Stepwise cleavage of the pro part of promelittin by dipeptidylpeptidase IV. Evidence for a new type of precursor--product conversion.

Melittin, the main constituent of honeybee venom, is derived from promelittin. In the amino acid sequence of the 'pro' region of this precursor, every second residue is either proline or alanine. The possibility has been investigated that activation of promellitin might proceed via sequential liberation of dipeptides catalyzed by a dipeptidylpeptidase IV. As substrates we used promelittin isolated from queen bees fed with radioactive proline, and enzymatic fragments of prepromelittin which contained the entire pro part and the NH2-terminal hexapeptide of melittin. It could first be demonstrated that pig kidney of dipeptidyleptides catalyzed by a dipeptidylpeptidase IV. As substrates we used promelittin isolated from queen bees fed with radioactive proline, and enzymatic fragments of prepromelittin which contained the entire pro part and the NH2-terminal hexapeptide of melittin. It could first be demonstrated that pig kidney of dipeptidylpeptidase IV releases dipeptides from the pro part. An enzyme of this type could then be detected in extracts from venom glands of q bees, which also contain a dipeptidase. After inhibiting the latter enzyme with mersalyl, a stepwise cleavage of dipeptides, starting at the amino end of the pro region could be demonstrated. This hydrolysis did not proceed into the melittin sequence. Furthermore, fragments with an extra residue at the amino end, which therefore had the wrong 'reading frame' for the dipeptidylpeptidase, were not hydrolyzed. With intact promelittin as substrate the rate of hydrolysis was always lower than with the fragments. The results presented in this paper suggest a new type of precursor-product conversion proceeding via stepwise cleavage of dipeptide units. Our experimental evidence also ascribes a biological function to a dipeptidylpeptidase IV, a type of enzyme widely distributed in animals tissues. The evidence, that the observed reaction in vitro reflects the mechanism of promelittin activation in vivo is discussed.