Gel filtration of marker enzymes of plasma membrane and endoplasmic reticulum from rat liver

1969 ◽  
Vol 193 (2) ◽  
pp. 468-471 ◽  
Author(s):  
Kenji Nakai ◽  
Shigehide Takemitsu ◽  
Toshisuke Kawasaki ◽  
Ikuo Yamashina
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Gel Filtration ◽  
Marker Enzymes

scholarly journals Subcellular localization of the enzyme that forms mannosyl retinyl phosphate from guanosine diphosphate [14C]mannose and retinyl phosphate

1979 ◽  
Vol 180 (3) ◽  
pp. 449-453 ◽  
Author(s):  
M J Smith ◽  
J B Schreiber ◽  
G Wolf
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Subcellular Localization ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Subcellular Distribution ◽  
Smooth Endoplasmic Reticulum ◽  
Marker Enzymes ◽  
Guanosine Diphosphate

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.

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

1970 ◽  
Vol 44 (3) ◽  
pp. 492-500 ◽  
Author(s):  
R. D. Cheetham ◽  
D. James Morré ◽  
Wayne N. Yunghans
Keyword(s):  
Endoplasmic Reticulum ◽  
Uric Acid ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Succinic Dehydrogenase ◽  
Acid Oxidase ◽  
Enzyme Substrate ◽  
Thiamine Pyrophosphatase ◽  
Cell Fractions

Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20–30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2–3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.

Taurocholate transport by basolateral plasma membrane vesicles isolated from developing rat liver

1985 ◽  
Vol 248 (6) ◽  
pp. G648-G654
Author(s):  
F. J. Suchy ◽  
S. M. Courchene ◽  
B. L. Blitzer
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Plasma Membrane Vesicles ◽  
Adult Rat ◽  
Basolateral Plasma Membrane ◽  
Taurocholate Transport

Taurocholate transport was characterized in basolateral plasma membrane vesicles prepared from the livers of 14-day-old Sprague-Dawley rats using a self-generating Percoll gradient method. Liver plasma membrane protein yield, intravesicular volume, and enrichments of various marker enzymes were similar to those obtained for vesicles from adult rat liver. The basolateral marker enzyme Na+-K+-ATPase was enriched 26-fold in the suckling rat basolateral membrane fraction while the bile canalicular marker enzymes alkaline phosphatase and Mg2+-ATPase were enriched only 3- and 5-fold, respectively. The activities of marker enzymes for endoplasmic reticulum, mitochondria, or lysosomes were not enriched compared with homogenate. In the presence of an inwardly directed 100 mM Na+ gradient, vesicle accumulation of taurocholate transiently reached a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot") in suckling and adult membrane vesicles, but the initial rate of taurocholate entry and peak intravesicular accumulation were markedly decreased in suckling compared with adult membrane vesicles. In the presence of an inwardly directed 100 mM K+ gradient, the rate of uptake was slower, and no overshoot occurred in either suckling or adult rat vesicles. The decreased rate of Na+-coupled taurocholate uptake by membrane vesicles from suckling rat liver could not be explained on the basis of more rapid dissipation of the transmembrane Na+ gradient. Kinetic studies demonstrated saturable, Na+-dependent taurocholate uptake for both suckling and adult vesicles. However, the Vmax for taurocholate uptake in suckling rat vesicles was less than half of the adult rate (2.46 +/- 0.13 vs. 5.25 +/- 0.22 nmol X mg prot-1 X min-1, respectively, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

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 A procedure for the rapid isolation from rat liver of plasma membrane vesicles exhibiting Ca2+-transport and Ca2+-ATPase activities

1984 ◽  
Vol 223 (3) ◽  
pp. 733-745 ◽  
Author(s):  
R J Epping ◽  
F L Bygrave
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Membrane Surface ◽  
Membrane Vesicles ◽  
Mitochondrial Fraction ◽  
Plasma Membrane Vesicles ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient

A technique is described for the isolation of a plasma membrane-enriched preparation from a rat liver post-mitochondrial fraction by using discontinuous Percoll density-gradient centrifugation. The procedure is simple, of high reproducibility and yield and requires a total isolation time of only 90 min. The preparation consists almost exclusively of membrane vesicles and is enriched approx. 26-fold in plasma membrane-localized enzymes with minor contamination (less than 10%) with membranes derived mainly from the endoplasmic reticulum and Golgi apparatus. Approx. 20% of the fraction comprises tightly-sealed vesicles in the inverted orientation which are capable of accumulating calcium ions and exhibiting vanadate-insensitive Ca2+-ATPase activity. The properties of these activities, including insensitivity to vanadate, oxalate, and to p-chloromercuribenzoate as well as a lack of requirement for added Mg2+, contrast markedly with the reported properties of Ca2+ transport by the endoplasmic reticulum isolated from rat liver. The technique may have wide application in the study of plasma membrane-associated activities in rat liver, particularly in relation to sinusoidal membrane surface-related events.

An improved procedure for the purification of plasma membranes from Dictyostelium discoideum

1977 ◽  
Vol 55 (12) ◽  
pp. 1233-1236 ◽  
Author(s):  
N. R. Gilkes ◽  
G. Weeks
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Plasma Membranes ◽  
Membrane Preparation ◽  
Preparation Procedure ◽  
Contamination Level ◽  
Marker Enzymes ◽  
Plasma Membrane Preparation

A novel procedure was recently described for the purification of plasma membranes of Dictyostelium discoideum (Gilkes, N. R. &Weeks, G. (1977) Biochim. Biophys. Acta 464, 142–156). Considerable enrichment of plasma membrane marker enzymes was achieved, but since purified mitochondrial and endoplasmic reticulum fractions were unavailable, it was not possible to accurately assess the contamination level of these organelles. We have therefore slightly modified the plasma membrane preparation procedure, improving purification, and have prepared partially purified mitochondrial and endoplasmic reticulum fractions. The data suggest that the contamination of the plasma membranes by endoplasmic reticulum membranes is no greater than 10%, and probably considerably less. No mitochondrial contamination is detectable.

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