Enzymatic characterization purified recombinant human renin L. Pilote, G. McKercher, D. Thibeault, and D. Lamarre Abstract: 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-'.h-' 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, ~ s ~ ' - ~ s n ' ~ , at their respective optimum pH of 5.5 and 6.8. Although there was a six-fold increase in both Kmand kc, values for the peptidic substrate (13.3 yM and 8.1 s-', respectively), when compared with values for the natural substrate (2.04 yM and 1.41 s-'), the catalytic efficiency (0.69 p~-'.s-') of the enzyme for both substrates was the same. However, the kcaJKm 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. Key words: recombinant human renin, aspartyl protease, enzyme lunetics. RCsumC : La rCnine, une aspartyl protkase spkcifique intervenant dans le systkme rknine-angiotensine, est synthCtisCe sous forme de prkprorknine. De la prorenine humaine recombinante a kt6 produite dans des unitis Multitray (cell factory) B partir d'une lignCe de cellules CpithCliales de chien B transfection stable, les cellules DAMP. Chacune de ces unites a fourni une quantitC Quivalant B10-15 mg de rCnine humaine recombinante. Une chromatographie dlaffinitC avec un inhibiteur de rknine comme ligand a permis de purifier la protCine par un facteur de 181 en conservant 81% de 1'activitC enzymatique initiale. Cette enzyme recombinante purifiCe ayant une activit6 spCcifique de 3,44 mg d'angiotensine I formCe.mg de protkines-'.h-' a kt6 utilisCe pour des Ctudes de cinktique enzymatique avec soit le substrat naturel, l'angiotensinogkne, soit un substrat synthktique contenant la portion N-terminale de l'angiotensinogkne, A S ~ ' - A S ~ ' ~ , aux pH optimum respectifs de 5,5 et 6,8. Les valeurs de Kmet kc,, mesurkes avec le substrat naturel (2,04 pM et 1,41 s-') sont six fois plus faibles que celles mesurkes avec le substrat peptidique (13,3 yM et 8,l s-'); il en rksulte cependant une meme efficacitk catalytique de 0,69 p ~ - ' , s - ' envers les deux substrats. Cette efficacitk catalytique (kc$Km) envers l'angiotensinogbne est 30 % plus faible au pH physiologique de 7,4 qu'au pH optimum de 5,5. Les paramktres cinktiques et le pH optimum de la rCnine humaine recombinante avec l'angiotensinogkne et le substrat peptidique sont similaires B ceux dCjB rapportks pour la rinine humaine de rein. Mots clks : :&nine humaine recombinante, aspartyl protkase, cinCtique enzymatique. Received December 12, 1994. Accepted April 26, 1995. Abbreviations: AI, angiotensin I; An, angiotensin 11; hANG, human angiotensinogen; hTDP, human tetradecapeptide; PCR, polymerase chain reaction; EDTA, ethylene diaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; TPCK, L-1-tosylamide-2-phenylethyl chlo- romethyl ketone; HSA, human serum albumin; ACHPA, (3s,4s)-4-amino-3-hydroxy-5-cyclohexylpentanoic acid; kDa, kilodalton(s); SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; bp, base pair(s); CMV IE, cytomegalovirus immediate early; LTR, long terminal repeat; MoMLV, Moloney murine leukemia virus; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle medium; RIA, radioimmunoassay; MES, 2-(N-morpho1ino)-ethanesulfonic acid; DMSO, dimethyl sulfoxide; PVDF, polyvinylidene difluoride. L. Pilote, G. McKercher, D. ~hibeault,' and D. Lamarre. Department of Biochemistry, Bio-MCgaoehringer Ingelheim Research Inc., Laval, PQ H7S 2G5, Canada. ' Author to whom all correspondence should be addressed. Biochem. Cell Biol. 73: 163-170 (1995). Printed in Canada 1 Imprim6 au Canada Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by 108.163.184.194 on 05/28/13 For personal use only.