scholarly journals The relationship of detergent-solubilization plasma-membrane components of rabbit alveolar macrophages to an isolated inhibitor of phagocytosis

1979 ◽  
Vol 179 (2) ◽  
pp. 305-314 ◽  
Author(s):  
R S Pratt ◽  
G M Cook
Keyword(s):  
Plasma Membrane ◽  
Alveolar Macrophages ◽  
Membrane Fraction ◽  
Lung Lavage ◽  
Membrane Glycoproteins ◽  
Rabbit Lung ◽  
Membrane Components ◽  
Triton X ◽  
Relationship Of ◽  
The Relationship

1. A plasma-membrane fraction prepared from rabbit alveolar macrophages by hyposmotic borate lysis is described. 2. Rabbit lung lavages, containing a glycoprotein inhibitor of phagocytosis, may be fractionated by preparative isoelectric focusing in the presence of Triton X-100. 3. Chemical analysis indicates that the glycoproteins of the lung lavage contain sialic acid, fucose, mannose, galactose, hexosamine and appreciable quantities of glucose. 4. The relationship of macrophage membrane glycoproteins, solubilized with Triton X-100 in the presence of borate, to the lung lavage glycoproteins is demonstrated immunoelectrophoretically.

Association of the hepatic IP3 receptor with the plasma membrane: relevance to mode of action

1993 ◽  
Vol 265 (6) ◽  
pp. C1588-C1596 ◽  
Author(s):  
L. Feng ◽  
N. Kraus-Friedmann
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Plasma Membrane Fraction ◽  
T Lymphoma Cells ◽  
T Lymphoma ◽  
Triton X ◽  
Brain Receptor ◽  
The Brain

Studies were carried out to characterize the interaction between inositol 1,4,5-trisphosphate (IP3) receptors and the plasma membrane fraction. Extraction of the membranes with the nonionic detergents Nonidet P-40 and Triton X-100, followed by centrifugation at 100,000 g, resulted in the doubling of the IP3 receptor in the pellets, whereas no detectable binding was found in the supernatants. These data indicate that the detergents did not solubilize the receptor, that it remained associated with membrane particles, and that it is likely to be associated with the cytoskeleton. The cytoskeleton proteins actin, ankyrin, and spectrin were identified in the plasma membrane fraction. However, comparison of the amount of these proteins in different fractions of the detergent, or otherwise treated plasma membrane fractions, showed no direct correlation between the presence of any of these proteins in the plasma membrane fraction and their ability to bind [3H]IP3. This is in contrast to the brain and T-lymphoma cells in which the IP3 receptor is attached to ankyrin (L. Y. W. Bourguigon, H. Jin, N. Iida, N. R. Brandt, and S. H. Zhang. J. Biol. Chem. 268: 6477-6486, 1993; and S. K. Joseph and S. Samanta. J. Biol. Chem 268: 6477-6486, 1993). Thus the hepatic IP3 receptor, which is different from the brain receptor, might attach to the cytoskeleton by anchoring to a different protein. Because cytochalasin D treatment of livers diminishes the ability of IP3 to raise cytosolic free Ca2+ levels, the attachment of the IP3 receptor to the cytoskeleton seems to involve an association with microfilaments.

scholarly journals Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets.

1976 ◽  
Vol 71 (2) ◽  
pp. 606-623 ◽  
Author(s):  
A Lernmark ◽  
A Nathans ◽  
D F Steiner
Keyword(s):  
Plasma Membrane ◽  
Wheat Germ Agglutinin ◽  
Membrane Fraction ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasma Membrane Fraction ◽  
Fresh Plasma ◽  
Rat Islets Of Langerhans ◽  
Membrane Components

Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5'-nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium-dependent ATPase and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific acivity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, 125I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.

The caveolar-mitochondrial interface: regulation of cellular metabolism in physiology and pathophysiology

2020 ◽  
Vol 48 (1) ◽  
pp. 165-177 ◽  
Author(s):  
Cerrone R. Foster ◽  
Shiho Satomi ◽  
Yuko Kato ◽  
Hemal H. Patel
Keyword(s):  
Plasma Membrane ◽  
Cellular Metabolism ◽  
Primary Factor ◽  
Cellular Organelle ◽  
Membrane Microdomain ◽  
Intracellular Communication ◽  
Intracellular Organelles ◽  
Relationship Of ◽  
The Relationship

The plasma membrane is an important cellular organelle that is often overlooked in terms of a primary factor in regulating physiology and pathophysiology. There is emerging evidence to suggest that the plasma membrane serves a greater purpose than a simple barrier or transporter of ions. New paradigms suggest that the membrane serves as a critical bridge to connect extracellular to intracellular communication particularly to regulate energy and metabolism by forming physical and biochemical associations with intracellular organelles. This review will focus on the relationship of a particular membrane microdomain — caveolae — with mitochondria and the particular implication of this to physiology and pathophysiology.

scholarly journals THE FINE STRUCTURAL LOCALIZATION OF ACETYLCHOLINESTERASE AT THE MYONEURAL JUNCTION

1962 ◽  
Vol 12 (2) ◽  
pp. 247-262 ◽  
Author(s):  
Russell J. Barrnett
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Axon Terminal ◽  
Myoneural Junction ◽  
Cytochemical Localization ◽  
Structural Localization ◽  
Terminal Axon ◽  
Esterolytic Activity ◽  
Relationship Of ◽  
The Relationship

A study of the cytochemical localization of acetylcholiriesterase activity, combining histochemistry with electron microscopy, showed that the final product of the reaction, which was deposited at or near enzyme sites, occurred at four places in the myoneural junction. These included: plasma membrane of the muscle covering the junctional folds, the primary and secondary synaptic clefts, parts of the plasma membrane covering the axon terminal, and vesicular structures in the terminal axoplasm. No reaction occurred in the presence of 10-4 eserine or DFP, whereas 10-5 DFP inhibited the reaction at all sites except in the vesicles of the terminal axon. These findings are discussed with reference to the histochemical method used and to the occurrence of esterolytic activity in the vesicles, as well as to some of the current hypotheses concerning the relationship of the site of acetylcholinesterase and synaptic transmission.

Platelet Glycoproteins in Platelet Aggregation

1979 ◽  
Author(s):  
David R. Phillips ◽  
Lisa K. Jennings ◽  
Harold H. Edwards
Keyword(s):  
Membrane Surface ◽  
Direct Interaction ◽  
Protein Composition ◽  
Actin Binding ◽  
Membrane Glycoproteins ◽  
Dense Cores ◽  
Insoluble Material ◽  
Filamentous Material ◽  
Membrane Components ◽  
Triton X

Thrombin stimulation alters the membrane surface of platelets so that specific components on the membrane surface interact. To identify such “aggregation factors”, tne exposed membrane proteins of washed platelets were labeled by lactoperoxidase-catalyzed iodination and tested for their association with cytoskeletal structures. Control, thrombin-stimulated (TS; nM thrombin in mM EDTA to prevent aggregation) and thrombin aggregated (TA; 2 mM Ca++) platelets were treated with 1% Triton X-100. The insoluble material (isolated by centrifugation) from TS platelets, but not unstimulated platelets, had clusters of filamentous material with dense cores about 1 μ in diameter. Each cluster appeared to arise from one platelet and contained proteins with the Mr of actin actin-binding protein and myosin plus a 56K and 90K protein. Triton extraction of TA platelets produced an insoluble material with a similar protein composition as that from TS platelets; however, the filamentous clusters remained- aggregration, indicating tnat membrane components which aggregate platelets were still present. Analysis of iodinated membrane components revealed that all were solubilized by Triton from control and TS platelets while two glycoproteins, termed IIb and III, remained with the filamentous material from TA platelets. This and the observation that platelets lacking IIb and III cannot aggregate [JCI. 60: 535 (1977)], indicate that one or both of these membrane glycoproteins are involved in the direct Interaction of platelets during aggregation.

Acid and alkaline phosphatases of Capnocytophaga species. III. The relationship of the enzymes to the cell wall

1983 ◽  
Vol 29 (10) ◽  
pp. 1369-1381 ◽  
Author(s):  
Thomas P. Poirier ◽  
Stanley C. Holt
Keyword(s):  
Cell Wall ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Marker Enzyme ◽  
Transmission Electron ◽  
Sds Page ◽  
Triton X ◽  
Relationship Of ◽  
The Relationship ◽  
Cell Fragments

Acid (AcP) and alkaline phosphatase (AlP) were localized by physicobiochemical techniques. Greater than 53% of the phosphatases were detected, following sonication, in a low speed centrifugation pellet while osmotically shocked and spheroplasted Capnocytophaga species released only 9–28% and 11–43% of the cellular phosphatases, respectively. French pressure cell disruption was more effective in releasing the phosphatases. Cell fragments were separated into cell wall, cytoplasmic membrane, and soluble fractions as determined by marker enzyme, chemical composition, transmission electron microscopy, sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE) protein analysis, and gel diffusion. AcP and AlP was partitioned between the isolated cell wall (31–46%) and soluble material (33–61%), with greater than 60% of the phosphatases remaining with the cell wall following Triton X-100 treatment. The amount of phosphatase at the surface of intact C. ochracea was quantitated by specific 125I-protein labelling.

Endotoxin-induced platelet aggregation and secretion. II. Changes in plasma membrane proteins

1977 ◽  
Vol 28 (1) ◽  
pp. 225-236
Author(s):  
K.J. Thorne ◽  
R.C. Oliver ◽  
D.E. MacIntyre ◽  
J.L. Gordon
Keyword(s):  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Membrane Proteins ◽  
Blood Platelets ◽  
Molecular Organization ◽  
Double Labelling ◽  
Membrane Composition ◽  
Plasma Membrane Proteins ◽  
Relationship Of ◽  
The Relationship

Responses of blood platelets to bacterial endotoxin lipopolysaccharide (LPS) have been correlated with changes in the molecular organization and composition of the platelet plasma membrane proteins. Binding of LPS, which occurred in the absence of Ca2+, was distinguished from platelet aggregation and degranulation, which required Ca2+ and plasma proteins. Changes in membrane organization were detected by double-labelling with [125I] and [131I] iodide, mediated by lactoperoxidase and hydrogen peroxide. Changes in total membrane composition were detected by gel electrophoresis of isolated membranes. Binding of LPS was associated with increased accessibility of a protein of mol. wt. 80000 to iodination. After aggregation and degranulation there was, in addition, increased accessibility of proteins of mol. wt. 68000 and 48000. Isolated membranes from LPS-stimulated platelets contained more of a protein of mol. wt. 200000 and less of a protein of mol. wt. 220000 than control membranes prepared from unstimulated platelets in the presence of cAMP and aminophylline. The relationship of the modified plasma membrane proteins to the contractile proteins of the platelet and their possible redistribution in the cell during aggregation and secretion is discussed.

scholarly journals Preferential degradation of the terminal carbohydrate moiety of membrane glycoproteins in rat hepatoma cells and after transfer to the membranes of mouse fibroblasts.

1983 ◽  
Vol 96 (1) ◽  
pp. 139-150 ◽  
Author(s):  
H Baumann ◽  
E Hou ◽  
G P Jahreis
Keyword(s):  
Plasma Membrane ◽  
Protein Fraction ◽  
Hepatoma Cells ◽  
Total Cell ◽  
Cell Protein ◽  
Two Dimensional ◽  
Membrane Glycoproteins ◽  
Membrane Components ◽  
Rat Hepatoma ◽  
Rat Hepatoma Cells

Glycoproteins in the plasma membrane of rat hepatoma cells were labeled at their externally exposed tyrosine residues with 131I and at their galactose and sialic acid residues with 3H. The degradation of both isotopes in the total cell protein fraction, in glycoproteins purified by concanavalin A, and in glycoproteins separated on two-dimensional gels was determined. Similarly, the total cellular membrane glycoproteins were metabolically labeled with [35S]methionine and [3H]fucose. The fate of both incorporated labels was followed by lectin chromatography or by precipitation of the proteins with specific antibodies followed by electrophoretic gel separation. In both labeling experiments, the carbohydrate markers were lost from the ligand-recognized fraction with similar kinetics as from the total cell protein fraction. In some glycoprotein species which were separated by two-dimensional gel electrophoresis, the polypeptide portion exhibited up to a twofold slower rate of degradation relative to that of the carbohydrate moiety. This difference is most pronounced in carbohydrate-rich glycoproteins. To corroborate this finding, double-labeled membrane glycoproteins were incorporated into reconstituted phospholipid vesicles which were then transferred via fusion into the plasma membrane of mouse fibroblasts. Both the polypeptide and carbohydrate moieties of the transferred membrane glycoproteins were degraded with the same relative kinetics as in the original hepatoma cells. The rate of degradation is mostly a function of the structural properties of the membrane components as shown by the preservation of metabolically stable fucogangliosides of Reuber H-35 hepatoma cells transferred onto the fibroblasts. The technique of insertion of membrane components into the plasma membrane of another cell should assist in the elucidation of the exact route and mechanism of membrane protein destruction.

scholarly journals Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes

1974 ◽  
Vol 140 (3) ◽  
pp. 469-478 ◽  
Author(s):  
Terry D. Butters ◽  
R. Colin Hughes
Keyword(s):  
Plasma Membranes ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Adenovirus Type ◽  
Insoluble Residue ◽  
Membrane Glycoproteins ◽  
Low Concentrations ◽  
Ceramide Trihexoside ◽  
Membrane Components ◽  
Triton X

1. A fraction enriched in plasma membranes of human tumour KB cell line, a permissive cell for adenovirus type 5, was obtained. 2. Electrophoresis of the membranes in polyacrylamide gels with buffers containing sodium dodecyl sulphate showed that the membranes after reduction with 2-mercaptoethanol contained over 20 polypeptide species. Three polypeptides were glycosylated and had apparent mol.wts. of 92000, 72000 and 62000. 3. The glycoproteins and the specific receptors responsible for adenovirus adsorption to the membranes were readily extracted into solutions containing low concentrations of Triton X-100. Glycolipids and proteins were also made soluble. A membranous residue obtained after Triton X-100 extraction was enriched in several proteins that appeared to consist of polypeptides of lower molecular weight than the average of KB membrane polypeptides. 4. Sphingomyelin, cholesterol and triglycerides were similarly concentrated in the insoluble residue remaining after successive extractions of KB membranes with Triton X-100. Further, ceramide trihexoside was significantly less easily extracted from KB membranes than lactosyl ceramide. 5. The differences noted in the ease of extraction of membrane components are discussed. 6. The components of membranes made soluble by detergent extraction and containing the large part of the KB membrane glycoproteins were subjected to chromatography on Sepharose 6B and DEAE-cellulose and to isoelectric focusing in the presence of buffers containing Triton X-100. In general, the degree of separation into fractions enriched in individual glycoproteins was disappointing. Possible reasons for the poor fractionation of membrane components by chromatographic systems conveniently used for purification of proteins and glycoproteins of non-membranous origin are briefly discussed.

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