Honeybees and magnetoreception.

immune sera, polyclonal rabbit antibodies to human IL-1 f3, or anti-p20 ICE and protein A-agarose. After extensive washes, the adsorbed proteins were eluted by boiling the beads in SDS-PAGE sample buffer and analyzed by SDS-PAGE and autoradiography. 13. The ICE homologs and p35 were expressed in E coli under the control of the X pL promoter. ICH-1 (7) and CPP32o (8) contained a polyhistidine linker and were purified by IMAC as described for ICH-2; p35 was purified by chromatography on Q-Sepharose, Mono0, and Superdex-75. 14. Colorimetric assays were performed in 96-well plates by incubating enzyme in assay buffer [100 mM Hepes (pH 7.5), 0.5 mM EDTA, 20% glycerol, and 5 mM dithiothreitol] with N-acetyl-Tyr-Val-Ala-Asp-pnitroanilide (AcYVAD-pNA) as substrate for ICE and ICH-2 or with N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (AcDEVD-pNA) (Quality Controlled Biochemicals, Hopkinton, MA) as substrate for ICH-1 and CPP32. Absorbance at 405 nm was monitored at 370C for 30 min. 15. L. Shi, C. M. Kam, J. C. Powers, R. Aebersold, A. H. Greenberg, J. Exp. Med. 176,1521 (1992). 16. Proteins were separated on gels containing a 10 to 20% gradient of acrylamide (Integrated Separations Science, Natick, MA) in tris-tricine buffer under reducing conditions. For immunoblot analysis, the proteins were electrophoretically transferred onto nitrocellulose and the membranes were blocked, washed, and incubated in purified rabbit immunoglobulin G (IgG) followed by donkey antibodies to horseradish peroxidase-conjugated rabbit IgG (Amersham). Immuno~ blots were developed by enhanced chemiluminescence (Amersham). Rabbit antisera to p35 were produced against residues 75 to 299 fused to trpE of E. coli. ICE has a kcat value of 0.5 mol aminomethylcoumarin (Amc) mol-1 s-1 compared to 1 mol Amc mol-1 s-1 reported previously (27). When approximately equal amounts of ICE and p35 protein were used, only about half of the p35 was cleaved (Fig. 2B, lane 3), consistent with the presence of some inactive enzyme in the preparations used. 17. N. Bump, unpublished results. 18. N. P. C. Walker et al., Cell 78, 343 (1994); K. P. Wilson et al., Nature 370, 270 (1994). 19. DNAs encoding p35 or human prolL-1,B were subcloned into pSVb. The TNT T7-coupled reticulocyte system (Promega) was used to generate proteins labeled with [35S]methionine (15 mCi/mi, Amersham). Radiolabeled p35 was incubated with ICE at 300C for 30 min, and the reactions were stopped by heating in SDSPAGE sample buffer. For the reassociation experiments, the mixtures that contained ICE, AcYVK(biotin)D-CHO, and 35S-labeled p35 or human prolL-1, were incubated with streptavidin-agarose. The beads were washed, adsorbed proteins were eluted by boiling in 10% SDS, and the labeled proteins were detected by autoradiography after SDS-PAGE. 20. Equimolar amounts of N-acetyl-Tyr-Val-Lys-Asp-aldehyde (Bachem Bioscience) and (biotinoyl-E-aminocaproyl)-E-aminocaproic acid N-hydroxysuccinimide ester (Molecular Probes) were combined in a 1 :1 mixture of ethanol and 100 mM sodium borate (pH 8.5) for 80 min at room temperature. The biotinylated product was separated from the starting peptidyl aldehyde by reversed-phase high-performance liquid chromatography (HPLC). The biotinylated peptide aldehyde AcYVK(biotin)D-CHO inhibited ICE with a Ki of less than 1 nM. 21. N. Bump, unpublished results. 22. Complementary DNA fragments encoding either the p32 or p45 forms of human ICE were inserted into pVL1 393 (Invitrogen). The transfer plasmid was cotransfected into SF-9 cells with linearized AcMNPV DNA by means of the BaculoGold system (Pharmingen). Recombinant virus expressing ICE (BV-ICE) was purified by several rounds of infection. 23. For production of ICE, SF-9 cells were infected at a multiplicity of infection of 2 to 5 with recombinant virus stock with a titer of 1 x 108 plaque-forming units per milliliter. The medium was harvested when cell viability was between 0 and 20% and concentrated on SPSepharose HP (Pharmacia). We added 10 mM dithiothreitol, 3 mM AcYVK(biotin)D-CHO, and streptavidinagarose to the eluted protein. Affinity-purified enzyme