scholarly journals Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment.

1995 ◽  
Vol 6 (7) ◽  
pp. 929-944 ◽  
Author(s):  
S Mayor ◽  
F R Maxfield
Keyword(s):  
Cell Surface ◽  
Tyrosine Kinases ◽  
Plasma Membranes ◽  
Cytoplasmic Proteins ◽  
Nonionic Detergent ◽  
Detergent Treatment ◽  
Carboxy Terminal ◽  
Almost All ◽  
Membrane Anchoring ◽  
Triton X

A diverse set of cell surface eukaryotic proteins including receptors, enzymes, and adhesion molecules have a glycosylphosphoinositol-lipid (GPI) modification at the carboxy-terminal end that serves as their sole means of membrane anchoring. These GPI-anchored proteins are poorly solubilized in nonionic detergent such as Triton X-100. In addition these detergent-insoluble complexes from plasma membranes are significantly enriched in several cytoplasmic proteins including nonreceptor-type tyrosine kinases and caveolin/VIP-21, a component of the striated coat of caveolae. These observations have suggested that the detergent-insoluble complexes represent purified caveolar membrane preparations. However, we have recently shown by immunofluorescence and electron microscopy that GPI-anchored proteins are diffusely distributed at the cell surface but may be enriched in caveolae only after cross-linking. Although caveolae occupy only a small fraction of the cell surface (< 4%), almost all of the GPI-anchored protein at the cell surface becomes incorporated into detergent-insoluble low-density complexes. In this paper we show that upon detergent treatment the GPI-anchored proteins are redistributed into a significantly more clustered distribution in the remaining membranous structures. These results show that GPI-anchored proteins are intrinsically detergent-insoluble in the milieu of the plasma membrane, and their co-purification with caveolin is not reflective of their native distribution. These results also indicate that the association of caveolae, GPI-anchored proteins, and signalling proteins must be critically re-examined.

scholarly journals Subcellular location and properties of bactericidal factors from human neutrophils.

1986 ◽  
Vol 164 (5) ◽  
pp. 1407-1421 ◽  
Author(s):  
J E Gabay ◽  
J M Heiple ◽  
Z A Cohn ◽  
C F Nathan
Keyword(s):  
Protein S ◽  
Subcellular Location ◽  
Inhibitory Effect ◽  
Human Neutrophils ◽  
E Coli ◽  
Fractionation Scheme ◽  
Percoll Gradients ◽  
Nonionic Detergent ◽  
Almost All ◽  
Triton X

We examined the subcellular location of bactericidal factors (BF) in human neutrophils, using an efficient fractionation scheme. Nitrogen bomb cavitates of DIFP-treated PMN were centrifuged through discontinuous Percoll gradients, each fraction extracted with 0.05 M glycine, pH 2.0, and tested for the killing of Escherichia coli. greater than 90% of BF coisolated with the azurophil granules. After lysis of azurophils, 98% of azurophil-derived BF (ADBF) sedimented with the membrane. ADBF activity was solubilized from azurophil membrane with either acid or nonionic detergent (Triton X-100, Triton X-114). Bactericidal activity was linear with respect to protein concentration over the range 0.3-30 micrograms/ml. 0.1-0.3 microgram/ml ADBF killed 10(5) E. coli within 30 min at 37 degrees C. At 1.4 micrograms/ml, 50% of 2 X 10(5) bacteria were killed within 5 min. ADBF was effective between pH 5-8, with peak activity at pH 5.5. Glucose (20 mM), EDTA (1-25 mM), and physiologic concentrations of NaCl or KCl had little or no inhibitory effect on ADBF. ADBF killed both Gram-positive and Gram-negative virulent clinical isolates, including listeria, staphylococci, beta-hemolytic streptococci, and Pseudomonas aeruginosa. Thus, under these conditions of cell disruption, fractionation, extraction, and assay, almost all BF in human PMN appeared to be localized to the membrane of azurophilic granules as a highly potent, broad-spectrum, rapidly acting protein(s) effective in physiologic medium. Some of these properties appear to distinguish ADBF from previously described PMN bactericidal proteins.

scholarly journals Crystallization of the light-harvesting chlorophyll a/b complex within thylakoid membranes.

1985 ◽  
Vol 100 (4) ◽  
pp. 1139-1147 ◽  
Author(s):  
M K Lyon ◽  
K R Miller
Keyword(s):  
Photosystem I ◽  
Crystallization Process ◽  
Light Harvesting ◽  
Coupling Factor ◽  
Membrane Bilayer ◽  
Lhc Ii ◽  
Ps Ii ◽  
Nonionic Detergent ◽  
Detergent Treatment ◽  
Triton X

We have found that treatment of the photosynthetic membranes of green plants, or thylakoids, with the nonionic detergent Triton X-114 at a 10:1 ratio has three effects: (a) photosystem I and coupling factor are solubilized, so that the membranes retain only photosystem II (PS II) and its associated light-harvesting apparatus (LHC-II); (b) LHC-II is crystallized, and so is removed from its normal association with PS II; and (c) LHC-II crystallization causes a characteristic red shift in the 77 degrees K fluorescence from LHC-II. Treatment of thylakoids with the same detergent at a 20:1 ratio results in an equivalent loss of photosystem I and coupling factor, with LHC-II and PS II being retained by the membranes. However, no LHC-II crystals are formed, nor is there a shift in fluorescence. Thus, isolation of a membrane protein is not required for its crystallization, but the conditions of detergent treatment are critical. Membranes with crystallized LHC-II retain tetrameric particles on their surface but have no recognizable stromal fracture face. We have proposed a model to explain these results: LHC-II is normally found within the stromal half of the membrane bilayer and is reoriented during the crystallization process. This reorientation causes the specific fluorescence changes associated with crystallization. Tetrameric particles, which are not changed in any way by the crystallization process, do not consist of LHC-II complexes. PS II appears to be the only other major complex retained by these membranes, which suggests that the tetramers consist of PS II.

scholarly journals Estimation of S-phase fraction in tumor tissue sections by immunohistochemical staining of PCNA.

1994 ◽  
Vol 42 (7) ◽  
pp. 957-960 ◽  
Author(s):  
K Sasaki ◽  
A Kurose ◽  
Y Ishida ◽  
M Matsuta
Keyword(s):  
Hela Cells ◽  
Immunohistochemical Staining ◽  
Tumor Tissue ◽  
Human Tumor ◽  
S Phase ◽  
Phase Fraction ◽  
Tissue Sections ◽  
Detergent Treatment ◽  
Almost All ◽  
Triton X

We describe a method for measuring the size of the S-phase fraction in human tumor tissue sections using an antibody to PCNA (PC10). Although treatment with Triton X-100 before fixation extracted a large amount of PCNA from the cells even in frozen tissue sections, PCNA remained exclusively in S-phase cells. Immunohistochemical staining of PCNA after the detergent treatment allowed estimation of the S-phase fraction in solid tumors. The validity of the method was directly proven by double staining of bromodeoxyuridine (BrdU) and PCNA in HeLa cells. The PCNA-positive cells were identical with BrdUrd-positive cells after the detergent treatment. In contrast, almost all HeLa cells in the exponentially growing phase were positive for PC10 without treatment with Triton X-100.

scholarly journals ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons.

1994 ◽  
Vol 126 (2) ◽  
pp. 391-401 ◽  
Author(s):  
S Tsukita ◽  
K Oishi ◽  
N Sato ◽  
J Sagara ◽  
A Kawai ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Family Members ◽  
Integral Membrane Protein ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Bhk Cells ◽  
Triton X

The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin, as well as moesin, are associated with CD44, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.

scholarly journals The arginine-rich carboxy-terminal domain of the hepatitis B virus core protein mediates attachment of nucleocapsids to cell-surface-expressed heparan sulfate

2005 ◽  
Vol 86 (1) ◽  
pp. 75-84 ◽  
Author(s):  
Peter Vanlandschoot ◽  
Freya Van Houtte ◽  
Benedikte Serruys ◽  
Geert Leroux-Roels
Keyword(s):  
Hepatitis B Virus ◽  
B Cells ◽  
Hepatitis B ◽  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Membranes ◽  
Core Proteins ◽  
B Virus ◽  
Carboxy Terminal

Binding of hepatitis B virus nucleocapsids to mouse B cells leads to production of nucleocapsid-specific antibodies, class II presentation of peptides and the generation of T helper-1 immunity. This T-cell-independent activation of B cells is thought to result from cross-linking of cell-surface immunoglobulin molecules, if these contain a specific motif in the framework region 1–complementarity determining region 1 junction. In the present study, it was observed that nucleocapsids bound to different B-cell lines, an interaction that was not dependent on cell-surface-expressed immunoglobulins. Furthermore, binding to several non-B-cell lines was observed. Capsids that lacked the carboxy-terminal protamine-like domains did not bind to cells. Treatment of nucleocapsids with ribonucleases enhanced the attachment of nucleocapsids to cells. Various soluble glycosaminoglycans inhibited attachment of nucleocapsids, while treatment of cells with heparinase I also reduced binding. These observations demonstrated that the arginine-rich protamine-like regions of the core proteins are responsible for the attachment of nucleocapsids to glycosaminoglycans expressed on the plasma membranes of cells.

scholarly journals Inactivation and Removal of Chikungunya Virus and Mayaro Virus from Plasma-derived Medicinal Products

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 234 ◽  
Author(s):  
Constanze Yue ◽  
Sebastian Teitz ◽  
Tomoyuki Miyabashi ◽  
Klaus Boller ◽  
Lia Lewis-Ximenez ◽  
...  
Keyword(s):  
Chikungunya Virus ◽  
Febrile Illness ◽  
Heat Inactivation ◽  
Medicinal Products ◽  
Emerging Viruses ◽  
Viral Inactivation ◽  
Detergent Treatment ◽  
Mayaro Virus ◽  
Almost All ◽  
Product Industry

Background: Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related members of the Semliki Forest complex within the genus alphavirus and are transmitted by arthropods, causing acute febrile illness in humans. CHIKV has spread to almost all continents, whereas autochthonous MAYV infections have been reported in South America and in the Caribbean. Nevertheless, there was concern about potential spread of MAYV to other regions similar to CHIKV in the past. The risk for transmission of emerging viruses by blood transfusion and the safety of plasma-derived medicinal products (PDMPs) are constant concerns. The manufacturing processes of PDMPs include procedures to inactivate/remove viruses. Methods: In this study, we investigated the reduction of MAYV and CHIKV by heat inactivation in various matrices, solvent/detergent treatment and nanofiltration. Results: Unexpectedly, MAYV was significantly more resistant to heat and solvent/detergent treatment compared to CHIKV. However, being similar in size, both MAYV and CHIKV were removed below the detection limit by 35 nm virus filters. Conclusions: The inactivation profiles of different alphavirus members vary considerably, even within the Semliki Forest Complex. However, robust dedicated viral inactivation/removal procedures commonly used in the plasma product industry are effective in inactivating or removing MAYV and CHIKV.

scholarly journals Globular and asymmetric acetylcholinesterase in frog muscle basal lamina sheaths.

1986 ◽  
Vol 102 (3) ◽  
pp. 762-768 ◽  
Author(s):  
M Nicolet ◽  
M Pinçon-Raymond ◽  
F Rieger
Keyword(s):  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Muscle Fibers ◽  
Frog Muscle ◽  
Motor Endplate ◽  
Molecular Forms ◽  
Nonionic Detergent ◽  
Plasmic Membrane ◽  
Triton X

After denervation in vivo, the frog cutaneus pectoris muscle can be led to degenerate by sectioning the muscle fibers on both sides of the region rich in motor endplate, leaving, 2 wk later, a muscle bridge containing the basal lamina (BL) sheaths of the muscle fibers (28). This preparation still contains various tissue remnants and some acetylcholine receptor-containing membranes. A further mild extraction by Triton X-100, a nonionic detergent, gives a pure BL sheath preparation, devoid of acetylcholine receptors. At the electron microscope level, this latter preparation is essentially composed of the muscle BL with no attached plasmic membrane and cellular component originating from Schwann cells or macrophages. Acetylcholinesterase is still present in high amounts in this BL sheath preparation. In both preparations, five major molecular forms (18, 14, 11, 6, and 3.5 S) can be identified that have either an asymmetric or a globular character. Their relative amount is found to be very similar in the BL and in the motor endplate-rich region of control muscle. Thus, observations show that all acetylcholinesterase forms can be accumulated in frog muscle BL.

scholarly journals Fluorescent antibodies and lectins stain intracellular structures in fixed cells treated with nonionic detergent.

1978 ◽  
Vol 26 (4) ◽  
pp. 251-257 ◽  
Author(s):  
P Laurila ◽  
I Virtanen ◽  
J Wartiovaara ◽  
S Stenman
Keyword(s):  
Binding Sites ◽  
Human Fibroblasts ◽  
T Antigen ◽  
Similar Distribution ◽  
Sv40 T Antigen ◽  
Con A ◽  
Acetone Treatment ◽  
Nonionic Detergent ◽  
Detergent Treatment ◽  
Scanning Electron

Nonionic detergent (NP40) treatment of paraformaldehyde-fixed normal and SV40-transformed human fibroblasts resulted in intracellular penetration of two chosen fluorescent antibodies and Concanavalin A (Con A). After the detergent treatment nuclear SV40 T antigen, cytoplasmic fibronectin glycoprotein and Con A binding sites could be visualized in fluorescence microscopy. The lowest NP40 concentration which made fixed cells permeable was 0.05%. The morphology of cells was preserved better by this new method than by conventional fixation methods, such as acetone treatment. In scanning electron microscopy the surface of the fixed NP40-treated cells had only small rugosities and fine pores. The subsurface cytoskeleton especially was well preserved and had a more distinct fine structure. The improved morphology made it possible to detect a similar distribution of fibronectin and Con A binding sites in the perinuclear endoplasmic reticulum regions.

scholarly journals The roles of phospholipase D and a GTP-binding protein in guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes

1990 ◽  
Vol 272 (3) ◽  
pp. 749-753 ◽  
Author(s):  
K M Hurst ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Rat Liver ◽  
Phospholipase D ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Gtp Binding ◽  
Liver Plasma Membranes ◽  
Low Concentrations ◽  
Rat Liver Plasma Membranes ◽  
Triton X ◽  
Hydrolysis Of

1. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5′-[beta gamma-imido]triphosphate and guanosine 5′-[alpha beta-methylene]triphosphate, but not adenosine 5′-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5′-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.

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