title: Isolation, biochemical characterization, and molecular modeling of American lobster digestive cathepsin D1.
abstract: An aspartic proteinase was isolated from American lobster gastric fluid. The purified cathepsin D runs as a single band on native-PAGE displaying proteolytic activity on a zymogram at pH 3.0, with an isoelectric point of 4.7. Appearance of the protein in SDS-PAGE, depended on the conditions of the gel electrophoresis. SDS treatment by itself was not able to fully unfold the protein. Thus, in SDS-PAGE the protein appeared to be heterogeneous. A few minute of boiling the sample in the presence of SDS was necessary to fully denature the protein that then run in the gel as a single band of ~50 kDa. The protein sequence of lobster cathepsin D1, as deduced from its mRNA sequence, lacks a 'polyproline loop' and β-hairpin, which are characteristic of some of its structural homologues. A comparison of amino acid sequences of digestive and non-digestive cathepsin D-like enzymes from invertebrates showed that most cathepsin D enzymes involved in food digestion, lack the polyproline loop, whereas all non-digestive cathepsin Ds, including the American lobster cathepsin D2 paralog, contain the polyproline loop. We propose that the absence or presence of this loop may be characteristic of digestive and non-digestive aspartic proteinases, respectively.
authors.affiliation: Centro de Investigaciones Biológicas del Noroeste (CIBNOR), La Paz, Mexico.
authors.forename: Liliana
authors.initials: L
authors.lastname: Rojo
authors.forename: Rogerio
authors.initials: R
authors.lastname: Sotelo-Mundo
authors.forename: Fernando
authors.initials: F
authors.lastname: García-Carreño
authors.forename: László
authors.initials: L
authors.lastname: Gráf
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002402:Cathepsin D / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008121:Nephropidae / Q000201:enzymology*; D010802:Phylogeny; D016415:Sequence Alignment; D013270:Stomach / Q000201:enzymology
chemical_list: D002402:Cathepsin D