scholarly journals Estimation of the free [Ca2+] gradient across endoplasmic reticulum membranes by a null-point method

1995 ◽  
Vol 310 (2) ◽  
pp. 371-374 ◽  
Author(s):  
A P Dawson ◽  
G T Rich ◽  
J W Loomis-Husselbee
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
External Medium ◽  
Microsomal Protein ◽  
Null Point ◽  
Similar Technique ◽  
Electrochemical Gradient ◽  
Efflux Rate ◽  
L1210 Cells ◽  
A Value

The rate of unidirectional efflux of 45Ca from rat liver microsomal vesicles loaded with 45Ca and then treated with thapsigargin is not inhibited by increased [Ca2+] in the external medium, although the net efflux rate is substantially inhibited. We have used this property to measure the electrochemical gradient of Ca2+ from the inside to the outside of the vesicles at a series of Ca2+ loadings, by measuring the external [Ca2+]free at which there is zero net efflux. At a loading of 7.9 +/- 0.6 nmol/mg of microsomal protein, the apparent internal [Ca2+]free is 21 +/- 1.6 microM. As the loading is increased, the internal [Ca2+]free increases linearly up to a value of 47 +/- 3.6 microM at a loading of 21.6 +/- 1.6 nmol/mg. Using a similar technique, the value for [Ca2+]free in the endoplasmic reticulum of permeabilized L1210 cells was found to be 12.5 microM.

scholarly journals The latency of rat liver microsomal protein disulphide-isomerase

1985 ◽  
Vol 228 (3) ◽  
pp. 635-645 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
High Speed ◽  
Microsomal Fraction ◽  
Membrane Integrity ◽  
Microsomal Protein ◽  
Permeability Barrier ◽  
Rat Liver Microsomes ◽  
Luminal Surface ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase (PDI) activity was not detectable in freshly prepared rat liver microsomes (microsomal fraction), but became detectable after treatments that damage membrane integrity, e.g. sonication, detergent treatment or freezing and thawing. Maximum activity was detectable after sonication. Identical latency was observed in microsomes prepared by gel filtration and in those prepared by high-speed centrifugation. PDI activity was latent in all particulate subcellular fractions, but not latent in the high-speed supernatant. When all fractions were sonicated to expose total PDI activity, PDI was found at highest specific activity in the microsomal fraction and co-distributed with marker enzymes of the endoplasmic reticulum. Washing of microsomes under various conditions that removed peripheral proteins and, in some cases, bound ribosomes did not remove significant quantities of PDI, nor did it affect the latency of PDI activity. Treatment of microsomes with proteinases, under conditions where the permeability barrier of the microsomal vesicles was maintained intact, did not inactivate PDI significantly or affect its latency. PDI was very readily solubilized from microsomal vesicles by low concentrations of detergents, which removed only a fraction of the total microsomal protein. In all these respects, PDI resembled nucleoside diphosphatase, a marker peripheral protein of the luminal surface of the endoplasmic reticulum, and differed from NADPH: cytochrome c reductase, a marker integral protein exposed at the cytoplasmic surface of the membrane. The data are compatible with a model in which PDI is loosely associated with the luminal surface of the endoplasmic reticulum, a location consistent with the proposed physiological role of the enzyme as catalyst of formation of native disulphide bonds in nascent and newly synthesized secretory proteins.

scholarly journals The long-chain sphingoid base of sphingolipids is acylated at the cytosolic surface of the endoplasmic reticulum in rat liver

1993 ◽  
Vol 290 (3) ◽  
pp. 751-757 ◽  
Author(s):  
K Hirschberg ◽  
J Rodger ◽  
A H Futerman
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Microsomal Protein ◽  
Fatty Acyl ◽  
Sphingolipid Metabolism ◽  
Free System ◽  
Protease Digestion ◽  
Liver Homogenates ◽  
Endogenous Activity

Ceramide, a key intermediate in sphingolipid metabolism, is synthesized by acylation of sphinganine followed by dehydrogenation of dihydroceramide to ceramide. Using radioactive sphinganine, we have examined the site and topology of dihydroceramide synthesis in well-characterized subcellular fractions from rat liver. [4,5-3H]Sphinganine was introduced as a complex with BSA and was metabolized to [4,5-3H]dihydroceramide upon incubation of rat liver homogenates or microsomes with fatty acyl CoA. Conditions were established in a detergent-free system in which dihydroceramide synthesis was not limited by either substrate availability or by amounts of microsomal protein or reaction time. The distribution of dihydroceramide synthesis was found to exactly parallel that of an endoplasmic reticulum (ER) marker upon subfractionation of microsomes, and no endogenous activity was detected in either purified Golgi apparatus or plasma membrane fractions. Limited protease digestion demonstrated that sphinganine N-acyltransferase is localized at the cytosolic surface of intact ER-derived vesicles. These results are discussed with regard to the subsequent transport of (dihydro)-ceramide from the ER to sites of further metabolism in a pre-Golgi apparatus compartment and in the cis and medial cisternae of the Golgi apparatus.

scholarly journals Release of calcium from the endoplasmic reticulum by bile acids in rat liver cells.

1988 ◽  
Vol 263 (5) ◽  
pp. 2299-2303 ◽  
Author(s):  
L Combettes ◽  
M Dumont ◽  
B Berthon ◽  
S Erlinger ◽  
M Claret
Keyword(s):  
Endoplasmic Reticulum ◽  
Bile Acids ◽  
Rat Liver ◽  
Liver Cells ◽  
Rat Liver Cells

scholarly journals Effects of taurolithocholate, a Ca2+-mobilizing agent, on cell Ca2+ in rat hepatocytes, human platelets and neuroblastoma NG108-15 cell line

1991 ◽  
Vol 273 (1) ◽  
pp. 153-160 ◽  
Author(s):  
J F Coquil ◽  
B Berthon ◽  
N Chomiki ◽  
L Combettes ◽  
P Jourdon ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Bile Acid ◽  
Cell Line ◽  
Rat Liver ◽  
Neuronal Cell ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Liver Cells ◽  
Human Platelets ◽  
Rat Liver Cells

The monohydroxy bile acid taurolithocholate permeabilizes the endoplasmic reticulum to Ca2+ in rat liver cells. To assess whether this action on the endoplasmic reticulum was restricted to this tissue, the effects of bile acid were investigated in two cell types quite unrelated to rat hepatocyte, namely human platelets and neuronal NG108-15 cell line. The results showed that taurolithocholate (3-100 microM) had no effect on free cytosolic [Ca2+] in human platelets and NG108-15 cells. whereas it increased it from 180 to 520 nM in rat hepatocytes. In contrast, in cells permeabilized by saponin, taurolithocholate initiated a profound release of the stored Ca2+ from the internal Ca2+ pools in the three cell types. The bile acid released 90% of the Ca2+ pools, with rate constants of about 5 min-1 and half-maximal effects at 15-30 microM. The results also showed that, in contrast with liver cells, which displayed an influx of [14C]taurolithocholate of 2 nmol/min per mg, human platelets and the neuronal cell line appeared to be resistant to [14C]taurolithocholate uptake. The influx measured in these latter cells was about 100-fold lower than in rat liver cells. Taken together, these data suggest that human platelets and NG108-15 cells do not possess the transport system for concentrating monohydroxy bile acids into cells. However, they show that human platelets and neuronal NG108-15 possess, in common with liver cells, the intracellular system responsible for taurolithocholate-mediated Ca2+ release from internal stores.

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