CALMODULIN-DEPENDENT CYCLIC-NUCLEOTIDE PHOSPHODIESTERASE IN ADULT ANDDEVELOPING CHICK SPINAL-CORD

We investigated the level and characteristics of ''low Km'' 3'-5' cycl ic nucleotide phosphodiesterase (PDE) activity in adult and embryo chi ck spinal cord, The DEAE cellulose chromatography elution profile of T riton X-100 extracts showed a single peak of calmodulin-dependent cAMP /cGMP PDE activity. After two additional purification steps, this acti vity showed a five-fold activation by calmodulin (Ka = 1.5 nM) for cGM P hydrolysis, and a linear kinetic behaviour with a Km of 1.3 mu M. Co nversely, the activity showed a biphasic behaviour for cAMP hydrolysis , with Km values of 3.1 and 18.5 mu M, The enzyme showed a Stokes radi us of 4.5 nm. Western blot analysis of the purified enzyme revealed tw o immunoreactive bands with molecular mass of 59 and 65 kDa, respectiv ely. Immunohistochemical staining showed motoneuron decoration both on cell soma and fibres. The developmental pattern of Ca2+-calmodulin-de pendent PDE expression in spinal cord was also studied; the hydrolytic activity for both substrates has been found to increase constantly fr om E5 to post-hatching stages, when it appears 5.6-fold higher as comp ared to the early embryo levels. Furthermore, in cultured spinal cord neurons from ES embryos, muscle extract has been shown to induce a two -fold increase of Ca2+-calmodulin-dependent cGMP activity. In conclusi on, the studies reported here present three relevant findings: (1) the presence in adult and embryo chick spinal cord of PDE activities With characteristics similar to those of the mammalian PDE I enzyme; (2) i ts localization in the ventral horn motoneurons; (3) its regulated exp ression during embryogenesis that is possibly related to soluble epige netic factors produced by the target cells. (C) 1997 Wiley-Liss, Inc.