scholarly journals Biosynthesis of liver membranes. Incorporation of [3H]leucine into proteins and of [14C]glucosamine into proteins and lipids of liver microsomal and plasma-membrane fractions

1971 ◽  
Vol 125 (2) ◽  
pp. 615-624 ◽  
Author(s):  
W. H. Evans ◽  
J. W. Gurd
Keyword(s):  
Plasma Membrane ◽  
Mouse Liver ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Protein Ratio ◽  
Glycolipid Synthesis ◽  
Liver Membranes ◽  
Liver Homogenates ◽  
Chloroform Methanol ◽  
Soluble Components

1. The smooth-and rough-microsomal and the light and heavy plasma-membrane fractions of mouse liver homogenates were prepared and characterized by using biochemical markers. 2. The hexosamine/protein ratio was threefold higher in the plasma membranes than in the smooth-microsomal fraction. Glucosamine was bound only to protein, and galactosamine was attached mainly to lipids. 3. [3H]-Leucine and [14C]glucosamine were injected into animals and the rates of incorporation of radioactivity into the fractions were determined. Both precursors were rapidly incorporated into the microsomal fractions, but plasma membranes showed a slower rate of synthesis which reached a maximum at 2–4h after intravenous administration. 4. The light- and heavy-plasma-membrane fractions showed similar patterns of incorporation, and therefore a precursor–product relationship appears unlikely. 5. Plasma membranes, especially the light subfraction, showed appreciable incorporation of hexosamine into chloroform–methanol-soluble components which were shown to be mainly glycolipids. 6. The results indicate that liver plasma-membrane proteins and glycoproteins are synthesized at similar rates. However, glycolipid synthesis in plasma membranes occurred more rapidly.

Fine-Structural Localization of Adenosine Triphosphatase in the Rectum of Calliphora

1968 ◽  
Vol 3 (1) ◽  
pp. 17-32
Author(s):  
M. J. BERRIDGE ◽  
B. L. GUPTA
Keyword(s):  
Plasma Membrane ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Fluid Transport ◽  
Microsomal Fraction ◽  
Adenosine Diphosphate ◽  
Structural Basis ◽  
Osmotic Gradient ◽  
Ph Optimum ◽  
Structural Localization

Adenosine triphosphatase (ATPase) activity in the rectal papillae of Calliphora has been studied by biochemical and histochemical techniques. The microsomal fraction contained a Mg2+-activated ATPase with a pH optimum of 8.0. The enzyme was not stimulated by the addition of Na+ plus K+ and was insensitive to ouabain. Histochemical studies using modifications of the Wachstein-Meisel method showed that at pH 7.2 this Mg2+-activated ATPase was specifically localized on the intracellular surface of the lateral plasma membranes. A similar though less intense reaction was obtained with adenosine diphosphate and inosine triphosphate, but not with guanosine triphosphate, uridine triphosphate or β-glycerophosphate as substrates. At an acid pH (6.6-6.8), very little reaction occurred on the lateral plasma membrane but some reaction product was present in mitochondria and nuclei. Very little enzyme activity was found in the flattened rectal epithelium. These results are discussed in relation to the available data on transport ATPases and on the structural basis of fluid transport by rectal papillae. It is proposed that the ATPase localized on the stacks of lateral plasma membrane may be involved with ion secretion into the intercellular spaces to create the osmotic gradient necessary to extract water from the lumen.

scholarly journals Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue

1984 ◽  
Vol 219 (1) ◽  
pp. 301-308 ◽  
Author(s):  
A A Davies ◽  
N M Wigglesworth ◽  
D Allan ◽  
R J Owens ◽  
M J Crumpton
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Insoluble Fraction ◽  
Insoluble Residue ◽  
Protein Ratio

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5′-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

Seasonal changes in plasma membrane glucose transporters enhance cryoprotectant distribution in the freeze-tolerant wood frog

1995 ◽  
Vol 73 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Patricia A. King ◽  
Mary N. Rosholt ◽  
Kenneth B. Storey
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Glucose Transport ◽  
Seasonal Changes ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Wood Frog ◽  
Maximal Velocity ◽  
Liver Membranes

One of the critical adaptations for freeze tolerance by the wood frog, Rana sylvatica, is the production of large quantities of glucose as an organ cryoprotectant during freezing exposures. Glucose export from the liver, where it is synthesized, and its uptake by other organs is dependent upon carrier-mediated transport across plasma membranes by glucose-transporter proteins. Seasonal changes in the capacity to transport glucose across plasma membranes were assessed in liver and skeletal muscle of wood frogs; summer-collected (June) frogs were compared with autumn-collected (September) cold-acclimated (5 °C for 3–4 weeks) frogs. Plasma membrane vesicles prepared from liver of autumn-collected frogs showed 6-fold higher rates of carrier-mediated glucose transport than vesicles from summer-collected frogs, maximal velocity (Vmax) values for transport being 72 ± 14 and 12.0 + 2.9 nmol∙mg protein−1∙s−1, respectively (at 10 °C). However, substrate affinity constants for carrier-mediated glucose transport (K1/2) did not change seasonally. The difference in transport rates was due to greater numbers of glucose transporters in liver plasma membranes from autumn-collected frogs. The total number of transporter sites, as determined by cytochalasin B binding, was 8.5-fold higher in autumn than in summer. Glucose transporters in wood frog liver membranes cross-reacted with antibodies to the rat GluT-2 glucose transporter (the mammalian liver isoform), and Western blots further confirmed a large increase in transporter numbers in liver membranes from autumn- versus summer-collected frogs. By contrast with the liver, however, there were no seasonal changes in glucose-transporter activity or numbers in plasma membranes isolated from skeletal muscle. We conclude that an enhanced capacity for glucose transport across liver, but not muscle, plasma membranes during autumn cold-hardening is an important adaptation that anticipates the need for rapid export of cryoprotectant from liver during natural freezing episodes.

scholarly journals Tyrosine phosphorylation of the receptor for insulin-like growth factor II is inhibited in plasma membranes from insulin-treated rat adipocytes

1988 ◽  
Vol 250 (1) ◽  
pp. 47-52 ◽  
Author(s):  
S Corvera ◽  
K A Yagaloff ◽  
R E Whitehead ◽  
M P Czech
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Plasma Membranes ◽  
Inhibitory Effect ◽  
Insulin Like Growth Factor ◽  
Receptor Phosphorylation ◽  
Adipocyte Plasma Membranes ◽  
Triton X ◽  
Soluble Components

Insulin action in intact adipocytes leads to a rapid increase in the concentration of receptors for insulin-like growth factor (IGF) II on the adipocyte cell surface, and to a decrease in the [32P]phosphate content of these receptors on the plasma membrane [Corvera & Czech (1985) Proc. Natl. Acad. Sci. U.S.A. 82. 7314-7318]. It has been previously shown that the receptor for IGF-II can be phosphorylated on tyrosine residues by a kinase activity which is expressed in isolated adipocyte plasma membranes. It is now shown that IGF-II-receptor phosphorylation in vitro, in plasma membranes derived from insulin-treated cells, is markedly decreased compared with the phosphorylation of the receptor in membranes from control cells. This effect of insulin cannot be attributed to an increase in the activity of phosphotyrosyl phosphatase in the membranes. The tyrosine kinase that catalyses the phosphorylation of IGF-II receptors is associated with a fraction of the plasma membrane which is insoluble in Triton X-100. Removal of the Triton X-100-soluble components of the membrane markedly enhances receptor phosphorylation. Moreover, the expression of the inhibitory effect of insulin requires the presence of one or several Triton X-100-soluble components of the plasma membrane.

scholarly journals The distribution of surface antigens during fractionation of mouse liver plasma membranes

1972 ◽  
Vol 130 (1) ◽  
pp. 271-280 ◽  
Author(s):  
James W. Gurd ◽  
W. H. Evans ◽  
Harold R. Perkins
Keyword(s):  
Plasma Membrane ◽  
Sequential Extraction ◽  
Mouse Liver ◽  
Plasma Membranes ◽  
Surface Membrane ◽  
Surface Antigens ◽  
Cross Reactivity ◽  
Antigenic Determinants ◽  
Total Plasma ◽  
Liver Plasma Membranes

1. Antiserum to purified mouse liver plasma membranes was prepared and the partially purified γ-globulin antibody fraction was iodinated with 125I. The reaction of the 125I-labelled γ-globulin antibody with isolated mouse liver plasma membranes was studied. 2. The γglobulin antibody bound specifically to mouse liver plasma membranes and there was little reaction with mouse liver intracellular membranes or with surface-membrane fractions from either rat liver or pig lymphocytes. 3. ‘Light’ and ‘heavy’ mouse liver plasma-membrane subfractions bound similar amounts of γ-globulin antibody, and this is consistent with a surface origin for the light fraction. 5. Plasma membranes were fractionated by sequential extraction with 50mm-NaHCO3–Na2CO3 buffer, pH10.2, containing 10mm-EDTA and aq. 33% (v/v) pyridine. The alkali-soluble and -insoluble fractions and the pyridine-soluble and -insoluble fractions all reacted with the antiserum, and the cross-reactivity among the various fractions and with the total plasma membranes was investigated. 5. The results are discussed in terms of the arrangement of the antigenic determinants within the membrane.

scholarly journals Heterogeneous distribution of enzymes among plasma-membrane fragments sedimenting with the microsomal fraction of rat liver

1974 ◽  
Vol 142 (3) ◽  
pp. 667-671 ◽  
Author(s):  
Kenneth A. Norris ◽  
Miloslav Dobrota ◽  
Faiz S. Issa ◽  
Richard H. Hinton ◽  
Eric Reid
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Heterogeneous Distribution ◽  
Similar Product ◽  
Liver Homogenates

Plasma-membrane fragments recovered in the microsomal fraction of rat liver homogenates were shown to be heterogeneous in density. It was demonstrated that 5′-nucleotidase, the most commonly used plasma-membrane marker, is concentrated in the lightest subfraction. Two of the published procedures for the isolation of plasma-membrane fragments from the microsomal fraction (Touster et al., 1970; Hinton et al., 1971) are shown to give products which are not representative of all the plasma-membrane fragments of microsomal size, and it is argued that a third procedure (House & Weidemann, 1970) is likely to give a similar product.

scholarly journals Isolation of plasma and nuclear membranes of thymocytes. II. Biochemical composition

1978 ◽  
Vol 77 (1) ◽  
pp. 232-245 ◽  
Author(s):  
A Monneron ◽  
J d'Alayer
Keyword(s):  
Plasma Membrane ◽  
Sialic Acid ◽  
Biochemical Composition ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
High Specific Activity ◽  
Live Cells ◽  
Protein Ratio ◽  
Nuclear Membranes

Thymocyte plasma and nuclear membranes obtained by the procedure described in the accompanying paper were analyzed for their biochemical composition. Plasma membranes were very rich in phospholipid, cholesterol, sialic aicd; they did not contain nucleic acids. In comparison, nuclear membranes had a lower phospholipid to protein ratio and contained much less sialic acid and cholesterol. 50% of the cellular cholesterol and of the membrane-bound sialic acid were found in the plasma membranes, 14% in the nuclear membranes. Live cells were labeled with 131I, and the acid-insoluble radioactivity was followed in the subfractions. A good correlation with the distribution and enrichment of plasma membrane market-enzymes was obtained. Label enrichment was about 50-fold in the two lightest of the three plasma membrane fractions. 60% of the label was contained in the plasma membranes, only 4% in the nuclear membranes. Cross-contamination of these two types of membranes was thus negligible. Sodium dodecyl sulfate-gel electrophoresis revealed three different patterns specific for, respectively, plasma membranes, the microsomal-mitochondrial fraction, and nuclear membranes. Each pattern was characterized by a set of proteins and glycoproteins, among which high molecular weight glycoproteins could be considered as marker-proteins of, respectively, 280,000, 260,000, and 230,000 daltons. 131I-labeling of live cells tagged with a very high specific activity three glycoproteins of mol wt 280,000, 200,000, and 135,000 daltons. Nuclear membranes prepared from labeled isolated nuclei had a set of labeled proteins completely different from plasma membranes.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

scholarly journals Hepatic endosome fractions contain an ATP-driven proton pump

1985 ◽  
Vol 225 (1) ◽  
pp. 51-58 ◽  
Author(s):  
T Saermark ◽  
N Flint ◽  
W H Evans
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Atpase Activity ◽  
Proton Pump ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Ph Gradients ◽  
Subcellular Organelle ◽  
Liver Homogenates

Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.

Pore-Spanning Plasma Membranes Derived from Giant Plasma Membrane Vesicles

2021 ◽  
Author(s):  
Nikolas K. Teiwes ◽  
Ingo Mey ◽  
Phila C. Baumann ◽  
Lena Strieker ◽  
Ulla Unkelbach ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles
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