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. Copyright (C) 1998 Elsevier Science Ltd.