Degradation of glial fibrillary acidic protein protein by a calcium dependent proteinase: an electroblot study

Abstract

In situ and in vitro degradation of glial fibrillary acidic (GFA) protein in mouse spinal cord was examined with electroblots stained for GFA protein by the peroxidase anti-peroxidase method. Non-degraded, intact GFA protein had a molecular weight of 48 Kdaltons and isoelectric points ranging from pH 5.8 to 6.4. The molecular weights to immunoreactive degradation products ranged from 47 to 28 Kdaltons. All of the degradation products had acid shifted isoelectric points (pH 5.8-5.2). Degradation was prevented by chelating calcium with EGTA. In contrast to in situ degradation, degradation in vitro with 3 mM CaCl₂ occured at a faster rate. The effect of pH and temperature on the degradation process were determined by incubating homogenized spinal cords in 3 mM CaCl₂ solutions varying in pH from 4 to 10 and at 4, 37, and 60°C. The greatest number of immunoreactive bands with the lowest molecular weights occurred at pH 8 and 37 °C. The results suggest that turnover of glial filaments is in part controlled by a calcium dependent proteinase active near neutral pH similar to that postulated for neurofilament turnover.