Proteolysis of bovine F-actin by cathepsin B

Abstract

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 Met 49–Gly 50, Thr 68–Leu 69 and Leu 107–Thr 108. 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: Gly 22–Phe 23, Ala 24–Gly 25, Arg 30–Ala 31, Lys 70–Tyr 71, His 75–Gly 76, Gly 76–Ile 77, Thr 79–Asn 80, Lys 86–Ile 87, Phe 92–Tyr 93, Arg 97–Val 98, Thr 105–Leu 106, Thr 251–Ile 252, Ala 321–Leu 322, Leu 322–Ala 323, Ile 329–Lys 330, Lys 330–Ile 331, and Glu 363–Tyr 364. 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.