In the polychete Hydroides norvegica there are two opercula with different degrees of organization: one is completely developed and functional, the other, present only in rudimentary outline. The latter only begins to develop when the functional operculum is removed. It was observed that in the whole body of the Hydroides, but particularly in the functional operculum, there is a substance which inhibits the development of the rudimental operculum. This substance was purified in order to determine its chemical nature.From preparation of opercular homogenates we chemically separated numerous fractions, each of which was tested for inhibitory activity and chemical and physical properties. The inhibitor under study proved to be soluble i n aqueous solutions, heat resistant, of low molecular weight, of an organic nature, anionic and aromatic. Finally it was chromatographically identified as 5' adenosine monophosphate.It is known that in the polychete Hydroides norvegica, immediately after the removal of the functional operculum, the rudimentary one begins to develop, reaching in about one week the shape and size of the one removed. The operculum which has thus developed constitutes the new functional operculum, while the stump left after the removal constitutes a new rudimentary operculum.This type of regeneration was first described by Zeleny ('02) and called "compensatory regeneration." Ludwig ('57), using homogenates of functional opercula, showed that the compensatory regeneration of the operculum is regulated by an inhibiting substance present in the functional operculum.In a previous paper Durante and Puccia ('70) showed that this inhibitory substance is present in the whole body gills, thorax and abdomen. The highest concentration, however, was found in the functional operculum. We further showed that this substance is soluble in aqueous solutions, resistant to heat (up to 80°C), has a low molecular weight and probably is of an aromatic nature.This research has been undertaken in order to investigate the chemical nature of the inhibiting substance.
METHODSThe soluble acid fraction containing the active substance was prepared as previously described (Durante and Puccia, '70). This fraction, when passed through active charcoal, loses its inhibitory capacity.The inhibitory substance is removed from the charcoal by treating it with a mixture of ethanol-water-ammonium hydroxide (2:1:2 in volume).The fraction thus obtained -the "charcoal eluted fraction" -was deprived of solvents by distillation and then divided into two parts. The first, after concentration, was re-suspended in sea-water and used for the inhibition test; the second, after alkalinization with ammonium hydroxide, was chromatographed on a "DEAE cellulose" resin column, 2 X 60 cm.The fractions were recovered by elution with cold ammonia bicarbonate solution pH 8.6 buffer (rate of flow 0.5 ml/minute) at an initial concentration of 2 mM and subsequently in a linear gradient from 2 mM to 0.25 M, a modification of Stahelin's method ('61) for the se...
