|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1991 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1990 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Renin is an key enzyme in the renin-angiotensi-system which regulates the blood pressure. Recently, it has been observed that prorenin, the inactive form of mature renin, existed in the blood-stream. The prorenin concentration has been, moreover, reported to be 5-1 0 times higher than that of mature renin in the plasma. However, the physiological role, activation mechanism has been little clarified. More recently, we established a rat prorenin expression system using CHO cells transfected with an vector including rat preprorenin cDNA, and observed prorenin releasing stimulator (PRS) in the rat submandibular gland. In this study, I tried to obtain a clue for elucidating the regulation mechanism of prorenin secretion through biochemical study on PRS using rat renal slices and the CHO cells. PRS (200 mu g) was isolated from 50 g of rat submandibular gland by benzamidine-Sepharose column, cation exchange column chromatography, preparative isoelectrofocusing electrophoresis, and high-perfor
mance gel chromatography. In this study, PRS with molecular size of 28, 000 was confirmed by SDS-PAGE in the each step preparation to make rapid for the purification procedure. The biological activity of PRS was only analyzed for the final PRS preparation. The pl value of PRS was determined at 8.7. PRS was negative for PAS-staining test so that it was thought to have probably no sugar chain. I am determining amino acid sequence of PRS completely. So far, N-terminal amino acid sequence of Leu-Ser-Glu-Glu has been determined.
The 5 x 10^<-9> M of PRS increased the release amount of prorenin up to 150% from rat renal slices, but never effected on the prorenin release from the CHO cells. On the other hand, verapamil, which is an antagonist of calcium channel and reported to stimulate prorenin release form the rabbit renal slices, increased prorenin release from the slices up to 150% and decrease the prorenin release by 20% at the concentration of 1 x 10^<-4>M.
From these results, it was elucidated that the specific activity of PRS had about 20, 000 times higher than that of verapamil, and PRS interacted more specifically to renal slice than to the CHO cells.