title: Proteolytic specificity of cathepsin D on bovine F-actin.
abstract: Proteolysis of bovine F-actin by cathepsin D (E.C. 3.4.23.5) in 50 mM Na acetate buffer, pH 5.5, at 37°C was investigated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and reverse-phase high performance liquid chromatography (RP-HPLC). Actin was hydrolyzed by cathepsin D during incubation to peptides detectable by RP-HPLC, although no degradation products were detected by SDS-PAGE. Peptides (2% trichloroacetic acid-soluble) from the hydrolyzate were isolated by RP-HPLC on a C(18) column using an acetonitrile/water gradient and identified from their N-terminal 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), Leu(107)-Thr(108), Thr(108)-Glu(109), Lys(120)-Met(121), Leu(144)-Tyr(145), Ile(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), Trp(342)-Ile(343), Arg(349)-Ser(350), Trp(358)-Ile(359), and Lys(375)-Cys(376). In general, cathepsin D preferentially cleaved bonds containing at least one hydrophobic amino acid residue. The results of this study showed that actin was degraded extensively by cathepsin D with peptides released from numerous locations in the protein molecule.
authors: Department of Food Science and Technology, University College, Cork, Ireland.
authors: M C
authors: MC
authors: Hughes
authors: A
authors: A
authors: Healy
authors: P L
authors: PL
authors: McSweeney
authors: E E
authors: EE
authors: O'Neill