scholarly journals The Characterization of Plasma Membrane-Bound Tubulin of Cauliflower Using Triton X-114 Fractionation

1997 ◽  
Vol 115 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
A. Sonesson ◽  
M. Berglund ◽  
I. Staxen ◽  
S. Widell
Keyword(s):  
Plasma Membrane ◽  
Membrane Bound ◽  
Triton X

scholarly journals Subcellular distribution and characterization of acetylcholinesterase activities from sheep platelets: relationships between temperature- dependence and environment

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 737-744
Author(s):  
J Sanchez-Yague ◽  
JA Cabezas ◽  
M Llanillo
Keyword(s):  
Acetylcholinesterase Activity ◽  
Break Point ◽  
Molecular Forms ◽  
Kinetic Properties ◽  
Multiple Molecular Forms ◽  
Key Enzyme ◽  
Membrane Bound ◽  
Hill Coefficients ◽  
Triton X

Acetylcholinesterase is a key enzyme in cholinergic neurotransmission for hydrolyzing acetylcholine and has been shown to possess arylacylamidase activity in addition to esterase activity. The enzyme is found at various loci, where its functional significance remains to be clarified, and it exists in multiple molecular forms. Sheep platelets have been shown to exhibit acetylcholinesterase activity associated with plasma membrane (Bp), endoplasmic reticulum (Cp), mitochondria granules (Dp), and soluble (As) fractions. These activities show differences in some physicochemical and kinetic properties. The soluble acetylcholinesterase is the most thermostable, and the enzyme from the Cp fractions shows the lowest affinity for the acetylthiocholine substrate and the strongest inhibition by fluoride. In all cases a noncompetitive inhibition of the enzyme by this ion is found. When membrane-bound acetylcholinesterases were assayed at temperatures between 12 degrees C and 33 degrees C, the Arrhenius plots of all activities exhibited a break point at about 17 degrees C. This discontinuity was abolished by addition of detergent to the assay medium (0.02% Triton X-100, final concentration). Their Hill coefficients were calculated in the presence of fluoride, showing unitary values in all cases, which points to a noncooperative effect and nonallosteric behavior in the particulate enzyme. These results suggest that the sheep platelet acetylcholinesterase associated with membrane-bound systems is modulated by the physical state of its environment, despite the fact that the enzyme might be lipid- or nonlipid-dependent.

scholarly journals Localization of calcium in red blood cells.

1983 ◽  
Vol 31 (9) ◽  
pp. 1109-1116 ◽  
Author(s):  
M Borgers ◽  
F J Thone ◽  
B J Xhonneux ◽  
F F De Clerck
Keyword(s):  
Plasma Membrane ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Potassium Phosphate ◽  
Human Red Blood Cells ◽  
A 23187 ◽  
Nonionic Detergent ◽  
Capillary Endothelial Cells ◽  
Membrane Bound ◽  
Triton X

The distribution of calcium is demonstrated in human red blood cells (RBC) with a combined phosphate-pyroantimonate technique (PPA). Freshly collected blood and tissue biopsies were initially fixed in potassium phosphate-glutaraldehyde and the complexed calcium was subsequently visualized on Vibratome sections with potassium pyroantimonate. The majority of cells, both in isolated as well as "in situ" preparations, show a fine granular precipitate located at the inner leaflet of the plasma membrane. A minority of cells lack these membrane-associated deposits, exhibiting instead a random distribution of very fine precipitate in their cytoplasm. Capillary endothelial cells and pericytes are devoid of plasma membrane-bound precipitate. When irreversible crenation of RBC is induced by exposure to ionophore A 23187 and calcium, the sphero-echinocytes loose their membrane-bound precipitate, whereas the cells that retain their discocyte shape demonstrate the usual pattern of membrane-bound deposits. Contrarily, cells showing reversible shape changes induced by either A 23187-Ca2+ challenge, by adenosine triphosphate depletion during aging, or contact with lysolecithin, retain or regain the membrane-bound calcium. This cytochemical demonstrable calcium at the inner leaflet of the plasma membrane is probably bound to acidic phospholipids, since it is readily extractable with the nonionic detergent Triton X-100.

scholarly journals Physical and kinetic properties of a plasma-membrane-bound β-d-glucosidase (cellobiase) from midgut cells of an insect (Rhynchosciara americana larva)

1983 ◽  
Vol 213 (1) ◽  
pp. 43-51 ◽  
Author(s):  
C Ferreira ◽  
W R Terra
Keyword(s):  
Plasma Membrane ◽  
Gradient Centrifugation ◽  
Soluble Form ◽  
Kinetic Properties ◽  
Carbonium Ion ◽  
Wide Range ◽  
Catalytic Constant ◽  
Rhynchosciara Americana ◽  
Membrane Bound ◽  
Triton X

The midgut caecal cells from Rhynchosciara americana larvae possess a plasma-membrane-bound beta-D-glucosidase (cellobiase, EC 3.2.1.21), which is recovered (75-95%) in soluble form both after treatment with Triton X-100 and after treatment with papain. The Triton X-100-solubilized beta-D-glucosidase displays Mr106000 and pI 5.4, whereas the papain-released beta-D-glucosidase shows Mr65000 and pI 4.7. Thermal inactivations of the detergent-solubilized and the papain-released forms of beta-D-glucosidase both follow apparent first-order kinetics with similar half-lives. The papain-released beta-D-glucosidase, after being purified by density-gradient centrifugation, hydrolyses beta-D-glucosides, beta-D-galactosides and beta-D-fucosides at the same active site, as inferred from experiments of competition between substrates. The beta-D-glucosidase seems to operate in accordance with rapid-equilibrium kinetics, since the Km (0.61 mM) for the enzyme is constant over a wide range of pH. The hydrolysis of the beta-D-glucosidic bond catalysed by the beta-D-glucosidase occurs without inversion of configuration, delta-gluconolactone is a strong (Ki 0.5 microM) inhibitor of the enzyme and substituents in the substrate aglycone affect the catalytic constant of the reaction. These data support the assumption that the mechanism of the reaction catalysed by the beta-D-glucosidase involves the intermediary formation of a carbonium ion, rather than a glucosyl-enzyme intermediate.

Characterization of plasma membrane bound cation-stimulated ATPase isolated from the roots of Cucumis sativus

1988 ◽  
Vol 66 (7) ◽  
pp. 1470-1473 ◽  
Author(s):  
Abdul Razaque Memon
Keyword(s):  
Plasma Membrane ◽  
Cucumis Sativus ◽  
Direct Interaction ◽  
Monovalent Cation ◽  
Phase System ◽  
Plasma Membrane Atpase ◽  
Cucumis Sativus L ◽  
Membrane Bound ◽  
Cucumber Roots

A membrane fraction enriched with Mg2+- or Mn2+-dependent, monovalent cation stimulated ATPase was isolated from cucumber roots (Cucumis sativus L.) by an aqueous two-polymer phase system of Dextran T500 (6.5%, w/w) and polyethylene glycol (PEG) 3350 (6.5%, w/w) at pH 7.8. The ATPase activity associated with the upper PEG-rich fraction (plasma membrane) was characterized. The optimum pH for the activation by Mg2+ and Mn2+ was in the range 5.8–6.0. The activity was substrate specific for ATP. Kinetics with Mg2+ or Mn2+ followed a simple Michaelis–Menten relationship. The apparent Km for Mg2+ activation (0.60 mM) of the ATPase was about twice that of the apparent Km for Mn2+ (0.38 mM). ATPase was stimulated by monovalent cations and showed an order of cation preference of [Formula: see text]. Calcium inhibited the plasma membrane ATPase, apparently by a direct interaction with ATPase rather than by disrupting the MgATP2− complex.

scholarly journals Identification and characterization of the 2D6 and Mr 23000 antigens on the plasma membrane of rat spermatozoa

1987 ◽  
Vol 241 (2) ◽  
pp. 353-360 ◽  
Author(s):  
R Jones ◽  
C R Brown
Keyword(s):  
Plasma Membrane ◽  
Antigenic Determinant ◽  
Egg Recognition ◽  
Western Blots ◽  
One Dimensional ◽  
Antigen Present ◽  
Membrane Bound ◽  
Species Specific ◽  
Identification And Characterization

Previous investigations [Jones, Brown, von Glos & Gaunt (1985) Exp. Cell Res. 156, 31-44] have demonstrated the appearance of a new antigenic determinant (recognized by monoclonal antibody 2D6) on the plasma membrane of rat spermatozoa during post-testicular maturation in the epididymis. Identification of the 2D6 antigen on Western blots from one-dimensional SDS/polyacrylamide gels revealed that it co-migrated with a membrane protein (designated Mr 23,000 antigen) present on testicular and immature germ cells, suggesting that one antigen might be a modified version of the other. In the present work, however, we demonstrate that, although they have similar Mr and are present in soluble and membrane-bound forms, the 2D6 and Mr 23,000 antigens are biochemically and immunologically distinct molecules. The properties of the antigens are described and compared. The Mr 23,000 antigen is present on both testicular and cauda epididymidal spermatozoa, has a pI of 6.1, contains no detectable carbohydrate, is not tissue-specific and is degraded by V8 protease. By contrast, the 2D6 antigen is glycosylated, has a broad pI from 4.5 to 6.1, is tissue- and species-specific and is resistant to digestion with V8 protease. Its role in sperm-egg recognition is discussed.

Characterization of Acetylcholinesterase in Caco-2 Cells

2002 ◽  
Vol 227 (7) ◽  
pp. 480-486 ◽  
Author(s):  
Lauren R. Plageman ◽  
Giovanni M. Pauletti ◽  
Kenneth A. Skau
Keyword(s):  
Active Site ◽  
Substrate Inhibition ◽  
Extracellular Fluid ◽  
Acetylcholinesterase Activity ◽  
Monomeric Form ◽  
Catalytic Center ◽  
Membrane Bound ◽  
Degree Of Differentiation ◽  
Triton X

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was solubilized from cultured Caco-2 cells. It was established that this enzyme activity is acetylcholinesterase by substrate specificity (acetylthiocholine, acetyl-β-methylthiocholine>propionylthiocholine>butyrylthiocholine), substrate inhibition, and specificity of inhibitors (BW284c51>iso-OMPA). The acetylcholinesterase activity increased proportional to the degree of differentiation of the cells. Most of the enzyme was membrane bound, requiring detergent for solubilization, and the active site faced the external fluid. Only one peak of activity, which corresponded to a monomeric form, could be detected on linear sucrose density gradients. The sedimentation of this form of the enzyme was shifted depending on whether Triton X-100 or Brij 96 detergent was used. These results indicate that the epithelial-derived Caco-2 cells produce predominantly an amphiphilic, monomeric form of acetylcholinesterase that is bound to the plasma membrane and whose catalytic center faces the extracellular fluid.

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