Proteolytic specificity of cathepsin D on bovine F-actin

Proteolysis of bovine F-actin by cathepsin D (E.C. 3.4.23.5) in 50 mM Na ac etate buffer, pH 5.5, at 37 degrees C was investigated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PACE) and reverse-phase h igh performance liquid chromatography (RP-HPLC). Actin was hydrolyzed by ca thepsin D during incubation to peptides detectable by RP-HPLC, although no degradation products were detected by SDS-PAGE. Pq,tides (2% trichloroaceti c acid-soluble) from the hydrolyzate were isolated by RP-HPLC on a C-18 col umn using an acetonitrile/water gradient and identified from their N-termin al sequence and mass. Cathepsin D cleavage sites were identified at Cys(12) -Asp(13), Gly(22)-Phe(23), Arg(30)-Ala(31), Thr(79)-Asn(80), Ile(87)-Trp(88 ), Thr(91)-Phe(92), Phe(92)-Tyr(93), Arg(97)-Val(98), His(103)-Pro(104), Le u(107)-Thr(108), Thr(108)-Glu(109), Lys(120)-Met(121), Leu(144)-Tyr(145), I le(153)-Val(154), Leu(155)-Asp(156), Ile(167)-Tyr(168), Leu(180)-Asp(181), Met(192)-Lys(193), Leu(195)-Thr(196), Arg(208)-Glu(209), Arg(212)-Asp(213), Leu(223)-Asp(224), Lys(240)-Ser(241), Thr(262)-Leu(263), TrP342-Ile(343), Arg(349)-Ser(350), Trp(358)-Ile(359), and Lys(375)-Cys(376). In general, ca thepsin D preferentially cleaved bonds containing at least one hydrophobic amino acid residue. The results of this study showed that actin was degrade d extensively by cathepsin D with peptides released from numerous locations in the protein molecule. (C) 2000 Published by Elsevier Science Ltd.