Enzyme distribution in mammalian membranes**The following abbreviations are used: ER, endoplasmic reticulum (excluding the Golgi); PM, plasma membrane (the equivalent term ‘cell membrane’ is not favoured). The usage of the terms ‘cytochemical’ (here restricted to EM studies), ‘microsome’ and ‘microsomal [or mitochondrial, lysosomal, etc.] fraction’ is indicated in the text. Membranes in apposition are best termed ‘paired', not ‘double’. A ‘vesicle’ is a ‘small bladder-like vessel’ or ‘sac’ (Oxford Dictionary). The centrifugal procedure commonly termed ‘differential centrifugation’ is here termed ‘differential pelleting’, in contrast with ‘banding’ or ‘zonal centrifugation’; the latter term does not necessarily imply use of a zonal rotor (Reid, 1972a; Reid and Williamson, 1974).

1976 ◽  
pp. 161-197 ◽  
Author(s):  
R.H. Hinton ◽  
E. Reid
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Membrane ◽  
Differential Centrifugation ◽  
Enzyme Distribution ◽  
Oxford Dictionary ◽  
Zonal Rotor

scholarly journals myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes

1986 ◽  
Vol 236 (1) ◽  
pp. 37-44 ◽  
Author(s):  
D M Delfert ◽  
S Hill ◽  
H A Pershadsingh ◽  
W R Sherman ◽  
J M McDonald
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Initial Rate ◽  
Membrane Vesicles ◽  
Heavy Fraction ◽  
Differential Centrifugation ◽  
Plasma Membrane Vesicles ◽  
Inositol 1,4,5 Trisphosphate ◽  
Uptake And Release

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.

scholarly journals Purification of an inositol 1,4,5-trisphosphate-binding calreticulin-containing intracellular compartment of HL-60 cells

1992 ◽  
Vol 281 (3) ◽  
pp. 651-656 ◽  
Author(s):  
C Van Delden ◽  
C Favre ◽  
A Spät ◽  
E Cerny ◽  
K H Krause ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Storage Protein ◽  
Binding Sites ◽  
Subcellular Fraction ◽  
Sucrose Density Gradient ◽  
Subcellular Fractions ◽  
Differential Centrifugation ◽  
Similar Extent ◽  
Inositol 1,4,5 Trisphosphate

To investigate the identity of Ins(1,4,5)P3-sensitive intracellular Ca2+ stores in myeloid cells, we have developed a method that yields subcellular fractions highly enriched in Ins(1,4,5)P3 binding. HL-60 cells were disrupted by nitrogen cavitation, and subcellular fractions were obtained by differential centrifugation, followed by Percoll- and sucrose-density-gradient separations. A subcellular fraction enriched 26-fold in Ins(1,4,5)P3-binding sites was obtained. This fraction showed no enrichment in plasma-membrane markers and only a comparatively moderate enrichment (7-fold) in endoplasmic-reticulum markers. The ratio between specific enrichment of Ins(1,4,5)P3 binding and endoplasmic-reticulum markers in the different fractions varied over 50-fold, from less than 0.1 to greater than 5. The purified Ins(1,4,5)P3-binding fraction was enriched to a similar extent (27-fold) in the putative intravesicular Ca(2+)-storage protein calreticulin. Our results favour the concept of a distinct Ins(1,4,5)P3-binding, calreticulin-containing compartment (i.e. the calciosome) in HL-60 cells.

scholarly journals Tissue-culture Cell fractionation. Zonal centrifugation of Lettrée cells homogenized by glycerol-induced lysis: subfractionation of membranes in sucrose gradients

1979 ◽  
Vol 182 (1) ◽  
pp. 165-171
Author(s):  
J M Graham ◽  
K H M Coffey
Keyword(s):  
Tissue Culture ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Cell Fractionation ◽  
Early Passage ◽  
Late Passage ◽  
Passage Cells ◽  
Zonal Rotor

1. Lettrée cells were grown intraperitoneally in MF-1 mice. 2. Cells that were loaded with glycerol were swollen in 0.1 M-sucrose and disrupted by Dounce homogenization. 3. Early-passage Lettrée cells were more easily disrupted than late-passage cells by this method, and the former produced larger fragments of plasma membrane. 4. The membranes were fractionated initially in sucrose gradients (on the basis of sedimentation rate) in a BXIV zonal rotor. 5. Fractions from this gradient were further resolved in isopycnic sucrose gradients. 6. Plasma-membrane and endoplasmic-reticulum fractions were recovered in good yield and high purity.

scholarly journals Tissue-culture cell fractionation. Fractionation of cellular membranes from 125I/lactoperoxidase-labelled Lettrée cells homogenized by bicarbonate-induced lysis: resolution of membranes by zonal centrifugation and in sucrose and metrizamide gradients

1979 ◽  
Vol 182 (1) ◽  
pp. 173-180 ◽  
Author(s):  
J M Graham ◽  
K H M Coffey
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Adenosine Triphosphatase ◽  
Gradient Centrifugation ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Cell Fractionation ◽  
Rate Gradient ◽  
Nadph Cytochrome C Reductase ◽  
Zonal Rotor

1. Lettrée cells were grown intraperitoneally in MF-1 mice and labelled extrinsically by the 125I/lactoperoxidase technique. 2. The cells were swollen in 1 mM-NaHCO3 and disrupted in a Dounce homogenizer. 3. Crude fractions of endoplasmic reticulum, plasma membrane and mitochondria were separated from a post-nuclear supernatant by sedimentation-rate gradient centrifugation in a BXIV zonal rotor. 4. Further resolution of these membranes was carried out in isopycnic sucrose gradients. 5. Bands of material from the latter were subfractionated in gradients of metrizamide. Some very pure subfractions of plasma membrane and endoplasmic reticulum were obtained. In addition, one subfraction containing 125I and NADPH-cytochrome c reductase but no Na++K+-stimulated adenosine triphosphatase and another containing these two enzymes but no 125I were resolved.

Triple Fixation For Electron Microscopy

1968 ◽  
Vol 26 ◽  
pp. 90-91 ◽  
Author(s):  
M. A. Hayat
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Beam ◽  
Plasma Membrane ◽  
Myelin Sheath ◽  
Good Deal ◽  
Granular Structure ◽  
Oxidizing Agent ◽  
Fixation Technique ◽  
Large Clusters

Potassium permanganate has been successfully employed to study membranous structures such as endoplasmic reticulum, Golgi, plastids, plasma membrane and myelin sheath. Since KMnO4 is a strong oxidizing agent, deposition of manganese or its oxides account for some of the observed contrast in the lipoprotein membranes, but a good deal of it is due to the removal of background proteins either by dehydration agents or by volatalization under the electron beam. Tissues fixed with KMnO4 exhibit somewhat granular structure because of the deposition of large clusters of stain molecules. The gross arrangement of membranes can also be modified. Since the aim of a good fixation technique is to preserve satisfactorily the cell as a whole and not the best preservation of only a small part of it, a combination of a mixture of glutaraldehyde and acrolein to obtain general preservation and KMnO4 to enhance contrast was employed to fix plant embryos, green algae and fungi.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

scholarly journals Septin-dependent compartmentalization of the endoplasmic reticulum during yeast polarized growth

2005 ◽  
Vol 169 (6) ◽  
pp. 897-908 ◽  
Author(s):  
Cosima Luedeke ◽  
Stéphanie Buvelot Frei ◽  
Ivo Sbalzarini ◽  
Heinz Schwarz ◽  
Anne Spang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Diffusion Barriers ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Protein Diffusion ◽  
Ultrastructural Studies ◽  
Bud Neck ◽  
Er Membrane

Polarized cells frequently use diffusion barriers to separate plasma membrane domains. It is unknown whether diffusion barriers also compartmentalize intracellular organelles. We used photobleaching techniques to characterize protein diffusion in the yeast endoplasmic reticulum (ER). Although a soluble protein diffused rapidly throughout the ER lumen, diffusion of ER membrane proteins was restricted at the bud neck. Ultrastructural studies and fluorescence microscopy revealed the presence of a ring of smooth ER at the bud neck. This ER domain and the restriction of diffusion for ER membrane proteins through the bud neck depended on septin function. The membrane-associated protein Bud6 localized to the bud neck in a septin-dependent manner and was required to restrict the diffusion of ER membrane proteins. Our results indicate that Bud6 acts downstream of septins to assemble a fence in the ER membrane at the bud neck. Thus, in polarized yeast cells, diffusion barriers compartmentalize the ER and the plasma membrane along parallel lines.

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