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Specificity of archaeal caspase activity in the extreme halophile Haloferax volcanii
Article first published online: 30 NOV 2012
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd
Environmental Microbiology Reports
Volume 5, Issue 2, pages 263–271, April 2013
How to Cite
Seth-Pasricha, M., Bidle, K. A. and Bidle, K. D. (2013), Specificity of archaeal caspase activity in the extreme halophile Haloferax volcanii. Environmental Microbiology Reports, 5: 263–271. doi: 10.1111/1758-2229.12010
- Issue published online: 5 MAR 2013
- Article first published online: 30 NOV 2012
- Accepted manuscript online: 31 OCT 2012 11:42PM EST
- Manuscript Accepted: 23 OCT 2012
- Manuscript Received: 19 MAY 2012
- NSF. Grant Numbers: OCE-0927829, OCE-1061883
Fig. S1. Panel of proteolytic activities in partially purified protein fractions. Hydrolysis rates were determined for cell extracts (CE) from exponentially growing H. volcanii cells, partially purified proteins obtained by sequential ammonium sulfate (AmSO4) precipitation (20%, 40%, 60%, 80% and 100%), and non-AmSO4-precipitable proteins (soluble). AmSO4 was sequentially added to cell extracts at 4°C with constant rocking and incubated for 30 min at each step. Prior to each AmSO4 additon, proteins were pelleted at 10 000 g. The supernatant, representing dissolved proteins, was transferred to a new tube and used for the next percent AmSO4 addition. Protein pellets were resuspended in 100 mM Tris-Cl (pH 7.4)/150 nM NaCl and activity was measured using the aforementioned assay conditions and panel of fluorogenic substrates. Note difference in scale bewteen caspase substrates (upper panel) and other protease substrates (lower panel). Hydroysis of Leu-AMC was also measured but no activity was detected in any fraction. Error bars represent standard deviations for triplicate measurements.
Fig. S2. Protease inhibitor efficacy on model protease substrates. To verify inhibitor effectiveness, purified proteases (recombinant human caspase-8, trypsin, papain, cathepsin B) were individually incubated with inhibitor treatments and compared with uninhibited control reactions. Each purified enzyme was pre-incubated with individual protease inhibitors at 37°C for 1 h at the aforementioned concentrations (see Fig. 3) prior to the addition of the preferred fluorogenic substrate for each respective enzyme: caspase-8 with IETD-, trypsin and cathepsin with ARR-, and papain with LLVY-AMC.
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