Some antagonists exhibit tissue selectivity in their pharmacological antagonism of muscarinic responses. However, the affinity constants for equilibrium binding of classical antagonists to muscarinic receptors in subcellular preparations have shown only small variations in different peripheral tissues and regions of the brain. The binding curves do not deviate significantly from the simple Langmuir isotherm, indicating apparent homogeneity of the receptor population in any given region. In contrast, heterogeneity has been detected by agonist binding studies but this may arise from different environmental or coupling restraints on the agonist-induced conformational change and cannot be taken as evidence for different receptor subtypes. We report here binding studies using a new anti-muscarinic drug, pirenzepine, in which we found heterogeneity of binding that correlates well with the pharmacological activity.

We have found that if suitable concentrations of toxin are employed a rapid and reproducible neuro-muscular block can be produced in the isolated rat phrenic nerve-diaphragm preparation of Bulbring (1946). This preparation is adaptable and has proved very suitable for the analysis of the action of the toxin. Torda & Wolff (1947) have claimed that choline acetylase is strongly inhibited by the toxin and that this might explain how the toxin produces neuromuscular block. The action of the toxin on this system and on the closely related aromatic amine acetylase has been investigated further. METHODSToxin. Type A toxin was obtained from Clostridium botulinum (strain 4587) grown in a medium consisting of 25% tryptic digest of casein, 10% yeast extract, and 1 % glucose. The ingredients were prepared as in CCY medium (Gladstone & Fildes, 1940). The toxin was purified by acid precipitation with HCl at pH 3 9, then rendered soluble by elution with 0-2M-Na2HPO4 to pH 6-0.The solution was freeze-dried and stored as a powder in vacuo over phosphorus pentoxide. As required, the toxin was dissolved in a buffer consisting of 1% Na2HPO4 and 0-2 % gelatin at pH 6-6 to give a concentration of 2 x 105 or 2 x 106 mouse LD50/ml. . (Lamanna, McElroy & Eklund, 1946), i.e. this material was c. 5% pure toxin.) In the text the amount of toxin used is expressed in terms of mouse LDr0/ml. of bath fluid which is for convenience called a unit.

The binding of one irreversible and two reversible radioactive antagonists to muscarinic receptors in synaptosome preparations of rat cerebral cortex has been studied. The ligands all bind to the same receptor pool and directly and competitively yield self-consistent binding constants closely similar to those obtained by pharmacological methods on intact smooth muscle. The binding process for antagonists seems to be a simple mass action-determined process with a Hill slope of 1.0. The quantitative correlations strongly support the view that the receptor studied by ligand binding corresponds to the receptor studied by pharmacological methods. Inhibition of antagonist binding by most agonists shows a reduced Hill slope which also applies to direct binding studies of [3H] acetylcholine. Mechansims that might account for the behavior of agonists are discussed but do not conclusively point to any single mechanism.

No abstract

The binding, or association, constants of NADP+ NADPH, and a series of structural analogues to dihydrofolate reductase from Lactobacillus casei MTX/R have been determined fluorometrically. Modification of the adenine or nicotinamide rings has little effect on the binding of the oxidized coenzyme, but the thionicotinamide and acetylpyridine analogues of the reduced coenzyme bind much more weakly than NADPH itself. In the presence of the substrate, folate, or the inhibitors methotrxate or trimethoprim, the oxidized coenzymes bind appreciably more tightly to the enzyme. The magnitude of this "cooperativity", which covers a range of 1-37 fold, depends markedly on the structure of both the coenzyme and the substrate or substrate analogue; the nicotinamide ring of the coenzymes is clearly important in these effects. The binding constants of the reduced coenzymes in the presence of methotrexate or trimethoprim were too high to be measured fluorometrically. The dissociation rate constants of the coenzymes from their ternary complexes were therefore measured and compared with the values for the binary complexes reported by Dunn and co-workers [Dunn, S. M. J., Bathchelor, J. G., & King, R. W.(1978) Biochemistry 17, 2356]. The presence of the inhibitors leads to very substantial decreases in the coenzyme dissociation rate constant--by factors of 300-2200. The binding constant of methotrexate in the ternary complex is calculated to be approximately 1.3 X 10(12) M-1. The structural origins of the differences in binding constant and cooperative behavior of the various coenzymes and coenzyme analogues are discussed in the light of information from crystallography and NMR spectroscopy.

No abstract

The resonances of the aromatic protons of trimethoprim [2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine] in its complexes with dihydrofolate reductases from Lactobacillus casei and Escherichia coli cannot be directly observed. Their chemical shifts have been determined by transfer of saturation experiments and by difference spectroscopy using [2',6'-2H2]trimethoprim. The complex of 2,4-diamino-5-(3',4'-dimethoxy-5'-bromobenzyl)pyrimidine with the L. casei enzyme has also been examined. At room temperature, the 2',6'-proton resonance of bound trimethoprim is very broad (line width great than 30 Hz); with the E. coli enzyme, the resonance sharpens with increasing temperature so as to be clearly visible by difference spectroscopy at 45 degrees C. This line broadening is attributed to an exchange contribution, arising from the slow rate of "flipping" about the C7-C1' bond of bound trimethoprim. The transfer of saturation measurements were also used to determine the dissociation rate constants of the complexes. In the course of these experiments, a decrease in intensity of the resonance of the 2',6'-proton resonance of free trimethoprim on irradiation at the resonance of the 6 proton of free trimethoprim was observed, which only occurred in the presence of the enzyme. This is interpreted as a nuclear Overhauser effect between two protons of the bound ligand transferred to those of the free ligand by the exchange of the ligand between the two states. The chemical shift changes observed on the binding of trimethoprim to dihydrofolate reductase are interpreted in terms of the ring-current shift contributions from the two aromatic rings of trimethoprim and from that of phenylalanine-30. On the basis of this analysis of the chemical shifts, a model for the structure of the enzyme-trimethoprim complex is proposed. This model is consistent with the (indirect) observation of a nuclear Overhauser effect between the 2',6' and 6 protons of bound trimethoprim.

2,4-Diaminopyrimidine and p-aminobenzoyl-L-glutamate can be regarded as "fragments" of methotrexate, a potent inhibitor of dihydrofolate reductase. The equilibrium constants for the binding of these "fragments" and of a series of structurally related compounds to Lactobacillus casei dihydrofolate reductase have been determined by fluorimetric methods. 2,4-Diaminopyrimidine and p-aminobenzoyl-Lglutamate bind cooperatively to the enzyme, in the sense that p-aminobenzoyl-L-glutamate binds 54-fold more tightly to the enzyme-2,4-diaminopyrimidine complex than to the enzyme alone. The reciprocal nature of this effect has been quantitatively confirmed. Alkyl substitution on the amino nitrogen of p-aminobenzoyl-L-glutamate leads to a marked (up to 130-fold) increase in the affinity for the enzyme alone, but is almost