Preservation and separation of endomembrane marker enzyme activity in potato leaf homogenates

1986 ◽  
Vol 64 (11) ◽  
pp. 2732-2737 ◽  
Author(s):  
Gerald Nagahashi ◽  
Thomas S. Seibles
Keyword(s):  
Plasma Membranes ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Membrane Degradation ◽  
Potato Leaf ◽  
Golgi Membranes ◽  
Green Tissue ◽  
Young Leaves ◽  
Specific Activities ◽  
Plastid Membranes

To minimize rapid browning and membrane degradation of crude microsomes, leaves of Solanum tuberosum (cv. Kennebec and cv. Katahdin) were initially homogenized in the presence of various inhibitors of polyphenol oxidase, phospholipase, and protease activity. To obtain and maintain marker enzyme activities used to identify plasma membranes, Golgi membranes, and endoplasmic reticulum, it was necessary to homogenize young leaves in the presence of sulfhydryls at pH 7.8. Further separation of these membranes, as determined by distribution of total activities of marker enzymes in linear sucrose density gradients, indicated a relatively pure plasma membrane fraction (1.15 g/cm3) free from contamination by thylakoids (1.19 g/cm3) and other endomembrane components. However, the distribution of specific activities across the gradient revealed that plasma membranes isolated from green tissue may be contaminated by Golgi membranes and not necessarily by plastid membranes.

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 LYSOSOMAL PHOSPHOLIPASES A1 AND A2 OF NORMAL AND BACILLUS CALMETTE GUERIN-INDUCED ALVEOLAR MACROPHAGES

1973 ◽  
Vol 56 (3) ◽  
pp. 621-627 ◽  
Author(s):  
Richard C. Franson ◽  
Moseley Waite
Keyword(s):  
Acid Phosphatase ◽  
Alveolar Macrophages ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Marker Enzymes ◽  
Sucrose Gradient Centrifugation ◽  
Control Distribution ◽  
Specific Activities ◽  
Selective Increase ◽  
Phospholipases A

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5–4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, ß-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A1 and A2 which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50–70% by 2.0 mM CaCl2. Homogenates of BCG-induced macrophages had specific activities of ß-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 107 cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.

Biogenesis of Plasma Membranes in Salt Glands of Salt-Stressed Domestic Ducklings: Localization of Acyltransferase Activity

1972 ◽  
Vol 11 (3) ◽  
pp. 855-873
Author(s):  
A. M. LEVINE ◽  
JOAN A. HIGGINS ◽  
R. J. BARRNETT
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Salt Gland ◽  
Secretory Cells ◽  
Salt Glands ◽  
Acyltransferase Activity ◽  
Structural Localization ◽  
Differentiated Cells ◽  
Golgi Membranes

In response to salt water stress there is a marked increase in the plasma membranes of the epithelial secretory cells of the salt glands of domestic ducklings. In the present study, the fine-structural localization of the acyltransferases involved in synthesis of phospholipids has been investigated in this tissue during this increased biogenesis of plasma membranes. The specific activity of the acyltransferases of the salt gland rose in response to salt stress, and this preceded the rapid increase in weight and cellular differentiation. After the weight increase of the gland became established, the specific activity of the acyltransferases declined, but the total activity remained constant. Salt gland tissue fixed in a mixture of glutaraldehyde and formaldehyde retained 35% of the acyltransferase activity of unfixed tissue. Cytochemical studies of the localization of acyltransferase activity in fixed and unfixed salt gland showed reaction product associated only with the lamellar membranes of the Golgi complex. This localization occurred in partially differentiated cells from salt-stressed glands to the greatest extent; and to only a small extent in cells of control tissue from unstressed salt glands. Omission of substrates resulted in absence of reaction product in association with the Golgi membranes. In addition, vesicles having limiting membranes morphologically similar to the plasma membrane occurred between the Golgi region and the plasma membrane in the partially differentiated cells. The phospholipid component of the plasma membrane appears therefore to be synthesized in association with the Golgi membranes and the membrane packaged at this site from which it moves in the form of vesicles to fuse with the pre-existing plasma membrane.

scholarly journals Mitochondrial-reticular cytostructure in liver cells

1983 ◽  
Vol 214 (3) ◽  
pp. 795-813 ◽  
Author(s):  
J Katz ◽  
P A Wals ◽  
S Golden ◽  
L Raijman
Keyword(s):  
Endoplasmic Reticulum ◽  
High Speed ◽  
Citrate Synthase ◽  
Respiratory Control ◽  
High Salt ◽  
Marker Enzyme ◽  
Salt Medium ◽  
Low Speed ◽  
Low Salt ◽  
Specific Activities

This study examines the structural relationship of mitochondria and the endoplasmic reticulum in liver. Livers of rat and Japanese quail were homogenized and fractionated in media of 0.25 M-sucrose, either 5mM or 50 mM in sodium Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid], pH 7.4 (2.2 mM or 22 mM in Na respectively), designated here as low- and high-salt media. Three particulate fractions were prepared by sequential centrifugation. A nuclear pellet sedimenting at 300 g was obtained as described by Shore & Tata [(1977) J. Cell Biol. 72, 714-725], and from the resulting supernatant thereof a low-speed pellet (1100-1500 g) and a high-speed pellet (8000-10 000 g) were prepared. In the low-salt medium the yields of mitochondrial matrix enzymes (citrate synthase, glutamate dehydrogenase, ornithine carbamoyltransferase) and their specific activities in the low-speed pellet were over twice those in the high-speed pellet. In the high-salt medium the yield of matrix enzymes was 4-5 times, and the specific activities were up to 3 times, higher in the low-speed pellet than in the high-speed pellet. Oxygen uptake and respiratory control ratio were also much higher in the low-speed pellets in both media. Some 50-65% of the microsomal marker enzyme glucose 6-phosphatase was in the supernatant from the high-speed pellet, and the rest sedimented with the mitochondria. Repeated washing with the high-salt medium removes only a limited amount of reticulum. Washing with salt-free sucrose removes most of the reticulum, but a fraction remains strongly bound to mitochondria. Homogenates from quail and rat liver were fractioned isopycnically on Percoll gradients in either 0.25 M-sucrose or 0.25 M-sucrose/50 mM-sodium Hepes. Up to five particulate bands were separated and assayed. Mitochondria were present in two to three bands and were associated with endoplasmic reticulum. As seen in the phase-contrast microscope the mitochondria prepared in the low-salt medium consist of separate organelles. In the high-salt medium the mitochondria appear as chains of from three to ten organelles not touching each other. On addition of univalent ions at concentrations above 20 mM, the mitochondria aggregate into chains, and at higher ionic strength larger multidimensional aggregates are formed. The dispersion and aggregation of mitochondria are reversible. Negatively stained electron micrographs reveal a branched mitochondrial structure, with mitochondria held together by strands of reticulum.(ABSTRACT TRUNCATED AT 400 WORDS)

Elevated 5'-nucleotidase and alkaline phosphodiesterase activities in trout gill localize to endothelial (pillar) cells

1998 ◽  
Vol 201 (13) ◽  
pp. 2011-2019
Author(s):  
JC Robertson ◽  
JR Hazel
Keyword(s):  
Plasma Membranes ◽  
Specific Activity ◽  
Apical Region ◽  
Pavement Cells ◽  
Pillar Cells ◽  
Secondary Lamellae ◽  
Histochemical Evidence ◽  
Tissue Homogenates ◽  
Specific Activities ◽  
Trout Gill

Tissue homogenates from rainbow trout gill had three- to fivefold higher specific activity for 5'-nucleotidase (5'NT) and more than twofold greater alkaline phosphodiesterase (APD) activity than liver or kidney homogenates. In isolated plasma membranes, gill 5'NT activity was 3-5 times greater than that of the kidney or liver; gill and kidney plasma membranes had similar APD specific activities, both more than five times that of liver. 5'NT and APD activities were localized by histochemistry to the endothelial (pillar) cells of trout gill secondary lamellae. Staining was consistent with the concentration of both activities at the apical plasma membranes of pillar cells (i.e. at the lamellar microvascular surfaces). This localization may reflect a capacity for processing nucleotide metabolites circulating in the blood, perhaps relating to purinergic regulation of local lamellar hemodynamics. There was no histochemical evidence of either 5'NT or APD activity in the gill epithelial (pavement) cells that interface directly with the environment. In contrast, in trout kidney, both enzyme activities localized to the apical region of tubule epithelial cells. The absence of 5'NT and APD activity in pavement cells reinforces the unique structural and functional character of the gill-environment epithelial barrier. The results indicate that 5'NT and APD activities have particular potential application as markers in efforts to isolate and characterize specific gill plasma membrane fractions.

scholarly journals Isolation and characterization of rabbit kidney brush borders

1972 ◽  
Vol 128 (5) ◽  
pp. 1319-1328 ◽  
Author(s):  
S. J. Quirk ◽  
G. B. Robinson
Keyword(s):  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Marker Enzymes ◽  
Isolation And Characterization ◽  
Adenosine Triphosphatases ◽  
Brush Borders

1. Brush borders were isolated from rabbit kidney-cortex homogenates by rate-zonal centrifugation through a sucrose density gradient in a B-XIV zonal rotor, followed by differential centrifugation. 2. The method of preparation gave brush borders of high purity with a reasonable yield. The morphological appearance supported the evidence from enzymic and chemical investigations, that the brush borders were only slightly contaminated with endoplasmic reticulum, mitochondria, lysosomes and nuclei. 3. The molar ratio of cholesterol to phospholipid lay within the range found in other plasma membranes, but the carbohydrate content was double that found in liver plasma membranes. 4. Alkaline phosphatase, maltase, trehalase and aminopeptidase were major enzymic constituents of the brush borders, and had an approximately equal yield and enrichment, but none of these enzymes fulfilled the criteria for marker enzymes. 5. Mg2+-dependent and Na+,K+-dependent adenosine triphosphatases, although found in brush borders, had low yields and low enrichments.

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)

Ionizing radiation stimulates muscarinic regulation of rat intestinal mucosal function

1998 ◽  
Vol 275 (6) ◽  
pp. G1333-G1340 ◽  
Author(s):  
F. Lebrun ◽  
A. Francois ◽  
M. Vergnet ◽  
L. Lebaron-Jacobs ◽  
P. Gourmelon ◽  
...  
Keyword(s):  
Ionizing Radiation ◽  
Enzyme Activities ◽  
Plasma Membranes ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Marker Enzyme ◽  
Γ Irradiation ◽  
Ussing Chambers ◽  
Radiation Induced ◽  
Total Body

The aim of this study was to determine whether ionizing radiation modifies muscarinic regulation of intestinal mucosal function. Rats exposed to total body 8-Gy γ-irradiation or sham irradiated were studied up to 21 days after irradiation. Basal and carbachol-stimulated short-circuit current ( I sc) and transepithelial conductance ( G t) of stripped ileum were determined in Ussing chambers. Muscarinic receptor characteristics using the muscarinic antagonist [3H]quinuclidinyl benzilate and three unlabeled antagonists were measured in small intestinal plasma membranes together with two marker enzyme activities (sucrase, Na+-K+-ATPase). Enzyme activities were decreased 4 days after irradiation ( day 4). Basal electrical parameters were unchanged. Maximal carbachol-induced changes in I sc and G t were increased at day 4 (maximal Δ I sc = 195.8 ± 14.7 μA/cm2, n = 19, vs. 115.4 ± 8.2 μA/cm2, n = 63, for control rats) and unchanged at day 7. Dissociation constant was decreased at day 4 (0.73 ± 0.29 nM, n = 10, vs. 2.14 ± 0.39 nM, n = 13, for control rats) but unchanged at day 7, without change in binding site number. Thus total body irradiation induces a temporary stimulation of cholinergic regulation of mucosal intestinal function that may result in radiation-induced diarrhea.

scholarly journals ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF BRUSH BORDERS FROM RABBIT KIDNEY

1970 ◽  
Vol 47 (3) ◽  
pp. 637-645 ◽  
Author(s):  
Sosamma J. Berger ◽  
Bertram Sacktor
Keyword(s):  
Brush Border ◽  
Renal Cortex ◽  
Biochemical Characterization ◽  
Rabbit Kidney ◽  
Renal Tubules ◽  
Marker Enzymes ◽  
Alkaline Phosphatases ◽  
Brush Borders ◽  
Specific Activities

A technique for the isolation of intact brush borders from rabbit renal cortex was evaluated. The procedure was monitored by phase and electron microscopy and marker enzymes, i.e. ATP:NMN adenylyl transferase, nuclear; cytochrome oxidase, mitochondrial; ß-glucuronidase, lysosomal; and glucose-6-Pase, microsomal; and indicated an essentially pure preparation of brush borders. The disaccharidase, trehalase, previously reported in renal tubules, was localized uniquely in brush borders. Maltase was also found; the specific activities of the two enzymes in the brush borders were increased 10- to 20-fold. Other disaccharidases, such as sucrase, isomaltase, lactase, and cellobiase, were absent. It is suggested that trehalase and maltase are appropriate candidates for marker enzymes of the renal brush border. Isolated brush borders possessed a ouabain-sensitive (Na+ + K+) ATPase, an oligomycin-insensitive Mg++ ATPase, and a Ca++-activated ATPase. Alkaline phosphatases, dephosphorylating ß-glycero-P, and trehalose-6-P were also present. The specific activities of these enzymes were increased three-to-five fold in the brush-border preparations; however, activities were found in other subcellular fractions of the renal cortex. Hexokinase, although evident in the isolated brush border, was found prominently associated with other membranous fractions. Phosphoglucomutase and UDPG pyrophosphorylase were localized in the soluble fraction of the renal cortex.

scholarly journals Isolation and partial characterization of the plasma membrane of the sea urchin egg.

1980 ◽  
Vol 87 (1) ◽  
pp. 248-254 ◽  
Author(s):  
W H Kinsey ◽  
G L Decker ◽  
W J Lennarz
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Sea Urchin ◽  
Plasma Membranes ◽  
Binding Property ◽  
Marker Enzyme ◽  
Surface Complex ◽  
Sea Urchin Egg ◽  
Peripheral Proteins ◽  
Cortical Vesicles

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.

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