Rat epidermal cathepsin B: purification and characterization of proteolytic properties toward filaggrin and synthetic substrates.
The aim of this study was to purify epidermal cathepsin B from rat skin and investigate its proteolytic activities on filaggrin and several synthetic substrates. The molecular weight of purified monomeric cathepsin B was estimated to be 30 kDa by SDS-polyacrylamide gel electrophoresis. The amino acid composition, similar to that of liver cathepsin B, indicated the enzyme to be an acidic protease. The enzyme had strong hydrolytic activity toward N-benzyloxy-carbonyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-MCA) (152 mU/mg) and N-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin (424 mU/mg), but had no proteolytic activity toward L-arginine-7-amido-4-methylcoumarin. The Km value for Z-Arg-Arg MCA was 0.34 mM and pH optimum was 5.5. Cathepsin B degraded rat epidermal filaggrin into small fragments at pH 4.0 and 5.5., and was inhibited by a specific cysteine proteinase inhibitor, N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)L-leucyl]- agmatin. This study demonstrated that filaggrin was susceptible to degradation by cathepsin B. Such an action may have relevance to skin differentiation in which acid proteases are thought to participate.
Kawada
A
A
Department of Dermatology, National Defense Medical College, Saitama, Japan.
Hara
K
K
Morimoto
K
K
Hiruma
M
M
Ishibashi
A
A
D000818:Animals; D000831:Animals, Newborn; D002401:Cathepsin B / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004151:Dipeptides / Q000138:chemical synthesis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004817:Epidermis / Q000201:enzymology*; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D004151:Dipeptides; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D002401:Cathepsin B