scholarly journals Hepatic endosome fractions contain an ATP-driven proton pump

1985 ◽  
Vol 225 (1) ◽  
pp. 51-58 ◽  
Author(s):  
T Saermark ◽  
N Flint ◽  
W H Evans
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Atpase Activity ◽  
Proton Pump ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Ph Gradients ◽  
Subcellular Organelle ◽  
Liver Homogenates

Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.

The Occurrence of Phenylalanine Ammonia-Lyase and Cinnamic Acid p-Hydroxylase on the Endoplasmic Reticulum of Cell Suspension Cultures of Glycine max

1978 ◽  
Vol 33 (1-2) ◽  
pp. 65-69 ◽  
Author(s):  
C. Postius ◽  
H. Kindi
Keyword(s):  
Endoplasmic Reticulum ◽  
Glycine Max ◽  
Golgi Apparatus ◽  
Cinnamic Acid ◽  
Time Course ◽  
Phenylalanine Ammonia Lyase ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Activity Increase ◽  
Membrane Bound

Abstract 1. The time course of activity of soluble and microsomal phenylalanine ammonia-lyase (PAL) was studied in dark grown cell cultures of soybean (Glycine max). A distinct activity increase of PAL in the soluble and microsomal fraction occurred prior to the stationary phase of the cell culture. Cinnamic acid p-hydroxylase and NADH : cytochrome c reductase, too, exhibited maximal activity in the log phase, 5 days after the transfer of soybean cells to fresh culture medium.2. Upon subfractionation of the once washed microsomal fraction by sedimentation velocity centrifugation on a sucrose gradient, membranes of the endoplasmic reticulum could be separated from fractions containing mainly membranes from the Golgi apparatus or plasma membranes, respectively. PAL and cinnamic acid p-hydroxylase were found in fractions of endoplasmic reticulum whereas no activity of either enzymes could be detected in fractions containing Golgi apparatus or plasma membranes.3. Repeated washing of microsomal fractions led to a residual membrane-bound PAL representing about 1% of the total PAL activity of the cells. This residual membrane-bound activity could be solubilized almost completely by Triton X-100 or digitonin at concentrations of 0.5 - 5%.

scholarly journals Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes?

1985 ◽  
Vol 101 (5) ◽  
pp. 1733-1740 ◽  
Author(s):  
A Yamamoto ◽  
R Masaki ◽  
Y Tashiro
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Subcellular Fraction ◽  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Cytochrome P 450 ◽  
Microscopic Techniques ◽  
Lysosomal Proteins

The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P-450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

1977 ◽  
Vol 55 (8) ◽  
pp. 876-885 ◽  
Author(s):  
Patricia L. Chang ◽  
John R. Riordan ◽  
Mario A. Moscarello ◽  
Jennifer M. Sturgess
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Golgi Membranes ◽  
Endomembrane Flow

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

scholarly journals The (Ca2+ + Mg2+)-stimulated ATPase of the rat parotid endoplasmic reticulum

1986 ◽  
Vol 235 (2) ◽  
pp. 491-498 ◽  
Author(s):  
P Thiyagarajah ◽  
S C Lim
Keyword(s):  
Endoplasmic Reticulum ◽  
Cyclic Amp ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Reductase Activity ◽  
Gradient Centrifugation ◽  
Fold Increase ◽  
Parotid Glands ◽  
Tissue Homogenates ◽  
Rat Parotid

A membrane fraction enriched in endoplasmic reticulum was prepared from rat parotid glands by using sucrose-gradient centrifugation. The fraction showed a 10-fold increase in specific activity of NADPH: cytochrome c reductase activity over that of tissue homogenates and minimal contamination with plasma membranes or mitochondria. The endoplasmic reticulum fraction possessed both Mg2+ -stimulated ATPase as well as Ca2+, Mg2+-ATPase [(Ca2+ + Mg2+)-stimulated ATPase]activity. The Ca2+, Mg2+-ATPase required 2-5 mM-Mg2+ for optimal activity and was stimulated by submicromolar concentrations of free Ca2+. The Km for free Ca2+ was 0.55 microM and the average Vmax. was 60 nmol/min per mg of protein. The Km for ATP was 0.11 mM. Other nucleotides, such as GTP, CTP or ADP, could not substitute for ATP in supporting the Ca2+-activated nucleotidase activity. Increasing the K+ concentration from 0 to 100 mM caused a 2-fold activation of the Ca2+, Mg2+-ATPase. Trifluoperazine, W7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide] and vanadate inhibited the enzyme. The concentration of trifluoperazine and vanadate required for 50% inhibition of the ATPase were 52 microM and 28 microM respectively. Calmodulin, cyclic AMP, cyclic AMP-dependent protein kinase and inositol 1,4,5-trisphosphate had no effect on the ATPase. The properties of the Ca2+, Mg2+ -ATPase were distinct from those of the Mg2+-ATPase, but comparable with those reported for the parotid endoplasmic-reticulum Ca2+-transport system [Kanagasuntheram & Teo (1982) Biochem. J. 208, 789-794]. The results suggest that the Ca2+, Mg2+-ATPase is responsible for driving the ATP-dependent Ca2+ accumulation by this membrane.

scholarly journals ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER

1971 ◽  
Vol 49 (3) ◽  
pp. 899-905 ◽  
Author(s):  
R. D. Cheetham ◽  
D. James Morré ◽  
Carol Pannek ◽  
Daniel S. Friend
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Specific Activity ◽  
Total Activity ◽  
Thiamine Pyrophosphate ◽  
Transferase Activity ◽  
Galactosyl Transferase ◽  
Thiamine Pyrophosphatase ◽  
Cell Components

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1–3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30–50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.

scholarly journals Quantitative assay and subcellular distribution of enzymes acting on dolichyl phosphate in rat liver

1981 ◽  
Vol 91 (3) ◽  
pp. 679-688 ◽  
Author(s):  
A Ravoet ◽  
A Amar-Costesec ◽  
D Godelaine ◽  
H Beaufay
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Golgi Complex ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Liver Microsomes ◽  
Reaction Medium ◽  
Previous Treatment ◽  
Reaction Products ◽  
Dolichyl Phosphate

To establish on a quantitative basis the subcellular distribution of the enzymes that glycosylate dolichyl phosphate in rat liver, preliminary kinetic studies on the transfer of mannose, glucose, and N-acetylglucosamine-1-phosphate from the respective (14)C- labeled nucleotide sugars to exogenous dolichyl phosphate were conducted in liver microsomes. Mannosyltransferase, glucosyltransferase, and, to a lesser extent, N- acetylglucosamine-phosphotransferase were found to be very unstable at 37 degrees C in the presence of Triton X-100, which was nevertheless required to disperse the membranes and the lipid acceptor in the aqueous reaction medium. The enzymes became fairly stable in the range of 10-17 degrees C and the reactions then proceeded at a constant velocity for at least 15 min. Conditions under which the reaction products are formed in amount proportional to that of microsomes added are described. For N- acetylglucosaminephosphotransferase it was necessary to supplement the incubation medium with microsomal lipids. Subsequently, liver homogenates were fractionated by differential centrifugation, and the microsome fraction, which contained the bulk of the enzymes glycosylating dolichyl phosphate, was analyzed by isopycnic centrifugation in a sucrose gradient without any previous treatment, or after addition of digitonin. The centrifugation behavior of these enzymes was compared to that of a number of reference enzymes for the endoplasmic reticulum, the golgi complex, the plasma membranes, and mitochondria. It was very simily to that of enzymes of the endoplasmic reticulum, especially glucose-6-phosphatase. Subcellular preparations enriched in golgi complex elements, plasma membranes, outer membranes of mitochondira, or mitoplasts showed for the transferases acting on dolichyl phosphate relative activities similar to that of glucose- 6-phosphatase. It is concluded that glycosylations of dolichyl phosphate into mannose, glucose, and N-acetylglucosamine-1-phosphate derivatives is restricted to the endoplasmic reticulum in liver cells, and that the enzymes involved are similarly active in the smooth and in the rough elements.

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 Electron Microscopy of the Development of Tracheoles in Drosophila Melanogaster

1963 ◽  
Vol s3-104 (65) ◽  
pp. 135-140
Author(s):  
S. AHMAD SHAFIQ
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Drosophila Melanogaster ◽  
Golgi Apparatus ◽  
Plasma Membranes ◽  
Developmental Stages ◽  
Membrane Structures ◽  
Flight Muscles

The tracheoblasts associated with the flight-muscles of Drosophila were studied by electron microscopy. During the developmental stages the cytoplasm of such tracheoblasts shows extensive membrane structures arranged in whorls. It seems that these membranes become aligned in pairs, spread out in tracheoblast cytoplasm, and form the walls of the new tracheolar vessels. The membranous whorls appear to have no obvious relationship with the usual endoplasmic reticulum, Golgi apparatus or plasma membranes. They are probably produced from certain large granules distributed irregularly in the cytoplasm of the young tracheoblasts. Membranes limiting the tracheoles from the tracheoblast cytoplasm (the so-called mestracheons) are not usually seen in the younger stages. They are sometimes seen after the cuticular lining of the tracheoles has been formed.

scholarly journals Membrane histochemistry of Physarum polycephalum myxamoebae.

1982 ◽  
Vol 30 (12) ◽  
pp. 1261-1267 ◽  
Author(s):  
H C Aldrich ◽  
J B Reiskind
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Physarum Polycephalum ◽  
Plasma Membranes ◽  
Phosphotungstic Acid ◽  
Chromic Acid ◽  
Hydrolytic Enzymes ◽  
Ruthenium Red ◽  
Food Vacuoles ◽  
Ultrathin Sections

Myxamoebae of Physarum polycephalum are the uninucleate, haploid stage of the organism. Histochemical studies were undertaken to characterize intracellular and plasma membranes, and to provide a basis for assaying subcellular fractions for enrichment in plasma membranes. Lead salts deposition techniques were employed for hydrolytic enzymes. Alcian blue-ruthenium red, osmium tetroxide-potassium ferrocyanide, and phosphotungstic acid-chromic acid stains were evaluated for specificity for plasma membranes. Glucose 6-phosphatase was localized in endoplasmic reticulum, Golgi apparatus, and perinuclear space. 5'-Nucleotidase was localized in food vacuoles, chromatin, and plasmalemma. Acid phosphatase was in food vacuoles and Golgi apparatus. Alkaline phosphatase was in food vacuoles and endoplasmic reticulum. We conclude that none of the above enzymes is suitable as a cytochemical marker for plasma membranes of Physarum myxamoebae, but recommend instead staining ultrathin sections of membrane pellets with phosphotungstic acid-chromic acid, which stains plasma membranes selectively.

Characterization of Mg-ATP-dependent Ca2+ transport in cat pancreatic microsomes

1983 ◽  
Vol 244 (5) ◽  
pp. G480-G490 ◽  
Author(s):  
A. Kribben ◽  
T. Tyrakowski ◽  
I. Schulz
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Atpase Activity ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Tissue Homogenate ◽  
Ionophore A23187 ◽  
Plasma Membrane Vesicles ◽  
Cytochrome C Reductase ◽  
Nadh Cytochrome

Mg-ATP-dependent 45Ca2+ uptake and Ca2+-ATPase activity have been examined in isolated microsomes obtained by differential centrifugation and in purified subcellular fractions obtained by Ficoll-sucrose density centrifugation in the presence of mitochondrial inhibitors. Mg-ATP-dependent 45Ca2+ uptake increased with increasing EGTA-buffered free [Ca2+], reaching a maximum of 2 nmol 45Ca2+ X 15 min-1 X mg prot-1 at 2 mumol/1 [Ca2+] in the incubation medium. Half-maximal 45Ca2+ uptake was at approximately 0.2 mumol/1 [Ca2+]. Maximal Ca2+ -Mg2+ -ATPase activity was 130 nmol X 15 min-1 X mg prot-1 at 2 mumol/l [Ca2+], with an apparent Km of approximately 0.3 mumol/l [Ca2+]. The Ca2+ ionophore A23187 (10(-6) mol/l), the mercurial compounds mersalyl (10(-5) mol/l) and CH3ClHg (10(-3) mol/l), as well as La3+ (10(-4) mol/l), vanadate (10(-4) mol/l), and saponin (50 micrograms/mg prot), abolished Mg-ATP-promoted 45Ca2+ uptake. In the absence of Mg2+, ATP did not provoke 45Ca2+ uptake. Using the purified smooth membrane fraction (F1) from the Ficoll-sucrose density gradient (enrichment of Na+-K+-ATPase specific activity by ninefold and of NADH-cytochrome c reductase by threefold as compared with total tissue homogenate), Mg-ATP-dependent 45Ca2+ uptake correlated better with Na+-K+-ATPase (r = 0.97) than with the smooth endoplasmic marker NADH-cytochrome c reductase (r = 0.52). No correlation was found with RNA, the marker for rough endoplasmic reticulum. We conclude that pancreatic plasma membranes contain a Ca2+-Mg2+-ATPase that represents the Ca2+ extrusion system from acinar cells. It is also possible that vesicular membrane structures associated with the plasma membrane, or endocytotic plasma membrane vesicles, take up Ca2+ and represent an intracellular Ca2+ pool.

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