Similarity between physicochemical properties of recombinant rat prorenin and native inactive renin

Abstract

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.

Adrenal and circulating renin-angiotensin system in stroke-prone hypertensive rats.

“…No difference was found between the two strains of rats with respect to the molecular mass, immunoreactivity, and affinity for con A. Furthermore, all these properties of adrenal active renin are similar to those observed for active renin from the kidney 31 -33 - 35 and plasma. 31 - 33 Thus, the increase in adrenal renin activity in SHRSP may be caused by the increased amount of active renin rather than the appearance of renin isoenzyme with higher enzymatic activity.…”

“…Inactive renin in each fraction was calculated as the difference between total and active renin. Preliminary experiments showed that the recovery of pure rat renal active renin 34 and purified recombinant rat prorenin 35 from a G3.000SW column was 91±3 (n=6) and 89±3% (n=6), respectively.…”

Biochemical properties of amphibian bone morphogenetic protein-4 expressed in CHO cells

“…which contains the mouse dhfr gene was separated on an agarose gel and ligated to the NruI-BamHI fragment (1.6 kb) derived from plasmid pCDM8 (Seed, 1987) harbouring the CMV promoter and XhoI site to yield the pCVD(X) plasmid. Figure l( b) shows the structure of the plasmid pSVD(X) which resulted from transformation of the BamHI site in pSVD (Hosoi et al, 1991) to the XhoI site. A 1.3 kb EcoRI-XhoII cDNA fragment containing the entire coding sequence of Xenopus BMP-4 (Nishimatsu et al, 1992) was blunt-ended with T4 DNA polymerase and ligated with XhoI linkers.…”