Biochemical approaches to the study of cytosolic calcium regulation in nerve endings

1981 ◽  
Vol 296 (1080) ◽  
pp. 115-122 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Steady State ◽  
Nerve Ending ◽  
Cytosolic Calcium ◽  
Endoplasmic Reticulum Membrane ◽  
Mitochondrial Inner Membrane ◽  
Voltage Dependent ◽  
Minor Significance

The nerve ending cytosol is bounded by the plasma membrane, the mitochondrial inner membrane and the endoplasmic reticulum membrane, transport across each of which is capable, in theory, of regulating the cytosolic free Ca 2+ concentration. By parallel monitoring of mitochondrial and plasma membrane potentials, ATP levels, Na + gradients and intrasynaptosomal Ca 2+ distribution in preparations of isolated synaptosomes, we conclude the following: ( a ) mitochondria in situ represent a major Ca 2+ pool, regulating the upper steady-state limit of the cytosolic free Ca 2+ concentration by sequestering Ca 2+ reversibly; ( b ) this limit is responsive to the cytosolic Na + concentration, but is below the concentration required for significant exocytosis; ( c ) plasma membrane Ca 2+ transport can be resolved into a constant slow influx, a voltage-dependent and verapamil-sensitive influx and an ATP-dependent efflux, while Ca 2+ efflux driven by the sodium electrochemical potential cannot be detected; ( d ) Ca 2+ regulation by intrasynaptosomal endoplasmic reticulum appears to be of minor significance in the present preparation.

scholarly journals Calcium efflux and cycling across the synaptosomal plasma membrane

1985 ◽  
Vol 226 (1) ◽  
pp. 225-231 ◽  
Author(s):  
R Snelling ◽  
D Nicholls
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Potential Gradient ◽  
Electrochemical Potential ◽  
Electrochemical Gradient ◽  
Calcium Efflux ◽  
Normal Medium ◽  
Voltage Dependent ◽  
Synaptosomal Plasma Membrane ◽  
Ca2 Channels

Ca2+ efflux from intact synaptosomes is investigated. Net efflux can be induced by returning synaptosomes from media with elevated Ca2+ or high pH to a normal medium. Net Ca2+ efflux is accelerated when the Na+ electrochemical potential gradient is collapsed by veratridine plus ouabain. Under steady-state conditions at 30 degrees C, Ca2+ cycles across the plasma membrane at 0.38 nmol . min-1 . mg-1 of protein. Exchange is increased by 145% by veratridine plus ouabain, both influx and efflux being increased. Increased influx is probably due to activation of voltage-dependent Ca2+ channels, since it is abolished by verapamil. The results indicate that, at least under conditions of low Na+ electrochemical gradient, some pathway other than a Na+/Ca2+ exchange must operate in the plasma membrane to expel Ca2+.

In Situ Topology of Cytochrome b5 in the Endoplasmic Reticulum Membrane

1996 ◽  
Vol 120 (4) ◽  
pp. 828-833 ◽  
Author(s):  
R. Kuroda ◽  
J.-y. Kinoshita ◽  
M. Honsho ◽  
J.-y. Mitoma ◽  
A. Ito
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome B5 ◽  
Endoplasmic Reticulum Membrane

scholarly journals The Arabidopsis AtGCD3 protein is a glucosylceramidase that preferentially hydrolyzes long-acyl-chain glucosylceramides

2019 ◽  
Vol 295 (3) ◽  
pp. 717-728 ◽  
Author(s):  
Guang-Yi Dai ◽  
Jian Yin ◽  
Kai-En Li ◽  
Ding-Kang Chen ◽  
Zhe Liu ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Fusion Proteins ◽  
Biosynthetic Pathway ◽  
Acyl Chain ◽  
Specific Inhibitor ◽  
Endoplasmic Reticulum Membrane ◽  
Growth Defects ◽  
The Common

Cellular membranes contain many lipids, some of which, such as sphingolipids, have important structural and signaling functions. The common sphingolipid glucosylceramide (GlcCer) is present in plants, fungi, and animals. As a major plant sphingolipid, GlcCer is involved in the formation of lipid microdomains, and the regulation of GlcCer is key for acclimation to stress. Although the GlcCer biosynthetic pathway has been elucidated, little is known about GlcCer catabolism, and a plant GlcCer-degrading enzyme (glucosylceramidase (GCD)) has yet to be identified. Here, we identified AtGCD3, one of four Arabidopsis thaliana homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant AtGCD3 has a low Km for the fluorescent lipid C6-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from Arabidopsis leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human β-glucosidase 2, N-butyldeoxynojirimycin, inhibits AtGCD3 more effectively than does a specific inhibitor of human β-glucosidase 1, conduritol β-epoxide. We also found that Glu-499 and Asp-647 in AtGCD3 are vital for GCD activity. GFP-AtGCD3 fusion proteins mainly localized to the plasma membrane or the endoplasmic reticulum membrane. No obvious growth defects or changes in sphingolipid contents were observed in gcd3 mutants. Our results indicate that AtGCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.

scholarly journals Subcellular localization of inositol lipids in blood platelets as deduced from the use of labelled precursors

1987 ◽  
Vol 244 (3) ◽  
pp. 757-761 ◽  
Author(s):  
G Mauco ◽  
P Dajeans ◽  
H Chap ◽  
L Douste-Blazy
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Blood Platelets ◽  
Blood Platelet ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Inositol Lipids ◽  
Hormone Sensitive ◽  
Reported Data ◽  
Phosphatidylinositol 4

1. By rapid fractionation of blood platelet lysates on Percoll density gradients at alkaline pH (9.6), a very pure plasma-membrane fraction was obtained, as well as discrimination between endoplasmic reticulum and lysosomes. 2. Labelling of intact platelets with [32P]Pi followed by subcellular fractionation showed an exclusive localization of all inositol lipids in the plasma membrane. 3. Preincubation of whole platelets with myo-[3H]inositol in a buffer containing 1 mM-MnCl2 allowed incorporation of the label into PtdIns (phosphatidylinositol) of both plasma and endoplasmic-reticulum membrane, whereas [3H]PtdIns4P (phosphatidylinositol 4-phosphate) and [3H]PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) were exclusively found on the plasma membrane. 4. It is concluded that PtdIns4P and PtdIns(4,5)P2 are exclusively localized in the plasma membrane, whereas PtdIns is present in both plasma and endoplasmic-reticulum membranes. This could provide an explanation for previously reported data on hormone-sensitive and -insensitive inositol lipid pools.

Protein translocation through the Sec61/SecY channel

2010 ◽  
Vol 30 (3) ◽  
pp. 201-207 ◽  
Author(s):  
Zhiliang Cheng
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Lipid Bilayer ◽  
Protein Translocation ◽  
Biological Membranes ◽  
Structural Data ◽  
Endoplasmic Reticulum Membrane ◽  
Primary Sequence ◽  
Membrane Channel ◽  
Final Destination

Special codes are embedded in the primary sequence of newly synthesized proteins to determine their final destination. Protein translocation across biological membranes requires co-operation between the targeting and translocation machineries. A conserved membrane channel, the Sec61/SecY complex, mediates protein translocation across or integration into the endoplasmic reticulum membrane in eukaryotes and the plasma membrane in prokaryotes. A combination of recent biochemical and structural data provides novel insights into the mechanism of how the channel allows polypeptide movement into the exoplasmic space and the lipid bilayer.

scholarly journals A comparative study of the molecular structures of the plasma membranes and the smooth and the rough endoplasmic-reticulum membranes from rat liver

1972 ◽  
Vol 129 (3) ◽  
pp. 781-788 ◽  
Author(s):  
F. Morin ◽  
S. Tay ◽  
H. Simpkins
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Structural Changes ◽  
Molecular Structures ◽  
Endoplasmic Reticulum Membrane ◽  
Marker Enzyme ◽  
Membrane Phospholipids

Plasma-membrane as well as smooth-, rough- and degranulated-endoplasmic-reticulum-membrane fractions were isolated from the microsomal pellet of rat liver. The purity of these fractions, as determined by marker-enzyme activities, electron microscopy, cholesterol content and RNA content, was found to be adequate for a comparative structural study. Major differences in lipid and protein composition were found to exist between the plasma membrane and the endoplasmic reticulum, but not between the smooth and the rough fractions of the endoplasmic reticulum. Differences in the location of membrane protein thiol groups and the mobility of the membrane phospholipids were observed between the plasma membranes and the endoplasmic reticulum, and these could be explained by differences in protein and lipid composition. However, by employing fluorescence and spin-labelling techniques structural changes were also observed between the smooth and the rough endoplasmic-reticulum fractions. These results suggest that the structural heterogeneity existing between the two latter membrane fractions occurs near or on their membrane surfaces and is not due to the greater number of ribosomes bound to the rough endoplasmic-reticulum fraction.

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