) or cytosol proteins (t1/2 75 h). The degradation of transplanted MOM proteins was inhibited to the same extent (30-45%) as that of endogenous mitochondrial and plasma membrane proteins by leupeptin and NH4Cl. No inhibition of HTC cell cytosol protein degradation by NH4Cl was observed. NH4Cl differentially inhibited the degradation of endogenous MOM and mitoplast protein subunits as shown after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Proteins in MOM transplanted into tissue culture cells were degraded either with t1/2 24-28 h (MRC-5, B82 and A549 cells) or with t1/2 55-70 h (CHO-K1 and 3T3-L1 cells) similar to that of proteins in MOM transplanted into rat hepatocytes [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The data suggest that membrane protein destruction is but the end part of a fundamental intracellular membrane recognition process.

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.

scholarly journals Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 63
Author(s):  
Raf Van Campenhout ◽  
Serge Muyldermans ◽  
Mathieu Vinken ◽  
Nick Devoogdt ◽  
Timo W.M. De Groof
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Transport ◽  
Transport Proteins ◽  
Therapeutic Drug ◽  
Cell Plasma Membrane ◽  
Plasma Membrane Proteins ◽  
Altered Expression ◽  
Membrane Transport Proteins ◽  
Cell Plasma

Cell plasma membrane proteins are considered as gatekeepers of the cell and play a major role in regulating various processes. Transport proteins constitute a subclass of cell plasma membrane proteins enabling the exchange of molecules and ions between the extracellular environment and the cytosol. A plethora of human pathologies are associated with the altered expression or dysfunction of cell plasma membrane transport proteins, making them interesting therapeutic drug targets. However, the search for therapeutics is challenging, since many drug candidates targeting cell plasma membrane proteins fail in (pre)clinical testing due to inadequate selectivity, specificity, potency or stability. These latter characteristics are met by nanobodies, which potentially renders them eligible therapeutics targeting cell plasma membrane proteins. Therefore, a therapeutic nanobody-based strategy seems a valid approach to target and modulate the activity of cell plasma membrane transport proteins. This review paper focuses on methodologies to generate cell plasma membrane transport protein-targeting nanobodies, and the advantages and pitfalls while generating these small antibody-derivatives, and discusses several therapeutic nanobodies directed towards transmembrane proteins, including channels and pores, adenosine triphosphate-powered pumps and porters.

Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

2009 ◽  
Vol 18 (6) ◽  
pp. 527-535 ◽  
Author(s):  
Andreas Lange ◽  
Claudia Kistler ◽  
Tanja B. Jutzi ◽  
Alexandr V. Bazhin ◽  
Claus Detlev Klemke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membrane Proteins ◽  
Subtractive Suppression Hybridization
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