Cathepsin D from the hepatopancreas of the cuttlefish (Sepia officinalis): purification and characterization.
Cathepsin D from the hepatopancreas of cuttlefish ( Sepia officinalis ) was purified to homogeneity by precipitation with ammonium sulfate (30-60%, w/v), Sephadex G-100 gel filtration, Mono-S cation-exchange chromatography, Sephadex G-75 gel filtration, and Mono-S FPLC with a 54-fold increase in specific activity and 17% recovery. The molecular weight of the purified cathepsin D was estimated to be 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of the native-PAGE and hemoglobin zymography, the purified protease appeared as a single band. The optimum pH and temperature for the cathepsin D activity were pH 3.0 and 50 °C, respectively, using hemoglobin as a substrate. The purified enzyme was completely inhibited by pepstatin A; however, no inhibition was observed with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Moreover, the activity was strongly inhibited by SDS and molybdate and enhanced by ATP. The purified cathepsin D was activated by Mg(2+), Ni(2+), Zn(2+), Cu(2+), Cd(2+), Sr(2+), and Co(2+) ions, whereas it was not affected by Na(+), K(+), and Ca(2+) ions. The N-terminal amino acid sequence of the first 13 amino acids of the purified cathepsin D was APTPEPLSNYMDA. S. officinalis cathepsin D, which showed high homology with cathepsin D from marine vertebrates and invertebrates, had a Pro residue at position 6 and a Ser residue at position 8, where Thr and Lys are common in all marine vertebrates cathepsins D. S. officinalis cathepsin D showed high efficiency for the hydrolysis of myofibrillar proteins extracted from cuttlefish muscle.
Balti
Rafik
R
Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d'Ingénieurs de Sfax, Route Soukra Km 3,5 BP 1173, 3038 Sfax, Tunisia.
Hmidet
Noomen
N
Jellouli
Kemel
K
Nedjar-Arroume
Naima
N
Guillochon
Didier
D
Nasri
Moncef
M
D000595:Amino Acid Sequence; D000818:Animals; D002402:Cathepsin D / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors; D043143:Hepatopancreas / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D049833:Sepia / Q000737:chemistry*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D002402:Cathepsin D