Purification And Characterization Of Rabbit Reticulocyte Cathepsin D (proteases).

Abstract

An acidic protease from rabbit reticulocyte membranes has been purified to homogeneity. The protease was purified from a reticulocyte dense membrane fraction by CHAPS extraction, followed by chromatography on immobilized pepstatin (pepstatin-aminohexyl Sepharose). The purified enzyme catalyzed both the degradation of hemoglobin and the cleavage of the hydrophobic tail of microsomal cytochrome b(,5). The molecular weight of the protease has been confirmed to be 45,000 by gel-filtration on Sephadex G-100 and by SDS-PAGE. The enzyme bound to concanavalin A-Sepharose and could be eluted from the gel with (alpha)-methylmannoside. The amino acid and sugar composition of the protease were determined, showing the enzyme to be a glycoprotein of the high-mannose type, apparently with two chains of oligosaccharides (each of them consisting of Man(,8)GlcNAc(,2) residues) per polypeptide chain. With hemoglobin as a substrate, the enzyme showed an apparent K(,M) of 3 x 10('-6); with cytochrome b(,5) as a substrate, the apparent K(,M) of the protease was estimated to be 1.8 x 10('-5) M. The K(,I) of the enzyme for pepstatin (a very strong competitive inhibitor of the protease) was determined to be 6 x 10('-9 M, )with hemoglobin as a substrate. Fe('3+) and urea also inhibited the acidic protease. At least five different forms of soluble cytochrome b(,5) were generated by the protease, in a time-dependent fashion. Slight decreases of pH below the pH optimum affected the relative amounts of the different soluble forms of cytochrome b(,5) generated by the protease. ATP and 2,3-DPG also affected the distribution of soluble forms of cytochrome b(,5) generated by the enzyme. At pH 3.5 and in the absence of detergents, but not at pH 5.8 and in the presence ot Triton X-100, the protease also degraded bovine serum albumin, insulin, lactic dehydrogenase, catalase, and cytochrome c, and generated TCA-soluble peptides from rabbit reticulocyte ribosomes. The properties of the reticulocyte protease were compared to those of bovine spleen cathepsin D. The comparison of the K(,M) values, the K(,I) values for pepstatin, the molecular weights, the sugar and amino acid compositions, the activity versus pH profiles, and the substrate specificity of both enzymes strongly suggests that the reticulocyte enzyme is cathepsin D.