On molecular sieve columns the neuropeptide head activator elutes at two distinct positions corresponding to apparent mol. wts of 700 and 1400 daltons. The low mol. wt component is stable only under high ionic conditions and represents the monomeric state of the head activator. Only this form is biologically active. The higher mol. wt component, which is reapidly formed under physiological conditions, is the dimeric head activator and is biologically inactive. We suggest that this dimerization is of biological relevance as a mechanism for inactivation of neuropeptides.

By exposing different sites of the ‘head activator’, different sets of antibodies were designed and produced which recognised either the amino or the carboxy terminus of the free ‘head activator’, which reacted with ‘head activator’ in a tissue‐fixed conformation, or which bound to the ‘head‐activator’ sequence, if it was part of a larger precursor‐like molecule. An enzyme‐linked immunosorbent assay (ELISA) was developed to characterise the antibodies and also to assay minute amounts of ‘head activator’ or ‘head‐activator’‐like immunoreactivities in animal or tissue extracts. A competitive ELISA is described which uses biotin‐avidin for enhancement. The assay is sensitive with an antibody specific for the amino terminus in the range of 0.5–50 fmol, with an antibody specific for the carboxy terminus in the range of 20–400 fmol. The ELISA specific for the amino terminus is 10‐times more sensitive than a radio‐immunoassay with tritiated ‘head activator’ [H. Bodenmüller and B. Zachmann (1983) FEBS Lett. 159, 237–240]. Previously no radioimmunoassay existed with specificity for the carboxy terminus.

A highly sensitive and specific radioimmunoassay for the head activator has been developed which utilises tritiated head activator. The assay is sensitive in the range of 40-200 fmol. Immunochemical studies showed that the antiserum recognised the intact molecule better than any of the fragments or derivatives produced by enzymatic treatment or chemical synthesis.

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Hydra forced to regenerate a head releases head activator and head inhibitor during the first hours after cutting to induce head‐specific growth and differentiation processes. Analysis of the size distribution demonstrated that the head‐activator peptide is co‐released with (a) large molecular weight carrier molecule(s) to which it is non‐covalently bound. The carrier‐bound head activator is fully active on Hydra indicating that a carrier does not hinder the interaction with receptors. In contrast to this the head inhibitor is released in its naked, low molecular mass form. The association or non‐association with a carrier molecule results in marked differences in biological properties. The head activator has a short range of action, but a long half‐life, the head inhibitor has a global range of action, but a short half‐life. These results provide a plausible explanation why two antagonistically acting substances, although they are released from the same site and simultaneously nevertheless can give rise to a well‐defined temporal and spatial pattern of differentiation as occurs, for example, during head regeneration in Hydra.