Both translocon and a cation channel are involved in the passive Ca2+ leak from the endoplasmic reticulum: A mechanistic study on rat liver microsomes

2007 ◽  
Vol 462 (1) ◽  
pp. 115-121 ◽  
Author(s):  
Roberta Giunti ◽  
Alessandra Gamberucci ◽  
Rosella Fulceri ◽  
Gábor Bánhegyi ◽  
Angelo Benedetti
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Cation Channel ◽  
Mechanistic Study ◽  
Rat Liver Microsomes

scholarly journals Evidence that β-hydroxyacyl-CoA dehydrase purified from rat liver microsomes is of peroxisomal origin

1992 ◽  
Vol 287 (1) ◽  
pp. 91-100 ◽  
Author(s):  
L Cook ◽  
M N Nagi ◽  
S K Suneja ◽  
A R Hand ◽  
D L Cinti
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Competitive Inhibition ◽  
Liver Microsomes ◽  
Carbon Chain ◽  
Enzymic Activity ◽  
Chain Elongation ◽  
Rat Liver Microsomes ◽  
Ph Optimum ◽  
Fatty Acid Chain

The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC 4.2.1.17), believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase.

scholarly journals 10-Undecynoic acid, an inhibitor of cytochrome P450 4A1, inhibits ethanolamine-specific phospholipid base exchange reaction in rat liver microsomes.

1999 ◽  
Vol 46 (1) ◽  
pp. 203-210 ◽  
Author(s):  
J Lenart ◽  
S Pikuła
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome P450 ◽  
Exchange Reaction ◽  
Rat Liver ◽  
Lauric Acid ◽  
Liver Microsomes ◽  
Specific Inhibitor ◽  
Rat Liver Microsomes ◽  
Dependent Manner ◽  
Base Exchange

1,12-Dodecanedioic acid, the end-product of omega-hydroxylation of lauric acid, stimulates in a concentration dependent manner, phosphatidylethanolamine synthesis via ethanolamine-specific phospholipid base exchange reaction in rat liver endoplasmic reticulum. On the other hand, administration to rats of 10-undecynoic acid, a specific inhibitor of omega-hydroxylation reaction catalyzed by cytochrome P450 4A1, inhibits the ethanolamine-specific phospholipid base exchange activity by 30%. This is accompanied by a small but significant decrease in phosphatidylethanolamine content in the endoplasmic reticulum and inhibition of cytochrome P450 4A1. On the basis of these results it can be proposed that a functional relationship between cytochrome P450 4A1 and phosphatidylethanolamine synthesis exists in rat liver. Cytochrome P450 4A1 modulates the cellular level of lauric acid, an inhibitor of phospholipid synthesis. In turn, ethanolamine-specific phospholipid base exchange reaction provides molecular species of phospholipids, containing mainly long-chain polyunsaturated fatty acid moieties, required for the optimal activity of cytochrome P450 4A1.

scholarly journals Cytochemical analysis of the reconstitution of endoplasmic reticulum after microinjection of rat liver microsomes into Xenopus oocytes.

1988 ◽  
Vol 36 (10) ◽  
pp. 1263-1273 ◽  
Author(s):  
J Paiement ◽  
F W Kan ◽  
J Lanoix ◽  
M Blain
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Light And Electron Microscopy ◽  
Liver Microsomes ◽  
Heat Denaturation ◽  
Rat Liver Microsomes ◽  
Dependent Manner ◽  
Lysosomal Activity

Fragments of rough and smooth endoplasmic reticulum purified from rat liver were injected into Xenopus oocyte cytoplasm. Light and electron microscopy, cytochemistry, immunocytochemistry, and enzyme assay were employed to determine the fate of heterologous membranes in the host cytoplasm. The in vivo-incubated microsomes disappeared in a time-dependent manner. Within 3 hr, rough microsomes were replaced by flattened ER cisternae and smooth microsomes were replaced by a network of anastomosing tubules. Polyclonal antibodies against rat liver microsomes and protein A-gold complexes were applied to glycol methacrylate sections of microinjected oocytes. Specific labeling was observed over discrete rough and smooth ER cisternae 3 hr after microinjection. Endogenous ER was not labeled by this technique, and label was not observed when sections were treated with pre-immune antibodies. Diaminobenzidene cytochemistry of microinjected rat lacrimal gland microsomes revealed enzyme activity in heterologous microsomes after 3 hr of in vivo incubation. Control injected microsomes (inactivated by heat denaturation) became associated with autophagic vacuoles, coincident with changes in lysosomal activity. Freshly isolated un-denatured microsomes did not provoke changes in lysosomal activity, and glucose-6-phosphatase activity associated with microinjected membranes could be detected 21 hr after in vivo incubation. Since rat liver microsomes reconstitute after in vivo incubation into cytoplasmic structures resembling those from which they were derived, we conclude that the microinjected membrane fragments act as templates for their own three-dimensional organization.

scholarly journals Evidence that a low-molecular-mass GTP-binding protein is required for store-activated Ca2+ inflow in hepatocytes

1997 ◽  
Vol 328 (2) ◽  
pp. 463-471 ◽  
Author(s):  
C. Kekulu FERNANDO ◽  
B. Roland GREGORY ◽  
Frosa KATSIS ◽  
E. Bruce KEMP ◽  
J. Greg BARRITT
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Mass ◽  
G Protein ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Brefeldin A ◽  
Rat Hepatocytes ◽  
Rat Liver Microsomes ◽  
Isolated Rat Hepatocytes ◽  
Gtp Binding

The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 μM intracellular) of guanosine 5ʹ-[γ-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 μM intracellular) of GTP[S] or guanosine 5ʹ-[βγ-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 μM) prevented the inhibition of Ca2+ inflow by GTP[S] and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4- (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5)P3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.

Hydroxylation of Aliphatic Compounds by Liver Microsomes

1971 ◽  
Vol 26 (4) ◽  
pp. 322-327 ◽  
Author(s):  
U. Frommer ◽  
V. Ullrich
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Liver Cell ◽  
Liver Microsomes ◽  
Steric Effects ◽  
Rat Liver Microsomes ◽  
Aliphatic Compounds ◽  
Trifluoroperacetic Acid ◽  
Microsomal Hydroxylation ◽  
Selective Mechanism

Aliphatic compounds are hydroxylated to the corresponding isomeric alcohols by an unspecific hydroxylation system in the endoplasmic reticulum of the liver cell. As models for this reaction the following hydroxylation systems were investigated: 1. trifluoroperacetic acid, 2. the photolysis of 2,2-dimethylquinoxaline-di-N-oxide and 3. the system Fe2⊖/2-mercaptobenzoic acid/O2. 2-Methylbutane, methylcyclohexane and n-pentane are hydroxylated by a selective mechanism probably involving an oxenoid species. The pattern of isomeric alcohols closely resembles that obtained of rat liver microsomes indicating a hydroxylation mechanism of similar selectivity. From comparison with the nonenzymatic systems it is possible to conclude on minor steric effects in the microsomal hydroxylation of these alkanes.

scholarly journals Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. I. Functional tests on rat liver microsomal subfractions.

1984 ◽  
Vol 99 (6) ◽  
pp. 2247-2253 ◽  
Author(s):  
A Amar-Costesec ◽  
J A Todd ◽  
G Kreibich
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Placental Lactogen ◽  
Translation System ◽  
Secretory Proteins ◽  
Rat Liver Microsomes ◽  
Functional Tests ◽  
Membrane Bound

A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation system programmed with mRNA coding for the precursor of human placental lactogen. Membranes from microsomes with the higher isopycnic density and a high ribosome content showed the highest translocation activity, whereas membranes derived from smooth microsomes had only a very low translocation activity. These results indicate the membranes of the rough and smooth portions of the endoplasmic reticulum are functionally differentiated so that sites for ribosome binding and the translocation of nascent polypeptides are segregated to the rough domain of the organelle.

scholarly journals Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. II. Rat liver microsomal subfractions contain equimolar amounts of ribophorins and ribosomes.

1984 ◽  
Vol 99 (6) ◽  
pp. 2254-2259 ◽  
Author(s):  
E E Marcantonio ◽  
A Amar-Costesec ◽  
G Kreibich
Keyword(s):  
Endoplasmic Reticulum ◽  
Western Blot ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Liver Microsomes ◽  
Molar Ratio ◽  
Rat Liver Microsomes ◽  
Radioimmune Assay ◽  
Membrane Bound ◽  
Translational Process

Ribophorins I and II, two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum (ER) are thought to be part of the translocation apparatus for proteins made on membrane bound polysomes. To study the stoichiometry between ribophorins and membrane-bound ribosomes we have determined the RNA and ribophorin content in rat liver microsomes or in microsomal subfractions of different density (i.e., ribosome content). The specificity of antibodies against the ribophorins was demonstrated by Western blot analysis of rat liver rough microsomes separated by 2-dimensional gel electrophoresis. The ribophorin content of microsomal subfractions was determined by indirect immunoprecipitation and for ribophorin I by a radioimmune assay. In the latter assay a molar ratio of ribophorin I/ribosomes approaching one was calculated for total microsomes as well as in the gradient subfractions. We therefore suggest that ribophorins mediate the binding of ribosomes to endoplasmic reticulum membranes or play a role in co-translational process which depend on this binding, such as the insertion of nascent polypeptides into the membrane or their transfer into the cisternal lumen.

scholarly journals ENZYME-STRUCTURE RELATIONSHIPS IN THE ENDOPLASMIC RETICULUM OF RAT LIVER

1962 ◽  
Vol 15 (3) ◽  
pp. 541-562 ◽  
Author(s):  
Lars Ernster ◽  
Philip Siekevitz ◽  
George E. Palade
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Membrane Fraction ◽  
Liver Microsomes ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Rat Liver Microsomes ◽  
Cytochrome C Reductase ◽  
Ribonucleoprotein Particles

Subfractionation of preparations of rat liver microsomes with a suitable concentration of sodium deoxycholate has resulted in the isolation of a membrane fraction consisting of smooth surfaced vesicles virtually free of ribonucleoprotein particles. The membrane fraction is rich in phospholipids, and contains the microsomal NADH-cytochrome c reductase, NADH diaphorase, glucose-6-phosphatase, and ATPase in a concentrated form. The NADPH-cytochrome c reductase, a NADPH (or pyridine nucleotide unspecific) diaphorase, and cytochrome b5 are recovered in the clear supernatant fraction. The ribonucleoprotein particles are devoid of, or relatively poor in, the enzyme activities mentioned. Those enzymes which are bound to the membranes vary in activity according to the structural state of the microsomes, whereas those which appear in the soluble fraction are stable. From these findings the conclusion is reached that certain enzymes of the endoplasmic reticulum are tightly bound to the membranes, whereas others either are loosely bound or are present in a soluble form within the lumina of the system. Some implications of these results as to the enzymic organization of the endoplasmic reticulum are discussed.

Sign in / Sign up

Export Citation Format

Share Document