Proteolysis of bovine F-actin by cathepsin B

The proteolytic specificity of cathepsin B on bovine F-actin was investigated. Actin (0.5 mg/ml) was incubated with cathepsin B (1.65 U/ml) for 6 h at 37°C and samples were taken periodically for analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). During incubation, actin was hydrolysed with the simultaneous appearance of three peptides detectable by SDS–PAGE with molecular masses of 35, 33, and 29 kDa. These peptides were electroblotted from SDS–PAGE gels onto polyvinylidene difluoride membranes and their N-terminal sequence determined by Edman degradation. Principal cleavage sites of cathepsin B activity on actin were identified at Met49–Gly50, Thr68–Leu69 and Leu107–Thr108. Reverse-phase high performance liquid chromatography (RP-HPLC) was performed on 2% trichloroacetic acid-soluble fractions of the 6 h hydrolysate. Thirteen peptides separated by RP-HPLC were collected and identified from their N-terminal sequence and, in some cases, from their mass (as determined by mass spectrometry). Cleavage sites were identified at: Gly22–Phe23, Ala24–Gly25, Arg30–Ala31, Lys70–Tyr71, His75–Gly76, Gly76–Ile77, Thr79–Asn80, Lys86–Ile87, Phe92–Tyr93, Arg97–Val98, Thr105–Leu106, Thr251–Ile252, Ala321–Leu322, Leu322–Ala323, Ile329–Lys330, Lys330–Ile331, and Glu363–Tyr364. The results of this study showed that actin was degraded extensively by cathepsin B with the majority of the peptides released arising from the N- and C-termini of the protein.