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 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 Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver

1989 ◽  
Vol 259 (3) ◽  
pp. 659-663 ◽  
Author(s):  
F Vanstapel ◽  
L Hammaker ◽  
K Pua ◽  
N Blanckaert
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Membrane System ◽  
Permeability Barrier ◽  
Membrane Bound ◽  
Marker Studies ◽  
High Degree ◽  
Regulatory Properties

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.

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 Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes.

1978 ◽  
Vol 78 (3) ◽  
pp. 874-893 ◽  
Author(s):  
E Rodriguez Boulan ◽  
G Kreibich ◽  
D D Sabatini
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Binding Sites ◽  
Microsomal Fraction ◽  
Lectin Binding ◽  
Membrane Glycoproteins ◽  
Con A ◽  
Microsomal Membranes ◽  
Carbohydrate Chains ◽  
Lectin Binding Sites

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.

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 Alteration of membrane barrier in stripped rough microsomes from rat liver on incubation with GTP: its relevance to the stimulation by this nucleotide of the dolichol pathway for protein glycosylation.

1983 ◽  
Vol 97 (2) ◽  
pp. 340-350 ◽  
Author(s):  
D Godelaine ◽  
H Beaufay ◽  
M Wibo ◽  
A M Ravoet
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Protein Glycosylation ◽  
Permeability Barrier ◽  
Endoplasmic Reticulum Membrane ◽  
Uridine Diphosphate ◽  
Guanosine Diphosphate ◽  
Membrane Barrier ◽  
Dolichol Pathway ◽  
Triton X

The membrane barrier of stripped rough microsomes from rat liver is markedly altered on incubation with GTP at 37 degrees C: after 30 min the structure-linked latency of mannose-6-phosphatase was considerably reduced, and esterase and nucleoside diphosphatase were partly released into the suspension medium. This phenomenon was already maximal with 30 microM GTP and was specific for this nucleotide. Similar conditions enhance the dolichol-mediated glycosylation of protein in microsomes incubated with uridine diphosphate N-acetylglucosamine and guanosine diphosphate mannose (Godelaine, D., H. Beaufay, M. Wibo, and A. Amar-Costesec, 1979, Eur. J. Biochem., 96:17-26; Godelaine, D., H. Beaufay, and M. Wibo, 1979, Eur. J. Biochem., 96:27-34). The GTP-induced permeability and glycosylation activities evolved in parallel in rough microsomes subjected to various treatments to detach the ribosomes and were maximal after removal of congruent to 60% of the RNA. In addition, GTP had no effect of this type in smooth microsome subfractions. Triton X-100, in spite of complex inhibitory effects on glycosylation reactions, mimicked the action of GTP by increasing the amount of microsomal dolichylphosphate that reacts with uridine diphosphate N-acetylglucosamine and by enhancing synthesis of dolichylpyrophosphoryl-chitobiose at concentrations greater than 2 mg/ml. Thus, GTP may activate dolichol-mediated glycosylation reactions in stripped microsomes by lowering the permeability barrier that prevents access of sugar nucleotides to the inner aspect of the membrane. The genuine role of GTP in the functioning of the endoplasmic reticulum membrane in situ remains unknown. Because GTP seems to act only on rough microsomes, we hypothesize that this role is somehow related to biosynthesis of protein by the rough endoplasmic reticulum.

scholarly journals Activation of a peroxisome-proliferating catabolite of cholic acid to its CoA ester

1993 ◽  
Vol 296 (1) ◽  
pp. 265-270 ◽  
Author(s):  
T Nishimaki-Mogami ◽  
A Takahashi ◽  
Y Hayashi
Keyword(s):  
Cholic Acid ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Liver Microsomes ◽  
Subcellular Fractionation ◽  
Fatty Acyl ◽  
Peroxisome Proliferator ◽  
Rat Liver Microsomes ◽  
Triton X ◽  
Long Chain

We have shown that a microbial cholic acid catabolite (4R)-4-(2,3,4,6,6a beta,7,8,9,9a alpha,9b beta-decahydro-6a beta-methyl-3-oxo- 1H-cyclopenta[f]quinolin-7 beta-yl)valeric acid (DCQVA), is a potent peroxisome proliferator. In this paper a possible key stage in DCQVA metabolism, the activation of DCQVA to its CoA ester, has been investigated in rat liver microsomes and particulate fractions. The microsomal reaction was dependent on CoA, ATP, DCQVA (0.2-1 mM) and protein content. The reaction was decreased by storage at 4 degrees C, preincubation of microsomes at 37 degrees C for 5 min, or inclusion of Triton X-100 in the reaction mixture. Such treatments also enhanced generation of long-chain fatty acyl-CoAs, as determined by h.p.l.c. analysis. The same effect was caused by exposing the microsomes to phospholipase A2, suggesting that endogenous fatty acids may compete with DCQVA for esterification with CoA. Subcellular fractionation of rat liver demonstrated that the activity of DCQVA-CoA synthesis was localized predominantly in the microsomal fraction, in contrast to long-chain fatty acyl-CoA synthetase, which was distributed among all particulate fractions. Administration of clofibrate of rats did not affect the distribution of DCQVA-CoA synthesis activity. In contrast to a 2-fold induction of long-chain fatty acyl-CoA synthetase by clofibrate treatment, the activity of DCQVA-CoA synthesis in the microsomal fraction decreased by 80%. These results suggest that DCQVA is activated by an enzyme distinct from long-chain fatty acyl-CoA synthetase. The resulting perturbation of fatty acid metabolism may be involved in the mechanism whereby DCQVA causes peroxisome proliferation.

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.

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