A kinetic study of the pH optimum of canine cardiac cathepsin D

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Abstract
An acid protease, most likely cathepsin D, was purified approximately 1000 fold to homogeneity from canine cardiac tissue using acetate precipitation, affinity chromatography and gel filtration. Molecular weight determinations of the isolated acid protease using gel filtration suggested the presence of a single polypeptide chain of molecular weight 42000. Similar studies using SDS polyacrylamide gel electrophoresis indicated the presence of two subunits of molecular weight 32000 and 14000. The purified acid protease exhibited a pH optimum of 3.4 (with haemoglobin as substrate), an amino acid composition similar to that known for cathepsin D and was markedly inhibited by pepstatin A. Apparent Km and Vmax (determined at 40°C and pH 3.4 using haemoglobin) were found to be 1.43 × 10−5 ± 0.18 × 10−5 mol·litre−1 (mean ± SD, n = 6) and 1.38 × 10−5 ± 0.16 × 10−5 mol tyrosine·litre−1·min−1 (mean ± SD, n = 6) respectively. Measurement of apparent Km and Vmax at higher pH values (4.2 and 4.5) yielded values of 0.68 × 10−5 ± 0.19 × 10−5 and 0.83 × 10−5 ± 0.08 × 10−5 (n = 4, pH 4.2) and 1.33 × 10−5 ± 0.23 × 10−5 and 0.49 × 10−5 ± 0.04 × 10−5 (n = 5, pH 4.5) respectively. Our results show that while the cathepsin D mediated hydrolysis of haemoglobin proceeds less efficiently at higher pH, the ability of the acid protease to bind haemoglobin at higher pH is not decreased. It is suggested that cathepsin D may be functional under similar conditions in vivo, that is at pH values less acidic than that indicated by the pH optimum of the enzyme.
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