SUBCELLULAR-DISTRIBUTION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE IN CEREBELLAR GRANULE CELLS UNDERGOING CYTOSINE ARABINOSIDE-INDUCED APOPTOSIS

We have previously shown that cytosine arabinoside (AraC)-induced apop tosis of cerebellar granule cells (CGCs) results in an increase of a 3 8-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresi s, identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1 .2.1.12). Antisense oligonucleotides to GAPDH mRNA afford acutely plat ed CGCs significant protection against AraC-induced apoptosis. We used differential centrifugation to examine which subcellular components a re affected, Treated and untreated cells were sonicated in 0.32 M sucr ose and sequentially centrifuged at 1,000, 20,000, and 200,000 g, to o btain crude nuclear, mitochondrial, microsomal, and cytosolic fraction s. Western blotting showed that the levels of GAPDH protein were marke dly increased in the 1,000- and 20,000-g pellets, The levels in the cy tosolic supernatant were decreased dramatically by AraC in acutely pla ted CGCs but not in cells 24 h after plating. It is noteworthy that al though GAPDH protein in the pellet fractions increased, the dehydrogen ase activity of GAPDH decreased. Two other dehydrogenases, lactate deh ydrogenase (EC 1.1.1.27)and glucose-6-phosphate dehydrogenase (EC 1.1. 1.49), were not similarly affected, suggesting that the effect was GAP DH specific. These observations suggest that GAPDH levels change in sp ecific organelles during apoptosis for reasons that are separate from its function as a glycolytic enzyme. The accumulation of GAPDH protein in specific subcellular loci may play a role in neuronal apoptosis.