Properties of the nicotinic acetylcholine receptor macromolecule of <i>Electrophorus electricus</i>

Fulpius, Bernard W.; Cha, Sungman; Klett, Robert P.; Reich, E.

doi:10.1016/0014-5793(72)80382-x

Cited by 52 publications

(25 citation statements)

References 8 publications

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“…the association of the pancreatic secretory trypsin inhibitor with trypsin (ASaO = 43.6 calxmol-l x K-l) [29] and the association of snake neurotoxins with the acetylcholine receptor (A&O = 74 cal [30]. The high value of A SaO could also be due in part to structural changes affecting the inhibitor and/or enzyme conformations within the complex.…”

Section: Discussionmentioning

confidence: 99%

The Interaction between α‐Chymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibitor)

Vincent¹,

Lazdunski²

1973

European Journal of Biochemistry

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1. The association constant, Ka, of the 1 : i complex formed between a-chymotrypsin and the pancreatic trypsin inhibitor is 110 pM-l a t pH 8.0 and 25 "C. The second-order rate constant for the association, Ea, is 110 mM-l s-l and the first-order rate constant for the dissociation, E d , is lo-, s-l under the same conditions. Thermodynamic parameters for complex formation a t 25 "C, pH 8.0 are A GaO = -li .O kcal x mol-l, A Ha'' = 3 kcal x mol-l and A 8 , ' ' = 48 cal.-mol-l * K-l. 2.Temperature and pH-dependences of the rate constants k, and k d have been studied. 3. The difference in stability between the a-chymotrypsin * inhibitor complex and the trypsin -inhibitor complex (Ka = 16 pM-l, d Q$ = -18.1 kcal x mol-l) is due essentially to differences of ka values. The dissociation of the a-chymotrypsin inhibitor complex is i.5 x 104 times faster than that of the trypsin -inhibitor complex. Differences in Ka and ka values are discussed in terms of differences in stabilizing interactions.4. The Cys,,-Cys,, disulfide bridge of the inhibitor, which is highly succeptible to reduction when the inhibitor is free, is masked in the a-chymotrypsin -inhibitor complex. Reduction of the Cys,,-Cys,, bridge in the free inhibitor does not prevent association with a-chymotrypsin. Values of k , and Ed for the formation of the 1 : 1 a-chymotrypsin * reduced-inhibitor complex and for the complex formed with the native inhibitor are very similar. This situation differs from that observed with the trypsin * pancreatic-inhibitor complex. I n that case, reduction of Cys,,-Cys,, bridge considerably decreases the stability of the complex formed with trypsin ; K , is decreased by a factor of 3 x lo4 and kd is increased by a factor of 8.6 x los.I n spite of their importance in control systems, only a limited number of interactions between heterologous proteins or between peptides and proteins have been extensively studied. However it is well known that many such associations are very tight. Protein and peptide hormones such as insulin and the thyroid-stimulating hormone [4] for example act a t very low doses. Dissociation constants for the complexes they form with their receptors are lower that 10 nM.We reported previously the results obtained for the formation of the complex between trypsin and pancreatic trypsin inhibitor. This association is one of the security devices which avoid accidental activation of trypsinogen in the pancreas. The association between trypsin and the pancreatic trypsin inhibitor is unusually strong. The dissociation constant, Kd, of the 1 : 1 complex is 60 fM at pH 8.0,We report here the results obtained for the formation of the complex between ol-chymotrypsin and pancreatic trypsin inhibitor. Each of the partners in the complex is well characterized. Both their covalent and three-dimensional structures in the crystalline state are available [6-111 and a considerable amount of work has been devoted to the analysis of the components of their active sites. The essential catalytic groups of ol-chymotrypsin are ; the ...

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Section: Discussionmentioning

confidence: 99%

The Interaction between α‐Chymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibitor)

Vincent¹,

Lazdunski²

1973

European Journal of Biochemistry

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“…In 15-ml, screw-top, plastic tubes (BectonDickinson), quadruplicate 50-ul samples containing 1-50 ,ug of protein were mixed with 50 ,ul of 0.4 mM dithiothreitol, in a final buffer composition as in the "slow-labeling" reduction. After 20 min at 25°, 10 Ml of 0.53 M PO4-3 (pH 6.7) was added, bringing the pH to 7.0. To two samples, 0.4 ml of 0.2% Triton-150 mM NaCl-10 mM PO4-3-1 mM EDTA (pH 7.0) was added; to two others, 0.4 ml of 3 upg of N. n. siamensis toxin per ml of the same buffer.…”

Section: Methodsmentioning

confidence: 99%

“…The dry filter was placed in a counting vial, 0.1 ml of H20 and 0.5 ml of NCS solubilizer were added, and the filter was kept at 500 for 1 hr. Finally, 10 ml of PPO-dimethyl POPOP-toluene scintillant was added and the vials were counted. The wash procedure reduces the background retention to about 0.05% of applied 3H activity.…”

Section: Methodsmentioning

confidence: 99%

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The Affinity-Labeling of Partially Purified Acetylcholine Receptor from Electric Tissue of Electrophorus

Karlin

Cowburn

1973

Proc. Natl. Acad. Sci. U.S.A.

125

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The receptor for acetylcholine was partially purified by affinity chromatography of an extract in Triton X-100 of membrane fragments from electric tissue. The receptor was assayed, after its reduction with dithiothreitol, by reaction with the affinity-alkylating agent, [methyl-'H] benzyltrimethylammonium iodide and subjected to electrophoresis on polyacrylamide gels in dodecyl sulfate and dithiothreitol, three major protein bands were observed. Only one of these, however, contained 3H activity; its mobility indicated a molecular weight of 40,000.

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“…Over the past few years, considerable effort has been directed towards understanding the molecular biology of the acetylcholine receptor (AChR), a large portion of which has been concerned with its purification (1)(2)(3)(4)(5)(6)(7)(8). Implicit in these efforts is the assumption that knowledge of the structural properties of purified receptor will provide insight into its function in situ.…”

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confidence: 99%

Studies on Purified Eel Acetylcholine Receptor and Anti-Acetylcholine Receptor Antibody

Patrick

Lindstrom

Culp

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

131

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Rabbit antiserum against purified Electrophorus electricus acetylcholine receptor is studied using an immunoprecipitin assay to measure either antibody titer or concentration of toxin-binding sites in solubilized receptor preparations. This antiserum, unlike control serum, blocks the electrophysiological response of the electroplax to carbamylcholine. Toxin (a-neurotoxin, Naja naja) and several cholinergic ligands produce partial inhibition of the reaction of antiserum with purified acetylcholine receptor. Evidence is presented that some of the toxin-binding sites on receptor, purified by affinity chromatography on toxin-agarose conjugates, are occluded by toxin. In addition, evidence is presented that antireceptor antiserum will cause precipitation of more toxinbinding sites present in an initial extract than in purified receptor preparations.Over the past few years, considerable effort has been directed towards understanding the molecular biology of the acetylcholine receptor (AChR), a large portion of which has been concerned with its purification (1-8). Implicit in these efforts is the assumption that knowledge of the structural properties of purified receptor will provide insight into its function in situ. In order to generate a probe of receptor structure and function which could be used to compare receptor purified and in situ, we stimulated anti-AChR antibody production in rabbits. As we have reported (9), the injection of rabbits with purified receptor resulted in the formation of antireceptor antibodies and subsequently the appearance of flaccid paralysis leading to death. In this paper, we present properties of one antiserum thus obtained and use this antiserum and antitoxin antiserum to study purified eel acetylcholine receptor. MATERIALS AND METHODSPreparation of Neurotoxin. Toxin was purified from the venom of the Indian Cobra N. Naja Naja, as previously described (10). Purified toxin was iodinated with Na 1251 (New England Nuclear Corp., carrier-free) to specific activities of 30-40 Ci/mmol (11).Preparation of Acetylcholine Receptor. Acetylcholine receptor was purified from the electric eel, Electrophorus electricus, by affinity chromatography on columns of toxin-agarose conjugates (6). Protein concentrations determined by the Lowry reaction (12) were corrected to dry-weight protein by stanAbbreviations: GAR, goat anti-rabbit immunoglobulin G antiserum; AChR, acetylcholine receptor; Toxin, a-neurotoxin,

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Properties of the nicotinic acetylcholine receptor macromolecule of Electrophorus electricus

Cited by 52 publications

References 8 publications

The Interaction between α‐Chymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibitor)

The Interaction between α‐Chymotrypsin and Pancreatic Trypsin Inhibitor (Kunitz Inhibitor)

The Affinity-Labeling of Partially Purified Acetylcholine Receptor from Electric Tissue of Electrophorus

Studies on Purified Eel Acetylcholine Receptor and Anti-Acetylcholine Receptor Antibody

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