Thermolysin-catalyzed peptide bond synthesis.

The rates of the thermolysin-catalyzed synthesis of peptides have been determined by means of HPLC. In the condensation of various N-substituted amino acids and peptides with L-leucinanilide, the enzyme exhibits preference for a hydrophobic L-amino acid as the donor of the carbonyl group of the newly formed bond. The presence of another hydrophobic amino acid residue adjacent to the carbonyl-group donor markedly enhances the rate of synthesis. In general, the effect of structural changes in both the carboxyl and amine components of the condensation reaction is in accord with the available data on the primary and secondary specificities of the thermolysin-catalyzed hydrolysis of oligopeptide substrates. A kinetic study of the condensation of benzyloxycarbonyl-L-phenylalanine with various amine components has given data on the apparent kcat and Km values for the entry of the acidic component into the condensation reaction. The results are consistent with the behavior of rapid-equilibrium random bi-reactant systems leading to ternary enzyme-substrate complexes, with a synergistic effect in the binding of the two reactants at the active site. Because the changes in the apparent kcat for the entry of the same acidic component into reaction with different amine components are greater than those in the apparent Km, it is suggested that this synergism is largely expressed at the level of the transition-state complex.

Specificity of pepsin-catalyzed peptide bond synthesis.

The rates of the pepsin-catalyzed synthesis of oligopeptides of the general type A-Phe-Leu-B by the condensation of A-Phe-OH with H-Leu-B have been determined by means of analytical high performance liquid chromatography. Variation of the A group led to large changes in the initial rates of the condensation reaction, and the effect of such changes was found to be similar to that previously found for the secondary specificity of pepsin in the hydrolysis of oligopeptide substrates. Replacement of the Phe and Leu residues of A-Phe-OH or H-Leu-B by other amino acid residues gave relative rates of synthesis in accord with the known primary specificity of the hydrolytic action of pepsin. Partially-acetylated pepsin, which exhibits enhanced hydrolytic activity, also catalyzed the condensation reaction more effectively. The results are discussed in relation to the potential utility and limitations of pepsin as a catalyst in the preparative synthesis of oligopeptides and to the problem of the mechanism of its action.

Chlorpromazine phototoxicity: growth inhibition and DNA-interaction in normal human fibroblasts.

Growth was impaired in normal human skin fibroblasts following treatment with chlorpromazine and long-wave ultraviolet light (UV-A). The degree of impairment was dose dependent to chlorpromazine within the concentration range tested, 2.5-20 microgram/ml, in the presence of UV-A, 1 J/cm2. Pre-irradiated chlorpromazine at a concentration of 20 microgram/ml had no effect on fibroblast growth. Chlorprotixene, a thioxanthene compound structurally similar to chlorpromazine at a concentration of 10 microgram/ml, was not phototoxic in this system. The effects of chlorpromazine and UV-A on fibroblast DNA were studied using the technique of zone sedimentation in alkaline sucrose. In the absence of light chlorpromazine did not affect sedimentation of DNA. After UV-A irradiation at 20 degrees or 0 degrees C in the presence of chlorpromazine, labeled DNA sedimented more slowly indicating that it had been reduced to smaller fragments. No evidence for interstrand DNA cross-links was found. Chlorpromazine alone or in combination with UV-A did not alter the size of the DNA. These results with cultured fibroblasts indicate that the phototoxic action of chlorpromazine at 366 nm is at least partially explained by interaction with DNA and is not due to the effects of cytotoxic photoproducts.