scholarly journals Characterization of calcium-ion-activated adenosine triphosphatase in the plasma membrane of rat mast cells

1976 ◽  
Vol 156 (3) ◽  
pp. 691-700 ◽  
Author(s):  
P H Cooper ◽  
D R Stanworth
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Adenosine Triphosphatase ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Ph Optimum ◽  
Peritoneal Mast Cells ◽  
Triton X ◽  
Calcium Ion Activated

The properties of a Ca2+ activated adenosine triphosphatase shown to be present in homogenates of purified rat peritoneal mast cells were investigated. The enzyme was activated by Ca2+, Mg2+, and to a lesser extent by Mn2+ and Co2+. Ca2+ alone was necessary for full activity and the further addition of Mg2+ did not have any effect. The chelating agents EGTA (ethanedioxybis(ethylamine)tetra-acetate) and EDTA completely inhibited the reaction. The pH optimum was 7.8. Reduced glutathione, cysteine, dithiothreitol, N-ethylmaleimide, urea, ADP, NaF, increasing ionic strength and Triton X-100 all inhibited the reaction. On subcellular fractionation of mast-cell homogenates by density-gradient centrifugation, the distribution of Ca2+ activated adenosine triphosphatase resembled that of 5′-nucleotidase, but differed from that of the other markers used, suggesting localization in the plasma membrane. Further experiments indicated that the enzyme is present on the external surface of the plasma membrane.

scholarly journals Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system

1975 ◽  
Vol 150 (3) ◽  
pp. 537-551 ◽  
Author(s):  
P H Cooper ◽  
J N Hawthorne
Keyword(s):  
Phosphatase Activity ◽  
Metal Ion ◽  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Sodium Deoxycholate ◽  
Subcellular Fractionation ◽  
Kidney Cortex ◽  
Ph Optimum ◽  
Triton X ◽  
Hydrolysis Of

Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.

scholarly journals Characterization of a salt-extractable phosphatidylinositol synthase from rat pituitary-tumour membranes

1989 ◽  
Vol 257 (3) ◽  
pp. 639-644 ◽  
Author(s):  
A B Cubitt ◽  
M C Gershengorn
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Pituitary Tumour ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Rat Pituitary ◽  
Phosphatidylinositol Synthase ◽  
Triton X

Solubilization of phosphatidylinositol (PtdIns) synthase (CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) from rat pituitary (GH3) tumours was investigated. PtdIns synthase activity was partially extracted from crude membranes by 3 M-KCl. Prior separation of membranes revealed that a greater proportion of plasma-membrane PtdIns synthase activity was salt-extractable than was endoplasmic reticulum activity. The activity of the salt-extracted enzyme was maximized by low concentrations of 3-(3-cholamidopropyl) dimethylammonio-1-propanesulphonate (CHAPS; 0.5 mM), Triton X-100 (0.1 mM) or a phospholipid mixture (0.05 mg/ml), but higher concentrations of detergents were inhibitory. The activity of salt-extracted PtdIns synthase was 0.25 +/- 0.08 nmol/min per mg of protein. Salt-extracted PtdIns synthase activity was dependent on Mg2+ (maximal at 0.1 mM) and Mn2+ (maximal at 5 mM), and its pH optimum was in the range 7.0-7.5. The apparent Km for myo-inositol (in the presence of 0.1 mM-CDP-diacylglycerol) was 0.06 mM, and that for CDP-diacylglycerol (at 0.1 mM-myo-inositol) was 0.21 mM. Salt-extracted PtdIns synthase activity was potently inhibited by Ca2+ (50% inhibition at 1 microM), with over 90% inhibition at 10 microM-Ca2+. These data imply the existence of two forms of membrane-associated PtdIns synthase, namely salt-extractable and salt-resistant, with different intracellular localizations. The salt-extractable form of this enzyme may be a useful preparation for further characterization and purification of mammalian PtdIns synthase.

scholarly journals Characterization of a retinylmonophosphatase in the plasma membrane of mouse brain

1986 ◽  
Vol 237 (3) ◽  
pp. 625-630 ◽  
Author(s):  
J V O'Fallon ◽  
B P Chew
Keyword(s):  
Plasma Membrane ◽  
Mouse Brain ◽  
Ph Optimum ◽  
Nitrophenyl Phosphate ◽  
Phosphorylated Compounds ◽  
Triton X ◽  
Thiamin Pyrophosphate ◽  
Substrate Concentrations ◽  
Distinct Role

Retinylmonophosphatase (RMPase) activity in mouse brain paralleled the subcellular distribution of the plasma-membrane marker Na+ + K+-dependent ATPase. The enzyme had a pH optimum between 5.5 and 7.0. The enzyme demonstrated linear kinetics with respect to time and both protein and substrate concentrations. RMPase was saturated by low retinyl monophosphate (RMP) concentrations and exhibited an apparent Km of 4.6 microM. The enzyme did not require MgCl2 for activity, and in fact assays were routinely run in the presence of 10 mM-Na2EDTA. In general, detergents inhibited the enzyme, with 0.05% Triton X-100 causing a 30% loss of activity. Phosphatidic acid was also inhibitory, but phosphatidylcholine and sphingomyelin stimulated phosphatase activity. RMPase was inhibited 35% by 5 mM concentrations of fluoride, phosphate or pyrophosphate. A series of other phosphorylated compounds, including glucose 6-phosphate, alpha-glycerophosphate, ATP, AMP, p-nitrophenyl phosphate and thiamin pyrophosphate, showed little or no inhibition. RMPase activity differed in several characteristics from that previously reported for dolichylmonophosphatase. It is concluded that RMP could play a distinct role in the plasma membrane.

scholarly journals Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-l-fucosidase from rat testis and epididymal spermatozoa

1996 ◽  
Vol 318 (3) ◽  
pp. 821-831 ◽  
Author(s):  
Manuel AVILÉS ◽  
Irene ABASCAL ◽  
José Angel MARTÍNEZ-MENÁRGUEZ ◽  
María Teresa CASTELLS ◽  
Sheri R. SKALABAN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Light And Electron Microscopy ◽  
Enzymic Activity ◽  
Epididymal Sperm ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Neutral Ph

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum α-l-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for α-l-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the α-l-fucosidase activity was associated with the 48000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of α-l-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH–activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm–egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm–egg interactions.

Fine-Structural Localization of Adenosine Triphosphatase in the Rectum of Calliphora

1968 ◽  
Vol 3 (1) ◽  
pp. 17-32
Author(s):  
M. J. BERRIDGE ◽  
B. L. GUPTA
Keyword(s):  
Plasma Membrane ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Fluid Transport ◽  
Microsomal Fraction ◽  
Adenosine Diphosphate ◽  
Structural Basis ◽  
Osmotic Gradient ◽  
Ph Optimum ◽  
Structural Localization

Adenosine triphosphatase (ATPase) activity in the rectal papillae of Calliphora has been studied by biochemical and histochemical techniques. The microsomal fraction contained a Mg2+-activated ATPase with a pH optimum of 8.0. The enzyme was not stimulated by the addition of Na+ plus K+ and was insensitive to ouabain. Histochemical studies using modifications of the Wachstein-Meisel method showed that at pH 7.2 this Mg2+-activated ATPase was specifically localized on the intracellular surface of the lateral plasma membranes. A similar though less intense reaction was obtained with adenosine diphosphate and inosine triphosphate, but not with guanosine triphosphate, uridine triphosphate or β-glycerophosphate as substrates. At an acid pH (6.6-6.8), very little reaction occurred on the lateral plasma membrane but some reaction product was present in mitochondria and nuclei. Very little enzyme activity was found in the flattened rectal epithelium. These results are discussed in relation to the available data on transport ATPases and on the structural basis of fluid transport by rectal papillae. It is proposed that the ATPase localized on the stacks of lateral plasma membrane may be involved with ion secretion into the intercellular spaces to create the osmotic gradient necessary to extract water from the lumen.

Diphosphatase Related to Lipid Metabolism and Gluconeogenesis in Cucumber Cotyledons: Localization in Plasma Membrane and Etioplasts but not in Glyoxysomes

1989 ◽  
Vol 44 (11-12) ◽  
pp. 937-945 ◽  
Author(s):  
Thomas Kirsch ◽  
Barbara Rojahn ◽  
Helmut Kindi
Keyword(s):  
Plasma Membrane ◽  
Gradient Centrifugation ◽  
Integral Membrane Protein ◽  
Inhibitory Effect ◽  
Inorganic Pyrophosphatase ◽  
Ph Optimum ◽  
Bivalent Cations ◽  
Isoelectric Points ◽  
Main Portion ◽  
Molecular Sieving

Abstract Diphosphatase (inorganic pyrophosphatase) activity was localized within compartments of cotyledons of germinating cucumber seeds during the stage of maximal conversion of fat into carbohydrates. At this stage, almost 2 mol pyrophosphate are produced during the formation of one mole sucrose from 0.28 mol triglyceride. When organelles of the 2000 x g pellet or 10,000 x g pellet were separated by density gradient centrifugation and gradient flotation, the diphosphatase activity paralleled the profiles of markers of the plastid stroma but was virtually absent from the glyoxysomes. Within the fraction of small vesicles and membranes, diphosphatase was attributed to the plasma membrane. The main portion of diphosphatase, contained in the plastids, was partially purified by chromatography on anion exchange resin and molecular sieving, leading to a 75-fold enrichment compared to the stroma fraction. Trace amounts of diphosphatase observed in the glyoxysomal fraction were analyzed in the same way. Comparison of the isoelectric points and the activity profile at different pH values and the inhibitory effect of the various cations indicated that the trace amounts of diphosphatase activity in the glyoxysome fraction represented contaminations originating from the plastids. The plasma membrane form of diphosphatase is an integral membrane protein which was solubilized with octylglucoside. It was shown to differ from the plastid form in pH optimum and sensitivity towards bivalent cations. All forms of diphosphatase were clearly distinguished from other phosphohydrolytic activities.

scholarly journals Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles

1987 ◽  
Vol 244 (1) ◽  
pp. 219-224 ◽  
Author(s):  
J M Jacobs ◽  
N J Jacobs
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chlorophyll Synthesis ◽  
Yeast Mitochondria ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Haem Biosynthesis ◽  
Triton X ◽  
Polypeptide Band

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.

scholarly journals Olopatadine Inhibits Exocytosis in Rat Peritoneal Mast Cells by Counteracting Membrane Surface Deformation

2015 ◽  
Vol 35 (1) ◽  
pp. 386-396 ◽  
Author(s):  
Asuka Baba ◽  
Masahiro Tachi ◽  
Yoshio Maruyama ◽  
Itsuro Kazama
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Lipid Bilayers ◽  
Surface Deformation ◽  
Lucifer Yellow ◽  
Membrane Surface ◽  
Water Soluble ◽  
Confocal Imaging ◽  
Peritoneal Mast Cells ◽  
Rat Peritoneal Mast Cells

Backgroud/Aims: Besides its anti-allergic properties as a histamine receptor antagonist, olopatadine stabilizes mast cells by inhibiting the release of chemokines. Since olopatadine bears amphiphilic features and is preferentially partitioned into the lipid bilayers of the plasma membrane, it would induce some morphological changes in mast cells and thus affect the process of exocytosis. Methods: Employing the standard patch-clamp whole-cell recording technique, we examined the effects of olopatadine and other anti-allergic drugs on the membrane capacitance (Cm) in rat peritoneal mast cells during exocytosis. Using confocal imaging of a water-soluble fluorescent dye, lucifer yellow, we also examined their effects on the deformation of the plasma membrane. Results: Low concentrations of olopatadine (1 or 10 µM) did not significantly affect the GTP-γ-S-induced increase in the Cm. However, 100 µM and 1 mM olopatadine almost totally suppressed the increase in the Cm. Additionally, these doses completely washed out the trapping of the dye on the cell surface, indicating that olopatadine counteracted the membrane surface deformation induced by exocytosis. As shown by electron microscopy, olopatadine generated inward membrane bending in mast cells. Conclusion: This study provides electrophysiological evidence for the first time that olopatadine dose-dependently inhibits the process of exocytosis in rat peritoneal mast cells. Such mast cell stabilizing properties of olopatadine may be attributed to its counteracting effects on the plasma membrane deformation in degranulating mast cells.

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