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 prorenin was produced in cell factories from stably transfected DAMP cells, a dog epithelial cell line. The equivalent of 10-15 mg of recombinant human renin was secreted in the supernatant from each cell factory. Following a single affinity chromatography step using a renin inhibitor as the ligand, a 181-fold purification was achieved with 81% recovery of the renin activity. This highly pure recombinant enzyme having a specific activity of 3.44 mg angiotensin I.mg protein-1.h-1 was used for kinetic analysis. The kinetic parameters were determined with the natural substrate angiotensinogen and a tetradecapeptide substrate corresponding to the amino terminus of angiotensinogen, Asp1-Asn14, at their respective optimum pH of 5.5 and 6.8. Although there was a six-fold increase in both Km and kcat values for the peptidic substrate (13.3 microM and 8.1 s-1, respectively), when compared with values for the natural substrate (2.04 microM and 1.41 s-1), the catalytic efficiency (0.69 microM-1.s-1) of the enzyme for both substrates was the same. However, the kcat/Km value with angiotensinogen at the physiological pH 7.4 was 30% lower than that observed at the optimum pH 5.5. The recombinant human renin displayed similar optimum pH and kinetic parameters with angiotensinogen and the tetradecapeptide substrate when compared with human kidney renin.
Pilote
L
L
Department of Biochemistry, Bio-Méga/Boehringer Ingelheim Research Inc., Laval, PQ, Canada.
McKercher
G
G
Thibeault
D
D
Lamarre
D
D
D000818:Animals; D001483:Base Sequence; D004285:Dogs; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012083:Renin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D012083:Renin