Procaspases and Apoptotic Protease-Activating Factor-1(APAF-1) in Bovine Thymus, Brain, and Liver Partial Purification and Tissue Distribution

Abstract

We fractionated and highly purified a terminal pro-DEVDase, procaspase 9, and Apafï¼ 1from bovine thymus extracts by three steps of column Chromatography. Using the purified factors, the essential factors of the mitochondriamediated(typeII) apoptotic pathway, we constructed a supplementation assay for each factor to examine the distribution in bovine thymus, brain, andliver. All the tissues expressed a class of procaspase 9 with a molecular size of 50 kDa. The distribution of proDEVDase, however, varied in the tissues. The chromatography of proDEVDase revealed the presence of four peaks in thymus, three in liver, and one in brain extracts. Among them, two major thymus proDEVDase(BT-prODEVDase-1 and-2) and a brain proDEVDase(BB-proDEVDase) were further purified. Although they were procaspase 3-1ike judging from the substrate specificity and reactivity with anti-human caspase 3 antibody, they were slgnificantly different from each otherin molecular size and in reactivity with anti-human caspase 7, Suggesting that they are different molecular species. All of these results revealed a diversity of tissue-specific expression proDEVDase. Among the three factors examined, tissue distribution of Apaf-1 showed the most distinctive feature. The brain extracts showed essentially no caspase activation when incubated with cytochrome c and dATP but restored the activity when purified thymus Apaf-1 was supplemented. Essentially no Apaf-1 activity was detected in the extracts of bovine brain, suggesting that mature bovine brain lacks type II apoptotic pathway in spite of the essential role in the programmed cell death during the normal development of the brain tissue. Biological significance of these findings will be discussed.