H. J. Smith

Inhibition of the muscarinic receptor by dibenamine

Abstract The rate of reaction of a group at the muscarinic receptor of isolated guineapig ileum and rat jejunum with the ethyleniminium ion derived from dibenamine is independent of pH over the range 6.9–8.9. This group may be either a carboxyl group or an imidazole residue. The muscarinic receptor is not identical with the active site of acetylcholinesterase as proposed since the enzyme reacts in a different manner with dibenamine over this pH range.

Inhibition of acetylcholinesterase by dibenamine and dibenzyline

Dibenamine and dibenzyline are irreversible inhibitors of acetylcholinesterase (AChE). Kinetic studies show that at pH 9.5 a fast reaction occurs between a group on the enzyme with pKa 9.1 and the ethyleniminium ion derived from the inhibitor. Either the ε‐amino‐group of a lysine residue is alkylated or else a lysine residue catalyses the alkylation of a non‐ionisable group (e.g. hydroxyl). At pH 6.5 there is a slow reaction between a carboxyl anion on the enzyme and the ethyleniminium ion. Studies of the alkylation reactions in the presence of the reversible competitive inhibitor of the enzyme, tetramethylammonium ion, show that alkylation occurs at some distance from the anionic site and probably on the borders of the active site.

Inhibition of α‐chymotrypsin by dibenamine

α‐Chymotrypsin is rapidly inhibited by an acid solution of dibenamine in an irreversible manner when the reactants are mixed in a buffer at near neutral pH. The degree of inhibition depends on the molar ratio of the reactants. The alkylating species is probably the ethyl‐eniminium ion from dibenamine, this ion being present at a higher level in the acid media than in the near neutral media used here.

Disulphide cross-linking of thiolated α-chymotrypsin to form macromolecules

Thiolation of α‐chymotrypsin with N‐acetyl homocysteine thiolactone (AHTL) occurs in the presence of Ag+ and imidazole with the introduction of 4–5·5 SH groups per mole. Oxidation of the thiolated enzyme with ferricyanide gave a macromolecule consisting of thiolated enzyme residues cross‐linked through intermolecular disulphide bonds. The number of residues linked in this manner varied with each batch of thiolated enzyme but was within the range 16–44 residues. The light scattering data indicated that the macromolecule was rod‐shaped.

Cross-linking of insulin with glutaraldehyde to form macromolecules.

In a search for a new type of sustained-release depot preparation for insulin, Mahbouba & Smith (1977) thiolated a proportion of the insulin units in the zincinsulin hexamer and cross-linked the hexamer through disulphide bridges to form macromolecules (n = 410708 monomer units) where native insulin was carried on a modified insulin skeleton. We describe here the formation of similarly structured macromolecules by the cross-linking of the zinc-insulin hexamer using glutaraldehyde.

Entrapment of proteins as disulphide cross-linked thiolated macromolecules within cross-linked dextran (“Sephadex”) gels

disposition of the naphthalene to the surface of silicate oxygen atoms. The surface occupied by the organic cation with this disposition is close to that available by exchange cation. Therefore it may be concluded that upon treating montmorillonite with aqueous solution of propranolol hydrochloride the propranolol-ammonium cations are adsorbed into the interlayer space of the silicate, giving rise t o the formation of a definite complex of 17.31 A basal spacing corresponding to the intercalation of a monomolecular cation layer. The formation of this complex is independent of the pH of the solution, within a pH margin of 3G8-0, but does depend on the concentration of the solution. The adsorption mechanism is one of cation exchange and the maximum amount adsorbed is 78 mequiv/100 g. These results are considered a valid base for the initiation of desorption studies in vitro and, later in vivo. We would like to thank Mr N. Skinner for his invaluable help in translating the manuscript.

Disulphide cross-linked macromolecules formed by thiolated insulin and globin

It has been considered by Mahbouba et a1 (1974) that thiolation (Benesch & Benesch 1958; 1962) of POlYpeptides and proteins of pharmacological interest, e.g. ACTH, asparaginase, followed by cross-linking through intermolecular disulphide bonds could give macromolecules in which the monomer units were afforded some protection from the action of tissue enzymes, provided that these units were closely packed together. Subsequent studies with insulin (Mahbouba & Smith 1977) and those reported here for globin have shown that the units in the macromolecules are more closely packed than expected from the earlier work with achymotrypsin macromolecules and have prompted an examination of the susceptibility of insulin macromolecules to attack by a protease. Formation of macromolecules. Globin was thiolated using N-acetyl homocysteine thiolactone (AHTL) at pH 1C11.5 by the method described for a-chymotrypsin and subsequently oxidized to the corresponding disulphide cross-linked macromolecule using potassium ferricyanide in the usual manner (Mahbouba et a1 1974). Thiolated globin was oxidized as soon as possible after its preparation and was not stored in the lyophilized state since freeze-drying was associated with loss in thiol content. The average molecular size of the oxidized thiolated globin macromolecule and the radius of gyration (R,) were calculated from light scattering measurements using the method of Mahbouba et a1 (1974). The aggregation number, n, of the macromolecules obtained was broadly related to the thiol content of the thiolated globin: n = 8 (4.1 -SH groups mol-l), 8.6 (0.5), 11 (3.3), 13 (3.1), 37 (7.4) and 65 (12). The shapes of the aggregates were estimated by plotting their particle scattering factors (Po) against sin (8/2) and comparing the curve shape with those of various model shapes (Doty & Steiner 1950). The results show that the plots for the rod and coil models were both similar to that for the globin solution. Macromolecules (n = 41C708 monomer units) containing native insulin carried by a modified insulin skeleton have been previously described (Mahbouba & Smith 1977) and were prepared by partially thiolating (05-0.7 -SH groups mol-l) the zinc-insulin hexamer and cross-linking using copper (11) ion-catalysed oxygenation. The macromolecule contained 2 Cu (11) and 4-6 Cu(1) atoms as determined by atomic absorption spectroscopy and titration with 2,21-biquinolyl (Felsenfeld 1960). The ESR spectrum of the macromolecule was consistent with replacement of zinc in

Application of mass spectrometry to enzyme inhibition studies

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A high content (−)‐deguelin concentrate from a commercial derris resin by steady‐state distribution

The isolation of a high content (‐)‐deguelin concentrate from a commercial derris resin using steady‐state distribution is described. The separation was followed by noting the change in ratio of extinctions at two wavelengths.

4-Substituted-2-anilinothiazolin-5-ones as substrates of α-chymotrypsin

a foundation for means of estimation of the risWbenefit Koreeda, M., Moore, P. D., Wislocki, P. G., Levin, W., ratio of its use compared to that of &MOP in the Conney, A. H., Yagi, H., Jerina, D. M. (1978) Science 199: 778-780 phototherapy of psoriasis. Pathak, M. A , , Biswas, R. K. (1977) J. Invest. Dermatol. 68: 236 We thank the B.C. Health Care Foundation for Scott, B. R., Path& M. A., Mohn, G. R. (1976) Mutat. financial support and Zyta Abramowski for technical Res. 39: 29-74 Song, P., Tapley, K. J . (1979) Photochem. Photobiol. 29: