Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography.
Cathepsin D, a lysosomal aspartic protease, has been purified from porcine liver using a combination of pepstatin-A agarose and Affi-Gel Blue affinity chromatography, followed by size-exclusion chromatography. The purified protein consists of two polypeptide chains of 15 and 30 kDa, and has an isoelectric point of 6.8. Porcine liver cathepsin D has maximum activity at pH 2.5-3.0 as determined by its activity against hemoglobin, with a Kcat of 14.3 s-1 and a kcat/KM of 2.70 x 10(6) s-1M-1 as determined by the hydrolysis of a fluorogenic peptide substrate.
Canduri
F
F
Departamento de Física, IBILCE/UNESP, São José do Rio Preto, Brazil.
Ward
R J
RJ
de Azevedo Júnior
W F
WF
Gomes
R A
RA
Arni
R K
RK
D000818:Animals; D002402:Cathepsin D / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010436:Pepstatins; D010455:Peptides / Q000378:metabolism; D013552:Swine; D014227:Triazines
D006454:Hemoglobins; D010436:Pepstatins; D010455:Peptides; D014227:Triazines; C014195:Streptomyces pepsin inhibitor; C012842:Cibacron Blue F 3GA; D002402:Cathepsin D; C031375:pepstatin