Renin purification

The renal pressor mechanism—renin and hypertensin—acts in acute hypertension and in acute renal ischemic states, but apparently not in chronic hypertension. The other mechanisms shown to be active in chronic hypertension are vasoexcitor-vasodepres-sor material relationship pherentasin, a pressor substance found only in human hypertension amines resulting from the insufficient oxidation of amino acids, which are increased in human hypertension and norepinephrine (Sympathin E), which largely reproduces the hemodynamic picture of chronic hypertension. Most of the known pressor substances, with the notable exception of norepinephrine, come from disturbances of, or are extracted from, the kidneys. The large number of pressor substances which have been obtained suggests that many may represent different stages of metabolism of certain parent substances, and that their effectors may be fewer in number and simpler in structure. The chemical identification and purification of most of these substances leave much to be desired, and their phafmacology has in most cases been inadequately studied. The whole problem, however, may soon become simplified. 

Heinrikson, R.L. (1988). Purification and characterization of recombinant proteins the example of human renin. In Therapeutic Peptides and Proteins Assessing the New Technologies. D.R.Marshak and D.T.Liu, eds. (New York Cold Spring Harbor Laboratory), pp. 23 38. 

Past studies of renin were severely hampered by the lack of a pure preparation of this important enzyme. Attempts at purification were hindered by the extremely low concentration of renin in the kidney and the increasing instability of the enzyme as it attained greater degrees of purity. 

It was fortunate that renin purification studies by our group began with an organ which contains extraordinary high concentration of renin. The submaxillary gland of the male adult mouse (5) and... 

As we mentioned, the purification of renal renin had presented insurmountable difficulties in the past (8-17). Recently, several special techniques have been devised and applied to this difficult problem, including affinity chromatography using synthetic inhibitor peptides of renin as affinity ligands (17,20). Burton, et al. synthesized specific renin inhibitors for this purpose (21). These synthetic peptides, however, did not possess sufficiently strong affinity for renin to sequester it from a crude kidney extract. 

There was another serious obstacle to the purification of renin. The reported instability of renin during its purification (12), particularly in its purer state, has been the major deterrent to the numerous attempts of renin purification in the past. We thought it likely that this instability was due to proteolytic destruction by contaminating proteases. Since elimination of these contaminants by physical separation methods was extremely difficult, they were selectively inactivated chemically by treating crude renal extracts with a cocktail of protease inactivators, diisopropyl-phosphoro-... 

Figure 2. Chromatographic elution pattern of mouse sub-maxillary gland renin at its final purification step from a carboxymethyl cellulose column at pH 5.4. Renin activity (-0-) was determined by the method of Reinharz and Roth (62), Protein concentration (—A--) was measured by absorbance at 280 nm. Ref. (54).

Figure 3. Structures of affinity gels used for renin purification.

The above purification method was applied with some modification to the isolation of rat renal renin to homogeneous states. Rat renin has a much weaker affinity for the pepstatin-aminohexyl-agarose gel than porcine renin (31). Human renin was also partially purified by a similar technique (32). Both rat and human renins are glycoproteins since they are bound by concanavalin A and released by 0-methyl-D-glucose. 

Figure 8. Chromatographic elution pattern of the stage of purification of porcine kidney big renin DEAE-cellulose column at pH 6.1. Renin activity ( and protein concentration (—) were determined as Figure 2. Ref. (60).

Further purification and characterization of the human and rabbit inactive renin is required to determine the biochemical nature of its activation. 

I. Rubin, E. Lauritzen and M. Lauritzen, University of Copenhagen, Denmark, "Purification of Renin from Rat Kidney by Affinity Chromatography Using the Substrate Analogy NH2-Leu-Leu-Val-Tyr-Ser-C00H". 

This shortcoming has been overcome, however, as illustrated in a synthesis of (5)-[ 1 - " C]-phenyManine (165) (Figure 11.55). Treatment of lithiated (5)-4-benzyl-3-(3-phenyl[l- C]propionyl)-l,3-oxazolidin-2-one (162) with DBAC followed by chromatographic purification afforded the hydrazinedicarboxylate 163 in 46% yield (yields up to 96% have been reported in the literature). Standard procedures converted 163 to the hydrazine 164. Hydrogenolytic N-N cleavage to the labeled amino acid was accomplished in this case under a more acceptable 50 psi H2 by using aqueous isopropanol as solvent instead of trifluoroacetic acid. The overall radiochemical yield of (5)-[l- " C]phenylalanine was 11%. It was used as shown for the synthesis of [ CJPD 132002 (166). a renin inhibitor. ... 

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