Transient ischemia depletes free ubiquitin in the gerbil hippocampal CA1 neurons

We investigated ubiquitin immunoreactivity in the postischemic gerbil hippocampus using two kinds of antibodies. Immunostaining for ubiquitin in the hippocampus was strongly dependent on the antibodies used. Using rabbit polyclonal antibody U-5379, immunoreactivity disappeared from the hippocampus in the early reperfusion period, and reappeared in the dentate granule cells and CA3 pyramidal cells, but never in the CA1 pyramidal cells. In contrast, rat monoclonal antibody DF2 showed sustained immunoreactivity in the CA1 during the 48-h reperfusion period. On the immunoblots of gerbil brain homogenates, U-5379 and DF2 exhibited similar specificities. Immunoprecipitation and immunoabsorption of these antibodies disclosed that, under nondenaturing conditions, U-5379 bound exclusively free ubiquitin, while DF2 had little affinity for free ubiquitin, but appeared to have more affinity for conjugated ubiquitin. Thus, transient ischemia depletes free ubiquitin but not conjugated ubiquitin in the CA1. This depletion may be caused by impaired conversion from conjugated to free ubiquitin and/or failure of de novo ubiquitin synthesis.