Similarity between physicochemical properties of recombinant rat prorenin and native inactive renin.
Rat prorenin was synthesized by Chinese-hamster ovary cells transfected with an expression vector containing rat preprorenin cDNA sequences, then purified by concanavalin A-Sepharose chromatography and h.p.l.c. on G3000SW. The molecular mass of purified prorenin was 46,000 Da, as determined by h.p.l.c. on G3000SW. Immunoblot analysis indicated that recombinant prorenin cross-reacted with anti-(mature renin) antibody and two kinds of antibodies recognizing the N-terminus and C-terminus of the prosegment of rat prorenin. Recombinant prorenin was bound to a Cibacron Blue-Sepharose column and eluted with 1.4 M-NaCl, but was not retained by an octapeptide renin inhibitor (H-77)-Sepharose column. Trypsin activation of prorenin increased the renin activity 110-fold, caused binding to an H-77-Sepharose column and nullified the reactivity to the above two kinds of anti-prosegment antibodies, findings indicating that the activation of prorenin with trypsin is due to the cleavage of the prosegment. Rat plasma inactive renin, partially purified by h.p.l.c. on G3000SW, had much the same physicochemical characteristics as the recombinant prorenin. These results provide evidence that rat plasma inactive renin is prorenin. Recombinant prorenin is a useful material for examining the physiological role of circulating prorenin.
Hosoi
M
M
Department of Pharmacology, Osaka City University Medical School, Japan.
Kim
S
S
Yamauchi
T
T
Watanabe
T
T
Murakami
K
K
Suzuki
F
F
Takahashi
A
A
Nakamura
Y
Y
Yamamoto
K
K
D000818:Animals; D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015151:Immunoblotting; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012083:Renin / Q000096:biosynthesis / Q000097:blood* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D004247:DNA; D014357:Trypsin; D012083:Renin