Intermediates in the degradation of abnormal globin. Bestatin permits the accumulation of the same peptides in cell-free extracts as in intact reticulocytes.

We have reported that Bestatin treatment results in accumulation of small peptide intermediates in the degradation of analog-containing globin by intact mouse reticulocytes. It was known that cell-free reticulocyte soluble extracts degrade analog-containing globin to amino acids. We now find that in this cell-free system, Bestatin also results in accumulation of peptide intermediates. The drug is effective at concentrations two orders of magnitude lower than those required to affect the process in intact cells. The intermediates are in the range of molecular weight corresponding to diand tripeptides. Removal of the NHz-terminal amino acid from L-[l-‘4C]leucinecontaining intermediates by one cycle of Edman degradation leaves a mixture of leucine and leucine-containing peptides, indicating that both diand tripeptides are represented in the intermediates. In the a and p chains of mouse globin, leucine can possibly be part of 28 different diand 83 different tripeptide sequences. Two-dimensional thin layer chromatography followed by autoradiography reveals, however, that most of the radioactivity is represented by eight distinct spots. Although some of these spots may include unresolved peptides, it is unlikely that 111 different sequences resulted in only 8 components. Therefore, the intermediates are not a random mixture of all possible sequences, and their chromatographic pattern must reflect the proteolytic pathways involved. Chromatographic analyses in two different solvent systems show strikingly similar fingerprints of peptide intermediates from intact cells and the cell-free system. These results are consistent with the hypothesis that the soluble cell-free system represents the process as it occurs in uiuo.