scholarly journals Purification, characterization and cellular localization of 5′-nucleotidase from Torpedo electric organ

1987 ◽  
Vol 245 (3) ◽  
pp. 805-810 ◽  
Author(s):  
E J M Grondal ◽  
H Zimmermann
Keyword(s):  
Enzyme Activity ◽  
Concanavalin A ◽  
Cellular Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Electric Organ ◽  
Immunohistochemical Analysis ◽  
Maximal Velocity ◽  
Nitrophenyl Phosphate ◽  
Torpedo Electric Organ ◽  
Triton X

5′-Nucleotidase was isolated from the electric organ of the electric ray Torpedo marmorata after solubilization in Triton X-100 and deoxycholate by affinity chromatography on concanavalin A-Sepharose and AMP-Sepharose. The purified enzyme has a Km for AMP of 38 microM, with a maximal velocity of 31 units/mg of protein. Of the purine and pyrimidine mononucleotides, AMP is hydrolysed most effectively. beta-Glycerophosphate, phosphoenolpyruvate and p-nitrophenyl phosphate are not substrates for the enzyme. Adenosine 5′-[alpha, beta-methylene]diphosphate, ADP and ATP are competitive inhibitors in this order of potency. Concanavalin A inhibits enzyme activity in a non-competitive manner. Whereas Mg2+, Ca2+ and Sr2+ activate enzyme activity in the millimolar range, Hg2+, and in particular Pb2+ and Zn2+, inhibit enzyme activity. On SDS/polyacrylamide-gel electrophoresis the enzyme has an apparent Mr of 62000, whereas that of the native deoxycholate-enzyme complex is 131000. An antiserum raised against the native enzyme inhibits enzyme activity. Inhibition studies suggest the presence of tissue-specific variants of the enzyme. By immunohistochemical analysis the enzyme can be localized to the ramifications of nerve terminals in the electric organ.

Sphingomyelinases in human tissues. IV. Purification of sphingomyelinase from human placenta and effect of Triton X-100

1978 ◽  
Vol 56 (9) ◽  
pp. 885-891 ◽  
Author(s):  
John W. Callahan ◽  
Prema Shankaran ◽  
Mary Khalil ◽  
Jacqualine Gerrie
Keyword(s):  
Enzyme Activity ◽  
Isoelectric Focusing ◽  
Concanavalin A ◽  
Human Placenta ◽  
Human Tissues ◽  
Hydrophobic Character ◽  
Nonionic Detergent ◽  
Triton X

Sphingomyelinase was purified about 1700-fold from human placenta. The major steps in the procedure included chromatography on Concanavalin A - Sepharose, Sepharose 6B, and carboxymethyl-Sepharose (CM-Sepharose). The final preparation was stable for at least 3 months when stored at 4 °C.The enzyme was found to be heterogeneous on CM-Sepharose and isoelectric focusing. Triton X-100 which was present in most buffers used during the purification appears to be partially responsible for the heterogeneity. When Triton X-100 is removed by treatment with Bio Beads, heterogeneity was reduced. However, removal of the detergent also leads to loss of enzyme activity which could not be restored by readdition of Triton X-100. The data suggest that sphingomyelinase has a high hydrophobic character and that both its stability and electrofocusing behaviour are influenced by interaction with the nonionic detergent.

Purification, characterization, and localization of two ATP diphosphohydrolase isoforms in bovine heart

1997 ◽  
Vol 273 (2) ◽  
pp. H673-H681 ◽  
Author(s):  
A. R. Beaudoin ◽  
J. Sevigny ◽  
G. Grondin ◽  
S. Daoud ◽  
F. P. Levesque
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Coronary Vessels ◽  
Protective Role ◽  
Cross Reactivity ◽  
Heat Inactivation ◽  
Maximal Velocity ◽  
Migration Patterns ◽  
Atp Diphosphohydrolase ◽  
Bovine Heart ◽  
Triton X

Two ATP diphosphohydrolase (ATPDase) isoforms have been purified from the bovine heart ventricle. The purification procedure includes the following steps: differential centrifugation, sucrose cushion centrifugation, solubilization with Triton X-100, DEAE agarose ion exchange, and Affi-Gel blue-Sepharose and concanavalin A (con A)-Sepharose chromatographies. The purified enzyme has an optimum pH of catalysis of 7.5 and requires Ca2+ or Mg2+. The apparent Michaelis constant of the enzyme, with ADP as the substrate, is 29 microM, and the apparent maximal velocity is 1.6 mumol.min-1.mg protein-1. Substrate specificity, heat-inactivation curves, and copurification of adenosinetriphosphatase (ATPase) and adenosinediphosphatase (ADPase) activities confirmed the identity of the purified enzyme as an ATPDase. In addition, polyacrylamide gel electrophoresis, under nondenaturing conditions, showed identical migration patterns for the protein involved in ATPase and ADPase activities. Western blot analysis, with an antibody that specifically recognizes the NH2-terminal sequence of pig pancreas ATPDase and specifically reacts with bovine and human ATPDases, showed cross-reactivity with the purified ATPDase isoforms from the bovine heart. Immunocytochemical localization in the ventricle produced strong reactions with the plasma membrane of Purkinje fiber cells and the majority of myocardial cells. Immunoreactivity was variable, producing a mosaic-like aspect. As expected, smooth muscle cells and endothelial cells of coronary vessels were highly reactive. This ectoenzyme could play a protective role against the potentially deleterious effects of extracellular ATP. In tandem with 5'-nucleotidase, it produces adenosine, a powerful vasodilator, especially in hypoxic or ischemic conditions that favor the release of ATP.

scholarly journals Physicochemical behaviour and structural characteristics of membrane-bound acetylcholinesterase from Torpedo electric organ. Effect of phosphatidylinositol-specific phospholipase C

1985 ◽  
Vol 226 (2) ◽  
pp. 369-377 ◽  
Author(s):  
A H Futerman ◽  
R M Fiorini ◽  
E Roth ◽  
M G Low ◽  
I Silman
Keyword(s):  
Phospholipase C ◽  
Structural Characteristics ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Electric Organ ◽  
Catalytic Subunit ◽  
Proteinase K ◽  
Short Amino Acid Sequence ◽  
Torpedo Electric Organ

Quantitative solubilization of the phospholipid-associated form of acetylcholinesterase (AChE) from Torpedo electric organ can be achieved in the absence of detergent by treatment with phosphatidylinositol-specific phospholipase C (PIPLC) from Staphylococcus aureus [Futerman, Low & Silman (1983) Neurosci. Lett. 40, 85-89]. The sedimentation coefficient on sucrose gradients of AChE solubilized in detergents (DSAChE) varies with the detergent employed. However, the coefficient of AChE directly solubilized by PIPLC is not changed by detergents. Furthermore, PIPLC can abolish the detergent-sensitivity of the sedimentation coefficient of DSAChE purified by affinity chromatography, suggesting that one or more molecules of phosphatidylinositol (PI) are co-solubilized with DSAChE and remain attached throughout purification. DSAChE binds to phospholipid liposomes, whereas PIPLC-solubilized AChE and DSAChE treated with PIPLC do not bind even to liposomes containing PI. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows that PIPLC-solubilized AChE, like unmodified DSAChE, is a catalytic subunit dimer; electrophoresis in the presence of reducing agent reveals no detectable difference in the Mr of the catalytic subunit of unmodified DSAChE, of AChE solubilized by PIPLC and of AChE solubilized by Proteinase K. The results presented suggest that DSAChE is anchored to the plasma membrane by one or more PI molecules which are tightly attached to a short amino acid sequence at one end of the catalytic subunit polypeptide.

Subcellular distribution and partial characterization of the three major classes of concanavalin A receptors associated with rat brain synaptic junctions

1980 ◽  
Vol 58 (10) ◽  
pp. 941-951 ◽  
Author(s):  
James W. Gurd
Keyword(s):  
Rat Brain ◽  
Concanavalin A ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Structural Heterogeneity ◽  
Molecular Weights ◽  
Receptor Sites ◽  
Synaptic Junctions ◽  
The Individual ◽  
Triton X

Synaptic junctional complexes from rat brain contain three major classes of glycoproteins which react with concanavalin A. They have apparent molecular weights of 110 000 (GP 110) 130 000 (GP 130), and 180 000 (GP 180). They are present in postsynaptic densities but are not found in microsomes, axolemma, synaptic vesicles, or myelin and are present in low concentrations in the Triton X-100 extract obtained during the preparation of synaptic junctions suggesting that they are uniquely localized to the postsynaptic apparatus. Reaction of the individual glycoproteins, partially purified by affinity chromatography on concanavalin A – agarose followed by polyacrylamide gel electrophoresis, showed that GP 130 contained the most receptor sites for concanavalin A per unit of protein followed by GP 180 and GP 110. Of the receptor sites for concanavalin A, 60–70% were subject to hydrolysis by endoglycosidase H indicating that the lectin reacts primarily with polymannose asparagine linked oligosaccharides. Each of the glycoproteins also reacted to varying degrees with the lectins from Lotus tetragonolobus (specific for α-L-fucose), wheat germ (N′-acetyl-D-glucosamine and (or) sialic acid), and lentils (mannose, N′-acetyl-D-glucosamine). Chromatography of 125I-labelled concanavalin A positive glycoproteins on wheat germ Sepharose resolved GP 110 and GP 180 into wheat germ positive and negative components indicating the presence of some structural heterogeneity within these molecular weight classes.

scholarly journals Purification and immunochemical characterization of monoamine oxidase from rat and human liver

1977 ◽  
Vol 161 (1) ◽  
pp. 167-174 ◽  
Author(s):  
R G Dennick ◽  
R J Mayer
Keyword(s):  
Enzyme Activity ◽  
Monoamine Oxidase ◽  
Enzyme Activities ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Triton X

1. Monoamine oxidase from rat and human liver was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The enzyme activity was extracted from mitochondrial preparations by Triton X-100. The enzyme was purified by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose, Sepharose 6B, spheroidal hydroxyapatite, and finally chromatography on diazo-coupled tyramine-Sepharose. 3. Distinct differences occur in the chromatographic behaviour of the two enzymes on both DEAE-cellulose and spheroidal hydroxyapatite. 4. It is unlikely that the purification of the enzymes on tyramine-Sepharose is due to affinity chromatography and reasons for this are discussed. 5. The purified enzymes did not oxidize-5-hydroxytryptamine and the relative activities of the enzymes with benzylamine were increased approx. 1.25-fold compared with the enzyme activities of mitochondrial preparations. 6. Immunotitration of enzyme activity in extracts of mitochondrial preparations from rat liver was carried out with 5-hydroxytryptamine, tyramine and benzylamine. The enzyme activities were completely immunoprecipitated by the same volume of antiserum. Similar results were obtained with the antiserum to the enzyme from human liver.

scholarly journals Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminal plasma membranes.

1983 ◽  
Vol 97 (6) ◽  
pp. 1737-1744 ◽  
Author(s):  
N Morel ◽  
M Thieffry ◽  
R Manaranche
Keyword(s):  
Nerve Terminal ◽  
Concanavalin A ◽  
Plasma Membranes ◽  
Acetylcholine Release ◽  
Motor Nerve ◽  
Biological Response ◽  
Electric Organ ◽  
Venom Glands ◽  
Torpedo Electric Organ ◽  
Glycera Convoluta

The crude extract of venom glands of the polychaete annelid Glycera convoluta triggers a large Ca2+-dependent acetylcholine release from both frog motor nerve terminals and Torpedo electric organ synaptosomes. This extract was partially purified by Concanavalin A affinity chromatography. The biological activity was correlated in both preparations to a 300,000-dalton band, as shown by gel electrophoresis. This confirmed previous determinations obtained with chromatographic methods. This glycoprotein binds to presynaptic but not postsynaptic plasma membranes isolated from Torpedo electric organ. Pretreatment of intact synaptosomes by pronase abolished both the binding and the venom-induced acetylcholine release without impairing the high K+-induced acetylcholine release. Pretreatment of nerve terminal membranes by Concanavalin A similarly prevented the binding and the biological response. Binding to Torpedo membranes was still observed in the presence of EGTA. An antiserum directed to venom glycoproteins inhibited the neurotoxin so we could directly follow its binding to the presynaptic membrane. Glycera convoluta neurotoxin has to bind to a ectocellularly oriented protein of the presynaptic terminal to induce transmitter release.

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.

scholarly journals Diadenosine polyphosphate hydrolase from presynaptic plasma membranes of Torpedo electric organ

1997 ◽  
Vol 323 (3) ◽  
pp. 677-684 ◽  
Author(s):  
Jesús MATEO ◽  
Pedro ROTLLAN ◽  
Eulalia MARTI ◽  
Inmaculada GOMEZ DE ARANDA ◽  
Carles SOLSONA ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Pyridoxal Phosphate ◽  
Electric Organ ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Ic50 Value ◽  
Neural Origin ◽  
Disulphonic Acid ◽  
Torpedo Electric Organ ◽  
Enzyme Activators

The diadenosine polyphosphate hydrolase present in presynaptic plasma membranes from the Torpedo electric organ has been characterized using fluorogenic substrates of the form di-(1,N6-ethenoadenosine) 5´,5‴-P1,Pn-polyphosphate. The enzyme hydrolyses diadenosine polyphosphates (Apn A, where n = 3–5), producing AMP and the corresponding adenosine (n-1) 5´-phosphate, Ap(n-1). The Km values of the enzyme were 0.543± 0.015, 0.478±0.043 and 0.520±0.026 μM, and the Vmax values were 633±4, 592±18 and 576±45 pmol/min per mg of protein, for the etheno derivatives of Ap3A (adenosine 5´,5‴-P1,P3-triphosphate), Ap4A (adenosine 5´,5‴-P1,P4 -tetraphosphate) and Ap5A (adenosine 5´,5‴-P1,P5-pentaphosphate) respectively. Ca2+, Mg2+ and Mn2+ are enzyme activators, with EC50 values of 0.86±0.11, 1.35±0.24 and 0.58±0.10 mM respectively. The fluoride ion is an inhibitor with an IC50 value of 1.38±0.19 mM. The ATP analogues adenosine 5´-tetraphosphate and adenosine 5´-[γ-thio]triphosphate are potent competitive inhibitors and adenosine 5´-[α,β-methylene]diphosphate is a less potent competitive inhibitor, the Ki values being 0.29±0.03, 0.43±0.05 and 7.18±0.8 μM respectively. The P2-receptor antagonist pyridoxal phosphate 6-azophenyl-2´,4´-disulphonic acid behaves as a non-competitive inhibitor with a Ki value of 29.7±3.1 μM, and also exhibits a significant inhibitory effect on Torpedo apyrase activity. The effect of pH on the Km and Vmax values, together with inhibition by diethyl pyrocarbonate, strongly suggests the presence of functional histidine residues in Torpedo diadenosine polyphosphate hydrolase. The enzyme from Torpedo shows similarities with that of neural origin from neurochromaffin cells, and significant differences compared with that from endothelial vascular cells.

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