Pepstatin fragments

To further study the mode of inhibition of pepsin and other acid proteases by pepstatin, we tried to produce various pepstatin fragments and measure their kinetics of inhibition. These pepstatin fragments are produced by three methods (a) complete acid hydrolysis yields free statine and other free amino acids (b) partial acid hydrolysis yields a mixture of peptides and (c) enzyme digestion with a-lytic protease yields the tetrapeptide, valyl-statyl-alanyl-statine. Purification by high-voltage paper electrophoresis from the above mixtures gives four statine-containing products in sufficient yield to allow further study statine, alanyl-statine, valyl-statine, and valyl-statyl-alanyl-statine. The isolated products are acetylated and quantified, and the completeness of their acetylation is determined prior to the initiation of inhibition studies (10). 

Fig. 4. Autoradiographs showing ADP ribosylation of fragmented poly(ADP-ribose) polymerase. Lymphocytes were treated with MNNG, permeabilized, and incubated with [ P]NAD as in Fig. 1. a No ATP 10 mM ATP. c-e Cells were incubated with 10 mM ATP, for 30 min at 0°C (c) or 37°C d, e). Then 5 mM PMSF and 5 mM pepstatin A were added to incubations followed by addition of [ P]NAD and cells were incubated for a further 15 min at 37°C. Cells in e also received 10 mM 3-AB before the addition of [ PJNAD. SDS gel electrophoresis and autoradiography was performed as in Fig. 1...

Results of the separation of the activation peptides, obtained as described above, are shown in Figure 3. Amino acid analysis of the peaks showed that the pepstatin was recovered in peak 1 and that peak 4 contained mainly peptide 1-16 (Table I). In addition to the amino acids listed, there were small amounts of histidine, glycine, and alanine. From the quantities of these amino acids and the known sequence of the total activation fragments (5,21,22), it was ascertained that the product was about 90% pure, the slightly high values for several amino acids being caused by contaimination with at least three other peptides. No further purification was considered considered necessary. 

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