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See detailChemical modification of the nicotinic cholinergic receptor of PC-12 nerve cell.
Leprince, Pierre ULg

in Biochemistry (1983), 22(24), 5551-6

The identity of the protein that mediates the nicotinic acetylcholine sensitivity in neuronal cells has been investigated by chemical modification and affinity labeling. When an ion flux assay is used, it ... [more ▼]

The identity of the protein that mediates the nicotinic acetylcholine sensitivity in neuronal cells has been investigated by chemical modification and affinity labeling. When an ion flux assay is used, it is possible to measure specifically the activity of the ionophore associated with the nicotinic acetylcholine receptor in cultured nerve cells (PC-12 pheochromocytoma). This activity is modulated by modification of the redox state of at least one disulfide bridge located at the vicinity of the agonist binding site. The oxidizing agent 5,5'-dithiobis(nitrobenzoic acid) restores the complete receptor response which had been inhibited by reduction with dithiothreitol. N-Ethylmaleimide and the nicotinic affinity labels [4-(N-maleimido)benzyl]-alpha-trimethylammonium iodide and bromoacetylcholine react also with the reduced receptor and irreversibly block the agonist-dependent response of the ionophore. The two affinity labels show strong affinities for the receptor, and apparent IC50 values of 20 and 560 nM can be respectively evaluated. Bromoacetylcholine, being an acetylcholine analogue, blocks the receptor function by desensitization, a process in which the constant interaction with the activator causes a shift into an inactive form of the receptor. Bromoacetylcholine can also be shown to activate untreated as well as reduced cells. In this case, the bound label induces a lasting response which is terminated by the irreversible desensitization of the modified receptor. These experiments thus show that the PC-12 nicotinic ionophore shares functional and structural similarities with peripheral receptors. They suggest that nicotinic affinity labels developed for the muscle receptor can also be used as specific markers of the nicotinic neural ionophore. [less ▲]

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See detailComparison of the interactions of a specific neurotoxin (alpha-bungarotoxin) with the acetylcholine receptor in Torpedo californica and Electrophorus electricus membrane preparations.
Leprince, Pierre ULg; Noble, R. L.; Hess, G. P.

in Biochemistry (1981), 20(19), 5565-70

alpha-Bungarotoxin, a snake neurotoxin, binds irreversibly and specifically to the acetylcholine receptor isolated from the electroplax of Electrophorus electricus and Torpedo species and has been an ... [more ▼]

alpha-Bungarotoxin, a snake neurotoxin, binds irreversibly and specifically to the acetylcholine receptor isolated from the electroplax of Electrophorus electricus and Torpedo species and has been an important tool in the study of the receptor-ligand binding mechanism. Two distinct kinetic processes have been observed in studies with membranes from E. electricus. A minimum mechanism for the toxin reaction involves (i) the reversible binding of two toxin molecules to the receptor prior to the irreversible formation of toxin receptor complexes and (ii) a toxin-induced conformational change of the receptor which leads to an increase in the affinity of the receptor binding sites for toxin [Hess, G. P., Bulger, J. E., Fu, J.-j. L., Hindy, E. F., & Silberstein, R. J. (1975) Biochem. Biophys. Res. Commun. 64, 1018-1027]. Only one process has been detected in Torpedo membranes. Here, we determine whether the receptors in Torpedo californica and E. electricus membranes have different properties or whether the measurements and their interpretation were responsible for the different results. Two methods which are frequently used in binding studies to separate free and bound toxin, a CM-52 cellulose minicolumn assay and DE-81 filter disk assay, have been compared. The results obtained indicate that the interaction of toxin with receptor from T. californica is similar to that observed with receptor from E. electricus. The apparent differences which have been reported in the literature are shown to have arisen from the design of the experiments in which T. californica membranes were used. [less ▲]

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See detailInactivation of rat liver RNA polymerases I and II and yeast RNA polymerase I by pyrodixal 5'-phosphate. Evidence for the participation of lysyl residues at the active site.
Martial, Joseph ULg; Zaldivar, J.; Bull, P. et al

in Biochemistry (1975), 14

Purified DNA-dependent RNA polymerase forms I (A) and II (B) from rat liver and form I from yeast are rapidly inactivated by pyridoxal 5'-phosphate at pH 8.0. The inhibition is relatively specific since ... [more ▼]

Purified DNA-dependent RNA polymerase forms I (A) and II (B) from rat liver and form I from yeast are rapidly inactivated by pyridoxal 5'-phosphate at pH 8.0. The inhibition is relatively specific since pyridoxamine 5'-phosphate is not an inhibitor and pyridoxal is about 12 times less effective than pyridoxal 5'-phosphate. The inactivation is reversed by high concentrations of amines, and can be made irreversible by reduction with NaBH4. Spectral analysis of the inhibited enzyme and its NaBH4 reduction product indicates that a Schiff base forms between the aldehyde group of pyridoxal 5'-phosphate and one or more amino groups of the protein. Nepsilon-Pyridoxyllysine was identified as the only product in acid hydrolysates of the reduced yeast RNA polymerase I-pyridoxal 5'-phosphate complex. Complete inactivation of yeast polymerase I results in the incorporation of 3-4 mol of pyridoxal 5'-phosphate/1 mol of enzyme. DNA and nucleotide substrates partially protect the enzymes from inactivation. These results suggest that one or more lysyl amino groups are critical for the activity of animal RNA polymerases and show that pyridoxal 5'-phosphate is a suitable probe for studying the active sites of these enzymes. Comparison of the present results with those previously obtained with Eschericha coli RNA polymerase in this laboratory suggest a new degree of structural homology between eucaryotic and procaryotic RNA polymerases. [less ▲]

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See detailOccurrence of D-alanyl-(D)-meso-diaminopimelic acid and meso-diaminopimelyl-meso-diaminopimelic acid interpeptide linkages in the peptidoglycan of Mycobacteria
Wietzerbin, Juana; Das, Bhupsh C.; Petit, Jean-François et al

in Biochemistry (1974), 13(17), 3471-3476

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See detailStructure of the wall peptidoglycan of streptomyces R39 and the specificity profile of its exocellular DD-carboxypeptidase-transpeptidase for peptide acceptors
Ghuysen, Jean-Marie ULg; Leyh-Bouille, Mélina; Campbell, James N. et al

in Biochemistry (1973), 12(7), 1243-1251

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the ... [more ▼]

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl2 buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10−6, 1.5×10−6 and 0.63×10−6s−1 (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [14C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [14C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10−5s−1 and 0.8×10−4s−1 respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site. [less ▲]

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See detailPenicillin-sensitive DD-carboxypeptidases from Streptomyces strains R39 and K11
Leyh-Bouille, Mélina; Nakel, Marlies; Frère, Jean-Marie ULg et al

in Biochemistry (1972), 11(7), 1290-1298

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See detailWall peptidoglycan in Aerococcus viridans strains 201 Evans and ATCC 11563 and in Gaffkya homari strain ATCC 10400
Nakel, Marlies; Ghuysen, Jean-Marie ULg; Kandler, Otto

in Biochemistry (1971), 10(11), 2170-2175

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See detailPenicillin-sensitive DD-carboxypeptidase from Streptomyces strain R 61
Leyh-Bouille, Mélina; Coyette, Jacques; Ghuysen, Jean-Marie ULg et al

in Biochemistry (1971), 10(11), 2163-2170

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See detailStructure of the walls of Lactobacillus acidophilus strain 63 AM Gasser
Coyette, Jacques; Ghuysen, Jean-Marie ULg

in Biochemistry (1970), 9(15), 2935-2943

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See detailWall autolysin of Lactobacillus acidophilus strain 63 AM gasser
Coyette, Jacqueline; Ghuysen, Jean-Marie ULg

in Biochemistry (1970), 9(15), 2952-2955

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See detailSubstrate requirements of the Streptomyces albus G DD carboxypeptidase
Leyh-Bouille, Mélina; Ghuysen, Jean-Marie ULg; Bonaly, Roger et al

in Biochemistry (1970), 9(15), 2961-2970

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See detailLL-diaminopimelic acid containing peptidoglycans in walls of Streptomyces sp. and of Clostridium perfringens (type A).
Leyh-Bouille, Mélina; Bonaly, Roger; Ghuysen, Jean-Marie ULg et al

in Biochemistry (1970), 9(15), 2944-2952

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See detailIsolation of DD carboxypeptidase from Streptomyces albus G culture filtrates
Ghuysen, Jean-Marie ULg; Leyh-Bouille, Mélina; Bonaly, Roger et al

in Biochemistry (1970), 9(15), 2955-2961

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See detailOn the Streptomyces albus G DD carboxypeptidase mechanism of action of penicillin, vancomycin, and ristocetin
Leyh-Bouille, Mélina; Ghuysen, Jean-Marie ULg; Nieto, Manuel et al

in Biochemistry (1970), 9(15), 2971-2975

The activity of the D-alanyl-D carboxypeptidase from the penicillin-resistant Streptomyces albus G is not or very little affected by penicillins and related antibiotics. The molecular basis for the ... [more ▼]

The activity of the D-alanyl-D carboxypeptidase from the penicillin-resistant Streptomyces albus G is not or very little affected by penicillins and related antibiotics. The molecular basis for the mechanism of action of penicillin is discussed. The Streptomyces albus G D-alanyl-D carboxypeptidase appears as a model for the study of a mechanism of penicillin resistance that does not involve the enzymatic degradation of the antibiotic. Vancomycin and ristocetin are shown to inhibit the hydrolysis of sensitive peptides by the Streptomyces albus G D-alanyl-D carboxypeptidase and the mechanism of inhibition is discussed. [less ▲]

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See detailStructure of the meso-diaminopimelic acid containing peptidoglycans in Escherichia coli B and Bacillus megaterium KM
Van Heijenoort, Jean; Elbaz, Lydia; Dezelee, Philippe et al

in Biochemistry (1969), 8(1), 207-213

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See detailCharacterization of the Micrococcus lysodeikticus type of peptidoglycan in walls of other Micrococcaceae
Campbell, J. N.; Leyh-Bouille, Mélina; Ghuysen, Jean-Marie ULg

in Biochemistry (1969), 8(1), 193-200

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See detailThe peptidoglycan in walls of Butyribacterium rettgeri
Guinand, Micheline; Ghuysen, Jean-Marie ULg; Schleifer, Karl H. et al

in Biochemistry (1969), 8(1), 200-207

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