Renin Substrate in Plasma of Various Mammalian Species: Electrophoresis on Polyacrylamide Gel

Abstract

In 1971, Nasjletti et al. (1) reported their studies of the distribution of renin substrate in sera of four different species of mammals following electrophoresis on starch gel. Subsequently, Sen et al. (2) and Menard et al.( 3 ) reported on the electrophoretic separation of rat renin substrate. Only Menard et al. used polyacrylamide gel as a supporting medium. We (4) independently developed a technique using polyacrylamide gel electrophoresis and radioimmunoassay in place of bioassay. This technique provides greater resolution and greater sensitivity. We have applied this technique to the plasma of eight mammalian species including the four originally reported by Nasjletti et al. (1). There are striking differences in the mobility of renin substrate of different mammals. Of the species tested, mouse renin substrate shows the greatest mobility, and sheep renin substrate shows the least.Methods. General description. The method we have developed consists of electrophoresis of plasma proteins on polyacrylamide gel, slicing the gel, incubation of each slice separately with renin and protective agents in a small volume of saline, and transfer of an aliquot of the saline to a test tube containing the necessary components for radioimmunoassay of angiotensin I. The radioimmunoassay technique and the determination of bound (B) and free (F) angiotensin I is described below. The B/F ratio is calculated for each gel slice. A significant lowering of this ratio indicates the presence of angiotensin I, which, in turn, indicates the presence of renin substrate. A standard doseresponse curve relating B/F values to the quantity of angiotensin I can be used, but we felt that the B/F values themselves provide equivalent information.Electrophoresis. Electrophoresis was carried out by a modification of the technique outlined by Nerenberg (5). Plasma was mixed with an equal volume of 0.5 A4 sucrose containing 0.5% bromphenol blue as a marker dye. The bromphenol blue travels in two bands during electrophoresis; the fast moving band indicates the electrophoretic "front" and the slower band indicates the position of albumin. Five microliters of the plasma mixture was applied to 5 % polyacrylamide gel cylinders of about 110 mm length and 5 mm diameter. A continuous buffer system was used with glycine-tris buffer adjusted to pH 7.9 with HC1 as the chamber buffer.This buffer was made with 2.9 g glycine, 0.6 g Tris (base) and approximately 0.5 ml 1 N HC1 per 100 ml and was diluted to 1000 ml before use. The gel buffer, pH 8.1, consisted of 2.9 g glycine and 0.6 g Tris (base) per 100 ml. We diluted 2.5 ml of this buffer to 20 ml by combination with the other ingredients making up the polyacrylamide gel. A constant voltage and time (300 V for 30 min) was used. In early experiments, room temperature was used (22-27"), but in later experiments a constant temperature of 25" was maintained.After electrophoresis, the gels were removed from the glass tubes and sliced manually into 2.5 mm sections with a scalpel, using lines on graph paper as a guide ...

Heterogeneity of Renin Substrate in Human Plasma: Effect of Pregnancy and Oral Contraceptives

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There have been few studies of the electrophoretic mobility of renin substrate in plasma and only two' reports of the behavior of renin substrate in human plasma (1,2). Both of these reports indicate that there may be, in human plasma, a small quantity of a second renin substrate with a lesser mobility than the obvious major component, which migrates to a position just behind albumin.

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“…Details of our technique are described in our previous publication (2). In brief, the procedure consists of electrophoresis of plasma proteins on a polyacrylamide-gel cylinder, slicing the gel, incubating each slice separately with human renin, EDTA, and BAL in a small volume of saline, and transferring an aliquot of this mixture to a test tube containing the necessary components for radioimmunoassay of angiotensin I.…”