ENZYMATIC CHARACTERIZATION OF PURIFIED RECOMBINANT HUMAN RENIN

Renin is a highly specific aspartyl protease of the renin-angiotensin system initially synthesized as preprorenin. Recombinant human proreni n was produced in cell factories from stably transfected DAMP cells, a dog epithelial cell line. The equivalent of 10-15 mg of recombinant h uman renin was secreted in the supernatant from each cell factory. Fol lowing a single affinity chromatography step using a renin inhibitor a s the ligand, a 181-fold purification was achieved with 81% recovery o f the renin activity. This highly pure recombinant enzyme having a spe cific activity of 3.44 mg angiotensin I . mg protein(-1). h(-1) was us ed for kinetic analysis. The kinetic parameters were determined with t he natural substrate angiotensinogen and a tetradecapeptide substrate corresponding to the amino terminus of angiotensinogen, Asp(1)-Asn(14) , at their respective optimum pH of 5.5 and 6.8. Although there was a six-fold increase in both K-m and k(cat) values for the peptidic subst rate (13.3 mu M and 8.1 s(-1), respectively), when compared with value s for the natural substrate (2.04 mu M and 1.41 s(-1)), the catalytic efficiency (0.69 mu M(-1). s(-1)) of the enzyme for both substrates wa s the same. However, the k(cat)/K-m value with angiotensinogen at the physiological pH 7.4 was 30% lower than that observed at the optimum p H 5.5. The recombinant human renin displayed similar optimum pH and ki netic parameters with angiotensinogen and the tetradecapeptide substra te when compared with human kidney renin.