BIOCHEMICAL CHARACTERISTICS OF CASPASE-3, CASPASE-6, CASPASE-7, AND CASPASE-8

The observation that the nematode cell, death effector gene product Ce d-3 is homologous to human interleukin-1 beta-converting enzyme (caspa se-1) has led to the discovery of at least nine other human caspases, many of which are implicated as mediators of apoptosis. Significant in terest has been given to aspects of the cell biology and substrate spe cificity of this family of proteases; however, quantitative descriptio ns of their biochemical characteristics have lagged behind. We describ e the influence of a number of environmental parameters, including pH, ionic strength, detergent, and specific ion concentrations, on the ac tivity and stability of four caspases involved in death receptor-media ted apoptosis. Based on these observations, we recommend the following buffer as optimal for investigation of their characteristics in vitro : 20 mM piperazine-N,N'-bis-(2-ethanesulfonic acid) (PIPES), 100 mM Na Cl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl) -dime thylammonio]-2-hydroxy-1-propanesulfonic acid (CHAPS), 10% sucrose, pH 7.2. Caspase activity is not affected by concentrations of Ca2+ below 100 mM, but is abolished by Zn2+ in the submicromolar range, a common characteristic of cysteine proteases. Optimal pH values vary from 6.8 for caspase-8 to 7.4 for caspase-3, and activity of all is relatively stable between 0 and 150 mM NaCl. Consequently, changes in the physio logic pH and ionic strength would not significantly alter the activity of the enzymes, inasmuch as all four caspases are optimally active wi thin the range of these parameters found in the cytosol of living and dying human cells.