Abstract 182

Background: Protein phosphorylation is involved in the signalling cascade after short term hypoxia. Aim: To monitor global protein tyrosine (Y) phosphorylation in synaptosomes from hypoxic and control animals, exploiting the resolving power of computer-aided 2-D gel electrophoresis (2-D PAGE) Materials and Methods: Cerebral cortical synaptosomes from three normoxic and three hypoxic newborn guinea pigs were prepared; brain tissue hypoxia in the fetus was documented biochemically by decreased levels of ATP and phosphocreatine. Synaptosomal samples were denatured and analysed by 2-D PAGE. Y-phosphorylated proteins were identified by 2-D immunoblotting with specific antiserum to phosphotyrosine residues. Silver-stained gels were scanned by computing densitometer. Proteins corresponding to immunoreactive spots were recognized on silver-stained gels by computer-aided matching carried out on a Sun SPARCstation using the MELANIE II software. Results: Hypoxia induces remarkable variations in the pool of tyrosine phosphorylated proteins: 45 electrophoretic units (including 31 single spots and 14 spots in 2 isoelectric series) were found in hypoxic synaptosomes only and 16 (including 6 single spots and 10 spots in 3 isoelectric series) in normoxic synaptosomes only; over 120 electrophoretic units (including 40 spots in 12 isoelectric series) were found in both samples. Conclusions: Our data suggests that hypoxia extensively remodels the signalling pathway switching on Y-phosphorylation in some proteins and switching it off in others.