Rat kidney renin and cathepsin D: purification and comparison of properties

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Abstract
Renin and cathepsin D were purified by 7 step procedures involving 5 steps common to both enzymes. These common five steps were extraction of freeze-dried kidney powder in 30 methoxyethanol-water, diethylaminoethylcellulose (DEAE-cellulose) batch adsorption and elution, pepstatin-aminohexyl-Sepharose chromatography, Sephadex G-100 chromatography, and DEAE-cellulose chromatography. The renin component was purified further by passage through an anti-rat spleen cathepsin D Ig G-Sepharose (IgG-Sepharose) column, followed by carboxymethyl-Sepharose (CM-Sepharose) chromatography which separated 2 renin components. Cathepsin D activity obtained by the fifth step was purified by passage through an anti-rat kidney renin IgG-Sepharose column followed by DEAE-Sephacel chromatography which separated three cathepsin D components. The homogeneity of renin and cathepsin D preparations was demonstrated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The 2 components of renins showed MW of 42,000 and 36,000 by gel filtration and 38,000 and 36,000 by SDS gel electrophoresis, respectively. They showed isoelectric points of 5.35 and 5.65 by electrofocusing in 5% polyacrylamide gels. Their optimum pH of enzyme activity were 6.5 as determined by using nephrectomized rat plasma as a substrate. Their specific angiotensin I (Ang I) generation activities were 158 and 146 .mu.g of Ang I (.mu.g of protein)-1 h-1, respectively, which correspond to 1100 and 1020 Goldblatt units (mg of protein)-1 h-1. The 3 cathepsins showed MW of 41,000, 43,000 and 41,000 by gel filtration and 46,000, 45,000 and 46,000 by SDS gel electrophoresis. They showed isoelectric points of 6.20, 6.05, and 6.00 by electrofocusing in 5% polyacrylamide gels. Each of the catehpsins showed two pH optima of 3.0 and 4.5 as examined by using bovine hemoglobin labeled with [14C]-glycine methyl ester as substrate. The cathepsins showed an optimal angiotensin I generating activity of 0.98, 1.27, and 1.22 .mu.g of Ang I (.mu.g of protein)-1 h-1, respectively, at pH 4.5. At pH 6.5, the cathepsin D showed a much diminished renin-like angiotensin I generation activity.

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