Molecular Forms of Purified Cytoplasmatic and Membrane Bound Bovine-Brain-Acetylcholinesterase Solubilized by Different Methods

1981 ◽  
Vol 36 (11-12) ◽  
pp. 968-972 ◽  
Author(s):  
K.-P. Rueß ◽  
M. Liefländer
Keyword(s):  
Molecular Form ◽  
Bovine Brain ◽  
Water Soluble ◽  
Molecular Forms ◽  
Enzyme Complex ◽  
Detergent Enzyme ◽  
Neuronal Tissues ◽  
Membrane Bound ◽  
Triton X ◽  
Brain Acetylcholinesterase

Abstract Membrane bound, Triton X-100 solubilized bovine nucleus caudatus acetylcholinesterase is sedimenting in presence of Triton X-100 concentrations higher than the CMC as a 10.5 S-detergent-enzyme complex. There is evidence that this complex does neither represent the molecular enzyme arrangement present in the membrane, nor the molecular form originally released from the membrane. The purified, cytoplasmatic acetylcholinesterase is sedimenting as a 10.5 S-form too. This form is clearly to be distinguished from the detergent enzyme complex, for it is obviously not capable of aggregating, whereas the 10.5 S-detergent-enzyme complex aggregates on detergent removal to defined water soluble oligomers with sedimentation coefficients of 16 S (700000 ± 10000), 20.6 S (960000 ± 60000) and 23.3 S (~ 1200000). In contrast to acetylcholinesterase from erythrocytes this aggregation is not easily reversibly by incubation with Triton X-100, reflecting differences in the hydrophobic part of the enzymes. Purified acetylcholinesterase solubilized without detergent under autolytic or tryptic conditions is mainly sedimenting as a 4.5 S-form. Such slow sedimenting forms detected in crude solubilisates of neuronal tissues, may originate at least partially form autolytic solubilization.

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 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

Abstract 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.

On the Membrane Association of Platelet Glycocalicin.

1979 ◽  
Author(s):  
N.O. Solum ◽  
I. Hagen ◽  
C. Filion-Myklebust ◽  
T. Stabæk
Keyword(s):  
Molecular Form ◽  
Water Soluble ◽  
Freezing And Thawing ◽  
Reactive Material ◽  
Calcium Dependent ◽  
Hydrophilic Nature ◽  
Sds Page ◽  
Complex Protein ◽  
Slow Moving ◽  
Triton X

More observations indicate that the hydrophilic protein glycocalicin found in soluble fractions after homogenization of platelets in the presence of calcium ions, exists in a different molecular form on the intact platelet. Thus, SDS-PAGE of SDS-dissolved whole platelets show glycocalicin to be derived from a more complex protein than the solubilized protein itself. Crossed immunoelectrophoresis of Triton x-100 extracts of platelets revealed anti-glycocalicin-reactive material existing as two components with different eloctrophoretic mobilities and with the fast-moving one corresponding to highly purified water-soluble glycocalicin. Crossed hydrophobic interaction immunoelectrophoresis confirmed the hydrophilic nature of this component, whereas the slow-moving component showed the characteristic retardation of an amphiphilic membrane protein. Glycocalicin is solubilized during freezing and thawing of platelets in buffer, immunoquantitation (Laurell) of solubilized glycocalicin showed that EDTA(3,6mM), NEM (4.0mM) and IAA(4.0mM)reduced solubilization by 87%, 65% and 57%, respectively, and also inhibited calcium-dependent protease of platelet homogenatos indicating that the solubilization may be enzyme-mediated.

scholarly journals Molecular forms of phosphatase and ribonuclease in phosphate deficient and N,N-dimethylmorpholinium chloride treated Spirodela oligorrhiza (Lemnaceae)

2015 ◽  
Vol 48 (1) ◽  
pp. 65-85 ◽  
Author(s):  
J. S. Knypl
Keyword(s):  
Gel Filtration ◽  
Molecular Forms ◽  
Membrane Material ◽  
Alkaline Phosphatases ◽  
Ph Optimum ◽  
Membrane Bound ◽  
Soluble Acid ◽  
Spirodela Oligorrhiza ◽  
Triton X ◽  
Membrane Bound Enzymes

Soluble, membrane bound, and extracellular phosphatases (EC 3.1.3.2 and 3.1.3.1) of control, N,N-dimethylmorphołinłum chloride (DMMC) treated, and phosphate deficient (-P) axenic <i>Spirodela oligorrhiza</i> plants were analysed by Sephadex G-150 gel filtration. Soluble, acid enzymes of control plants were separated into two molecular forms with apparent MW ≥ 400 000 and 85 000. Phosphatase with MW 34 000 replaced the latter isoenzyme in the presence of DMMC. Two alkaline enzymes with apparent MW 210 000 and 36 000 were detected in -P plants. Triton X-100 solubilized a number of acid and alkaline phosphatases from membrane material. DMMC caused the appearance of two membrane bound enzymes (MW 48 000 and 14 000) which were not detected in the control. Senescimg control and DMMC treated plants released an acid phosphatase (MW 48 000; pH optimum 5.2) into the nutrient medium. -P plants released, in addition ,an alkaline phosphatase (MW 170,000; pH optimum 7.8-8.2). Ribonucleases (EC 2.7.7.17.) with apparent MW 31000 and 28 000 daltons were induced by DMMC and -P, respectively.

The occurrence of pituitary prolactin depletion–transformation in lactating rats: dependence on strain of rats, homogenization conditions and method of assay

1987 ◽  
Vol 113 (1) ◽  
pp. 71-80 ◽  
Author(s):  
D. M. Lawson ◽  
D. J. Haisenleder ◽  
R. R. Gala ◽  
J. A. Moy
Keyword(s):  
Molecular Form ◽  
Gel Filtration ◽  
Molecular Forms ◽  
Sprague Dawley ◽  
Rapid Changes ◽  
Prolactin Content ◽  
Pituitary Prolactin ◽  
Triton X ◽  
Benzamidine Hydrochloride ◽  
Molecular Nature

ABSTRACT The objectives of this study were to determine (1) whether pre-release transformation (depletion) of pituitary prolactin occurs as the result of suckling to the same extent in several strains of lactating rats, (2) the molecular nature of the transformed hormone, (3) whether the quantity of transformed (depleted) prolactin recovered is dependent upon the type of homogenization buffer used and (4) whether the method of assay influences the extent to which transformed prolactin is detected. During the course of these experiments other factors such as the methods of handling and storing pituitaries and homogenates were also found to influence the amount of prolactin recovered. The results indicated that transformation of prolactin is a very labile event which is affected by many factors. Strain and supplier of rats was critical to the observation of suckling-induced depletion of prolactin, with Spartan- and Holtzman-derived Sprague–Dawley strains exhibiting the most consistent responses. When transformation was observed, it mattered little which buffer was used for homogenization; however, alkaline or acidic buffers extracted more prolactin than did neutral buffers. Triton X-100 also significantly enhanced the efficiency of extraction by neutral buffers. Maintaining pituitaries on dry ice immediately upon removal from the animal increased the amount of prolactin recovered, as did freezing the homogenate for 1–5 weeks before assay. The addition of the protease inhibitor, benzamidine hydrochloride, did not affect the pituitary content of prolactin. Assay of prolactin by polyacrylamide electrophoresis and densitometry yielded more prolactin than either radioimmunoassay or the Nb2 lymphoma bioassay. The molecular nature of pituitary prolactin, extracted at neutral pH, as judged by gel filtration was altered slightly but consistently by suckling, such that large molecular forms increased at the expense of the smallest molecular form. We conclude from these studies that great care must be exercised when attempting to characterize dynamic changes in pituitary prolactin content in lactating rats. Strain and supplier of rats, methods of handling and storing pituitaries, types of buffers used for homogenization and methods of assay all influence the amount of prolactin recovered and can influence the extent to which rapid changes in pituitary prolactin are detected. J. Endocr. (1987) 113, 71–80

Dissociation of imino proton(s) of a highly water-soluble metal-free phthalocyanine and determination of its pKa value in water

2013 ◽  
Vol 17 (06n07) ◽  
pp. 447-453 ◽  
Author(s):  
Hiroaki Isago ◽  
Harumi Fujita
Keyword(s):  
Aqueous Media ◽  
Thermal Reaction ◽  
Water Soluble ◽  
Acid Dissociation ◽  
Acid Dissociation Constant ◽  
Metal Free ◽  
Imino Proton ◽  
Pka Value ◽  
Triton X

Dissociation of imino proton(s) in the cavity of the macrocycle of a highly water-soluble, metal-free phthalocyanine ( H 2( H 4 tsppc ); where H 4 tsppc denotes tetrakis{(2′,6′-dimethyl-4′-sulfonic acid)phenoxy}phthalocyaninate) in ethanolic and aqueous solutions has spectrophotometrically been investigated. The spectral changes associated with reaction with NaOH have been found to involve one-proton transfer process in aqueous media while two-protons process in ethanolic media. The acid-dissociation constant of the first imino proton in water (in the presence of Triton X-100) has been determined to be 12.5 ± 0.2 (as pKa) at 25 °C. The doubly deprotonated species in EtOH has been easily converted to its corresponding cobalt(II) derivative by thermal reaction with anhydrous CoCl 2.

scholarly journals Fast Dissolving Electrospun Nanofibers Fabricated from Jelly Fig Polysaccharide/Pullulan for Drug Delivery Applications

Polymers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 241
Author(s):  
Thangavel Ponrasu ◽  
Bei-Hsin Chen ◽  
Tzung-Han Chou ◽  
Jia-Jiuan Wu ◽  
Yu-Shen Cheng
Keyword(s):  
Drug Delivery ◽  
Water Vapor Permeability ◽  
Electrospun Nanofibers ◽  
Average Diameter ◽  
Xrd Analysis ◽  
Water Soluble ◽  
Vapor Permeability ◽  
Uv Spectrum ◽  
Sem Images ◽  
Triton X

The fast-dissolving drug delivery systems (FDDDSs) are developed as nanofibers using food-grade water-soluble hydrophilic biopolymers that can disintegrate fast in the oral cavity and deliver drugs. Jelly fig polysaccharide (JFP) and pullulan were blended to prepare fast-dissolving nanofiber by electrospinning. The continuous and uniform nanofibers were produced from the solution of 1% (w/w) JFP, 12% (w/w) pullulan, and 1 wt% Triton X-305. The SEM images confirmed that the prepared nanofibers exhibited uniform morphology with an average diameter of 144 ± 19 nm. The inclusion of JFP in pullulan was confirmed by TGA and FTIR studies. XRD analysis revealed that the increased crystallinity of JFP/pullulan nanofiber was observed due to the formation of intermolecular hydrogen bonds. The tensile strength and water vapor permeability of the JFP/pullulan nanofiber membrane were also enhanced considerably compared to pullulan nanofiber. The JFP/pullulan nanofibers loaded with hydrophobic model drugs like ampicillin and dexamethasone were rapidly dissolved in water within 60 s and release the encapsulants dispersive into the surrounding. The antibacterial activity, fast disintegration properties of the JFP/pullulan nanofiber were also confirmed by the zone of inhibition and UV spectrum studies. Hence, JFP/pullulan nanofibers could be a promising carrier to encapsulate hydrophobic drugs for fast-dissolving/disintegrating delivery applications.

scholarly journals Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 508-514 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Time Dependent ◽  
Binding Studies ◽  
Fibrinogen Binding ◽  
Human Thrombin ◽  
Membrane Bound ◽  
Independent Association ◽  
Triton X

Abstract Previous studies indicated a correlation between the formation of EDTA- resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA- resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA- resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet- fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

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