Activation mechanism of erythrocyte cathepsin E. evidence for the occurrence of the membrane-associated active enzyme.
Activation of the erythrocyte cathepsin E located on the cytoplasmic surface of the membrane in a latent form was studied in stripped inside-out membrane vesicles prepared from human erythrocyte membranes. Incubation of the vesicles at 40 degrees C at pH 4 resulted in increased degradation of the membrane proteins, especially band 3. This proteolysis was selectively inhibited by the inclusion of pepstatin (isovaleryl-Val-Val-statyl-Ala-statine) or H 297 [Pro-Thr-Glu-Phe(CH2-NH)Nle-Arg-Leu] in the incubation mixtures, indicating that cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis. Two potential active-site-directed inhibitors of aspartic proteinases, pepstatin and H 297, were used to prove the occurrence of the membrane-associated active enzyme. To minimize potential errors arising from non-specific binding, the concentrations of the inhibitors used in the binding assay (pepstatin, 5 x 10(-8) M; H 297, 1 x 10(-5) M) were determined by calibration for purified and membrane-associated cathepsin E. The inhibition of the membrane-associated cathepsin E by each inhibitor, which showed the binding of the inhibitor to the activated enzyme, was temperature- and time-dependent. The binding of each inhibitor to the enzyme on the exposed surface of the membrane at pH 4 was highly specific, saturable, and reversible. The present study thus provides the first evidence that cathepsin E tightly bound to the membrane is converted to the active enzyme in the membrane-associated form, and suggests that this enzyme may be responsible for the degradation of band 3.
Ueno
E
E
Department of Pharmacology, Nagasaki University School of Dentistry.
Sakai
H
H
Kato
Y
Y
Yamamoto
K
K
D001711:Biotransformation; D020059:Cathepsin E; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000097:blood*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D010436:Pepstatins / Q000494:pharmacology; D011487:Protein Conformation
D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D002403:Cathepsins; D020059:Cathepsin E; C031375:pepstatin