Abstract
Brain renin has been purified 1,670,000-fold to homogeneity from bovine anterior pituitary in seven steps, including affinity chromatography on pepstatin-aminohexyl-agarose. The molecular weight of this enzyme is 36,000 as determined by gel filtration on Ultrogel AcA 44 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an isoelectric point of 5.25 and works best at physiological pH. In contrast to renal renin, bovine brain renin fails to bind to concanavalin A and four other lectins. The angiotensin I-forming activity of the purified brain renin was completely neutralized by anti-hog renal renin antibody. Rabbit antisera against pure brain renin showed a low degree of species and tissue specificity, reacting readily with hog brain renin and bovine and hog kidney renins. These results provide definite evidence for the presence of a functional brain renin-angiotensin system.
Highlights
Brain renin has been purified 1,670,000-fold to ho- were used instead of rat or hog pituitaries because large mogeneity frombovineanteriorpituitary in seven quantities of starting material were needed to obtain a signifsteps, including affinity chromatographoyn pepstatin- icant amount of pure enzyme.Biochemical and antigenic aminohexyl-agarose.The molecular weight of this en- properties of the purified enzymeare presented. zyme is 36,000 as determined by gel filtration on Ultrogel AcA 44 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The enzyme has an isoelec
The angioten- DEAE-cellulose (DE52) and CM-cellulose (CM52) were purchased sin I-forming activity of the purified brain renin was from Whatman; DEAE-Sephacel, Sepharose-CL 4B, and wheat germ completely neutralized by anti-hog renal renin antiagglutinin-Sepharose from Pharmacia; Ampholine and Ultrogel AcA
Affinity Chromatography-Pepstatin affinity chromatography, which has been successfully used for the purification of aspartyl proteases from a variety of sources, was employed for the purification of bovine anterior pituitary renin
Summary
Materials contrast to renal renin, bovine brain renfainils to bind Bovine pituitaries were obtained from Shibaura Zoki, Tokyo. to concanavalinA and four otherlectins. Renin activity was eluted from the DE52 as follows: the washed filter cake was resuspended in 6 liters of 0.1 M acetate (pH 5.0) containing 0.2M NaCl, stirred for 30 min, and suction-fdtered again. The supernatant (15 liters) was passed through a pepstatin-aminohexyl-agarose column (4.4 X 11 cm) previously equilibrated with 20 m~ imidazole buffer (pH 6.0) containing 0.6 M NaC1, a t a flow rate of 250 ml/h. The fractions containing renin activity were pooled (200 ml), concentrated to 30 ml by ultrafitration on a Toyo UK-10 filter, and dialyzed against 10 liters of 50 m~ pyridine buffer, pH 5.5, for 18h with two changes. The column was eluted with the Same buffer at a flow rate of 6 ml/h and 1.1-ml fractions were collected.
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