Mannosyl carrier functions of retinyl phosphate and dolichyl phosphate in rat liver endoplasmic reticulum.
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ABSTRACT:
SUBMITTER: Creek KE
PROVIDER: S-EPMC1154255 | biostudies-other | 1983 Feb
REPOSITORIES: biostudies-other
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Project description:The four most important non-specific carboxylesterases from rat liver were assayed for their ability to hydrolyse retinyl esters. Only the esterases with pI 6.2 and 6.4 (= esterase ES-4) are able to hydrolyse retinyl palmitate. Their specific activities strongly depend on the emulsifier used (maximum rate: 440 nmol of retinol liberated/h per mg of esterase). Beside retinyl palmitate, these esterases cleave palmitoyl-CoA and monoacylglycerols with much higher rates, as well as certain drugs (e.g. aspirin and propanidid). However, no transacylation between palmitoyl-CoA and retinol occurs. Retinyl acetate also is a substrate for the above esterases and for another one with pI 5.6 (= esterase ES-3). Again the emulsifier influences the hydrolysis by these esterases (maximum rates: 475 nmol/h per mg for ES-4 and 200 nmol/h per mg for ES-3). Differential centrifugation of rat liver homogenate reveals that retinyl palmitate hydrolase activity is highly enriched in the plasma membranes, but only moderately so in the endoplasmic reticulum, where the investigated esterases are located. Since the latter activity can be largely inhibited with the selective esterase inhibitor bis-(4-nitrophenyl) phosphate, it is concluded that the esterases with pI 6.2 and 6.4 (ES-4) represent the main retinyl palmitate hydrolase of rat liver endoplasmic reticulum. In view of this cellular localization, the enzyme could possibly be involved in the mobilization of retinol from the vitamin A esters stored in the liver. However, preliminary experiments in vivo have failed to demonstrate such a biological function.
| S-EPMC1148208 | biostudies-other
Project description:The subcellular distribution of the enzyme catalysing the conversion of retinyl phosphate and GDP-[14C]mannose into [14C]mannosyl retinyl phosphate was determined by using subcellular fractions of rat liver. Purity of fractions, as determined by marker enzymes, was 80% or better. The amount of mannosyl retinyl phosphate formed (pmol/min per mg of protein) for each fraction was: rough endoplasmic reticulum 0.48 +/- 0.09 (mean +/- S.D.); smooth membranes (consisting of 60% smooth endoplasmic reticulum and 40% Golgi apparatus), 0.18 +/- 0.03; Golgi apparatus, 0.13 +/- 0.03; and plasma membrane 0.02.
| S-EPMC1161080 | biostudies-other
Project description:Mannose-phosphate-dolichol (MPD) is a multifunctional glycolipid that is synthesized on the cytoplasmic face of the endoplasmic reticulum (ER) and used on the opposite side of the membrane in the ER lumen as a mannose donor for protein N-glycosylation, glycosylphosphatidylinositol-anchoring, and C- and O-mannosylation. For this, it must be translocated, i.e., flipped, across the ER membrane. The molecular identity of the MPD translocator (MPD flippase) is not known. Here we show that MPD-flippase activity can be reconstituted in large unilamellar proteoliposomes prepared from phosphatidylcholine and Triton X-100-solubilized rat liver ER-membrane proteins. Using carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl NO(+) as a topological probe to selectively oxidize MPD molecules in the outer leaflet of the reconstituted vesicles, we demonstrate rapid, protein-dependent, ATP-independent transbilayer translocation of MPD from the inner to the outer leaflet. MPD flipping is highly specific. A stereoisomer of MPD was weakly translocated (> 10-fold lower rate) compared with natural MPD. Competition experiments with water-soluble isoprenyl monophosphates showed that MPD flippase recognizes the dolichol chain of MPD, preferring a saturated alpha-isoprene to unsaturated trans- or cis- alpha-isoprene units. Chromatography of the detergent-solubilized ER protein mixture prior to reconstitution indicated that MPD flippase (i) is not a Con A-binding glycoprotein and (ii) can be resolved from the oligosaccharide-diphosphate dolichol flippase that translocates Man(5)GlcNAc(2)-PP-dolichol, a lipid intermediate of N-glycosylation. These data provide a mechanistic framework for understanding MPD flipping, as well as a biochemical basis for identifying MPD flippase.
| S-EPMC2895134 | biostudies-literature
Project description:In the absence of detergent, the transfer of mannose from GDP-mannose to rat liver microsomal vesicles was highly stimulated by exogenous retinyl phosphate in incubations containing bovine serum albumin, as measured in a filter binding assay. Under these conditions 65% of mannose 6-phosphatase activity was latent. The transfer process was linear with time up to 5min and with protein concentration up to 1.5mg/0.2ml. It was also temperature-dependent. The microsomal uptake of mannose was highly dependent on retinyl phosphate and was saturable against increasing amounts of retinyl phosphate, a concentration of 15mum giving half-maximal transfer. The uptake system was also saturated by increasing concentrations of GDP-mannose, with an apparent K(m) of 18mum. Neither exogenous dolichyl phosphate nor non-phosphorylated retinoids were active in this process in the absence of detergent. Phosphatidylethanolamine and synthetic dipalmitoylglycerophosphocholine were also without activity. Several water-soluble organic phosphates (1.5mm), such as phenyl phosphate, 4-nitrophenyl phosphate, phosphoserine and phosphocholine, did not inhibit the retinyl phosphate-stimulated mannosyl transfer to microsomes. This mannosyl-transfer activity was highest in microsomes and marginal in mitochondria, plasma and nuclear membranes. It was specific for mannose residues from GDP-mannose and did not occur with UDP-[(3)H]galactose, UDP- or GDP-[(14)C]glucose, UDP-N-acetyl[(14)C]-glucosamine and UDP-N-acetyl[(14)C]galactosamine, all at 24mum. The mannosyl transfer was inhibited 85% by 3mm-EDTA and 93% by 0.8mm-amphomycin. At 2min, 90% of the radioactivity retained on the filter could be extracted with chloroform/methanol (2:1, v/v) and mainly co-migrated with retinyl phosphate mannose by t.l.c. This mannolipid was shown to bind to immunoglobulin G fraction of anti-(vitamin A) serum and was displaced by a large excess of retinoic acid, thus confirming the presence of the beta-ionone ring in the mannolipid. The amount of retinyl phosphate mannose formed in the bovine serum albumin/retinyl phosphate incubation is about 100-fold greater than in incubations containing 0.5% Triton X-100. In contrast with the lack of activity as a mannosyl acceptor for exogenous dolichyl phosphate in the present assay system, endogenous dolichyl phosphate clearly functions as an acceptor. Moreover in the same incubations a mannolipid with chromatographic properties of retinyl phosphate mannose was also synthesized from endogenous lipid acceptor. The biosynthesis of this mannolipid (retinyl phosphate mannose) was optimal at MnCl(2) concentrations between 5 and 10mm and could not be detected below 0.6mm-MnCl(2), when synthesis of dolichyl phosphate mannose from endogenous dolichyl phosphate was about 80% of optimal synthesis. Under optimal conditions (5mm-MnCl(2)) endogenous retinyl phosphate mannose represented about 20% of dolichyl phosphate mannose at 15min of incubation at 37 degrees C.
| S-EPMC1163574 | biostudies-other
Project description:BACKGROUND & AIMS:Disruption to endoplasmic reticulum (ER) calcium homeostasis has been implicated in obesity, however, the ability to longitudinally monitor ER calcium fluctuations has been challenging with prior methodologies. We recently described the development of a Gaussia luciferase (GLuc)-based reporter protein responsive to ER calcium depletion (GLuc-SERCaMP) and investigated the effect of a high fat diet on ER calcium homeostasis. METHODS:A GLuc-based reporter cell line was treated with palmitate, a free fatty acid. Rats intrahepatically injected with GLuc-SERCaMP reporter were fed a cafeteria diet or high fat diet. The liver and plasma were examined for established markers of steatosis and compared to plasma levels of SERCaMP activity. RESULTS:Palmitate induced GLuc-SERCaMP release in vitro, indicating ER calcium depletion. Consumption of a cafeteria diet or high fat pellets correlated with alterations to hepatic ER calcium homeostasis in rats, shown by increased GLuc-SERCaMP release. Access to ad lib high fat pellets also led to a corresponding decrease in microsomal calcium ATPase activity and an increase in markers of hepatic steatosis. In addition to GLuc-SERCaMP, we have also identified endogenous proteins (endogenous SERCaMPs) with a similar response to ER calcium depletion. We demonstrated the release of an endogenous SERCaMP, thought to be a liver esterase, during access to a high fat diet. Attenuation of both GLuc-SERCaMP and endogenous SERCaMP was observed during dantrolene administration. CONCLUSIONS:Here we describe the use of a reporter for in vitro and in vivo models of high fat diet. Our results support the theory that dietary fat intake correlates with a decrease in ER calcium levels in the liver and suggest a high fat diet alters the ER proteome. Lay summary: ER calcium dysregulation was observed in rats fed a cafeteria diet or high fat pellets, with fluctuations in sensor release correlating with fat intake. Attenuation of sensor release, as well as food intake was observed during administration of dantrolene, a drug that stabilizes ER calcium. The study describes a novel technique for liver research and provides insight into cellular processes that may contribute to the pathogenesis of obesity and fatty liver disease.
| S-EPMC6122848 | biostudies-literature
Project description:Uptake and metabolism of UDP-glucuronic acid (UDPGlcA) by rough-endoplasmic-reticulum (RER)-derived vesicles was studied. Analysis of the molecular species, double-labelling experiments and trans-stimulation experiments revealed that initial uptake represented entry into microsomes of predominantly intact UDPGlcA, concomitant with rapid hydrolysis of the internalized nucleotide sugar. The uptake constituted effective translocation from the medium into the lumen of the vesicles. Thus the amount of vesicle-associated label at equilibrium uptake was directly proportional to the volume of the intravesicular space. Permeabilized microsomes were unable to retain UDPGlcA. The microsomal uptake of UDPGlcA met the criteria of bidirectional carrier-mediated translocation. Transport was time- and temperature-dependent, saturable, selective, capable of trans-stimulation, and operational against a concentration gradient. Microsomal uptake was inhibited by N-ethylmaleimide that was presented at the cytosolic side of the endoplasmic-reticulum (ER) membrane. Uptake studies performed in membrane preparations that were highly enriched in RER, smooth ER or Golgi revealed that UDPGlcA was taken up by the ER as well as by the Golgi apparatus. Our findings demonstrate the existence in rat liver ER of a carrier system mediating proper translocation of intact UDPGlcA across the membrane.
| S-EPMC1137218 | biostudies-other
Project description:The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation.
| S-EPMC1163782 | biostudies-other
Project description:Pex19p, a soluble cytoplasmic transport protein, is required for the traffic of the peroxisomal membrane proteins Pex3p and Pex15p from the endoplasmic reticulum (ER) to the peroxisome. We documented Pex15p traffic from the ER using a chimeric protein containing a C-terminal glycosylation acceptor peptide. Pex15Gp expressed in wild-type yeast cells is N-glycosylated and functions properly in the peroxisome. In contrast, pex19?-mutant cells accumulate the glycoprotein Pex15Gp in the ER. We developed a cell-free preperoxisomal vesicle-budding reaction in which Pex15Gp and Pex3p are packaged into small vesicles in the presence of cytosol, Pex19p, and ATP. Secretory vesicle budding (COPII) detected by the packaging of a SNARE protein (soluble N-ethylmaleimide-sensitive attachment protein receptor) occurs in the same incubation but does not depend on Pex19p. Conversely a dominant GTPase mutant Sar1p which inhibits COPII has no effect on Pex3p packaging. Pex15Gp and Pex3p budded vesicles sediment as low-buoyant-density membranes on a Nycodenz gradient and copurify by affinity isolation using native but not Triton X-100-treated budded vesicles. ER-peroxisome transport vesicles appear to rely on a novel budding mechanism requiring Pex19p and additional unknown factors.
| S-EPMC3003071 | biostudies-literature
Project description:A remarkable and immediate decrease in GDP-mannose:retinyl phosphate mannosyltransferase activity was found on pre-incubation of rat liver postnuclear membranes with phospholipase A2 or phospholipase C. Under the same conditions of pre-incubation (1 min at 37 degrees C) trypsin did not affect the enzyme activity, whereas pre-incubation for 30 min with trypsin and Pronase abolished enzyme activity. The lipid extract of untreated rat liver membranes partially restored enzyme activity after phospholipase treatment. Sphingomyelin was as active as the endogenous lipids. Other phospholipids were less active in the following order: phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol = phosphatidylserine. Dolichyl phosphate mannose synthesis was inhibited less (33%) by phospholipase C than was Ret-P-Man synthesis (98.5%) under identical conditions of incubation, which included 0.025% Triton. However, retinyl phosphate mannose synthesis by purified endoplasmic reticulum was found to be resistant to phospholipase C. Mixing experiments failed to demonstrate an inhibitory effect of the phospholipase-treated postnuclear membrane fraction on the synthetic activity of the endoplasmic reticulum, thus excluding the release of an inhibitory factor from the postnuclear membranes.
| S-EPMC1152568 | biostudies-other
Project description:1. The transfer of mannose from GDP-(U-14-C)mannose into endogenous acceptors of bovine adrenal medullla and rat parotid was studied. The rapidly labelled product, a glycolipid, was partially purified and characterized. 2. It was stable to mild alkaline hydrolysis but yielded (14-C)mannose on mild acid hydrolysis. It co-chromatographed with mannosyl phosphoryl dolichol in four t.l.c. systems and on DEAE-cellulose acetate. Addition of dolichol phosphate or a dolichol phosphate-enriched fraction prepared from pig liver stimulated mannolipid synthesis. 3. The formation of mammolipid appeared reversible, since addition of GDP to a system synthesizing the mannolipid caused a rapid loss of label from the mannolipid. UDP-N-acetylglucosamine did not inhibit mannolipid synthesis except at high concentrations (2 mM), even though in the absence of GDP-mannose, N-acetylglucosamine was incorporated into a lipid having the properties of a glycosylated polyprenyl phosphate. 4. Mannose from GDP-mannose was also incorporated into two other acceptors, (2y being insoluble in chloroform-methanol (2:1, v/v) but soluble in choloroform-methanol-water (10:10:3, by vol.) and (ii) protein. These are formed much more slowly than the mannolipid. 5. Exogenous mannolipid served as a mannose donor for acceptors (i) and (ii), and it is suggested that transfer of mannose from GDP-mannose to mannosylated protein occurs via two intermediates, the mannolipid and acceptor (i).
| S-EPMC1165354 | biostudies-other