Proteolysis of bovine F-actin by cathepsin B

The proteolytic specificity of cathepsin B on bovine F-actin was investigat ed. Actin (0.5 mg/ml) was incubated with cathepsin B (1.65 U/ml) for 6 h at 37 degrees C and samples were taken periodically for analysis by sodium do decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). During incuba tion, actin was hydrolysed with the simultaneous appearance of three peptid es detectable by SDS-PAGE with molecular masses of 35, 33, and 29 kDa. Thes e peptides were electroblotted from SDS-PAGE gels onto polyvinylidene diflu oride membranes and their N-terminal sequence determined by Edman degradati on. Principal cleavage sites of cathepsin B activity on actin were identifi ed 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% trich loroacetic acid-soluble fractions of the 6 h hydrolysate. Thirteen peptides separated by RP-HPLC were collected and identified from their N-terminal s equence and, in some cases, from their mass (as determined by mass spectrom etry). 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. (C) 1999 Elsevier Science Ltd. All rights reserved.