Biochimica et Biophysica Acta, 427 (1976) 208-217

© Elsevier Scientific Publishing Company, Amsterdam --Printed in The Netherlands BBA 37255 P U R I F I C A T I O N A N D PARTIAL C H A R A C T E R I Z A T I O N OF H U M A N ANGIOTENSINOGEN

PETER EGGENA, CHUNG L. CHU, JACK D. BARRETT and MOHINDER P. SAMBHI Hypertension Division, Medical Service, Veterans Administration Hospital, Sepulveda, California and Department of Medicine, University of California at Los Angeles (U.S.A.)

(Received July 28th, 1975)

SUMMARY Renin substrate was initially extracted from human plasma by (NH4)zSO4 followed by chromatography on Sephadex G-150, DEAE cellulose, calcium phosphate gel, isoelectric focusing and preparative polyacrylamide gel electrophoresis. On the basis of one mol of angiotensin per mol of substrate, the purity of the preparation is in excess of 95 %. On analytical polyacrylamide gel electrophoresis in the presence of 1% sodium dodecyl sulfate or 8 M urea, the protein appears homogenous. In addition, the purified protein shows only one preciptin line against anti-normal human serum on either Ouchterlony immunodiffision or immunoelectrophoresis. The biological activity appears similar to "native" renin substrate since the Km is the same as that reported for the renin reaction in whole plasma. The molecular weight was determined as 110 000 by gel filtration and polyacrylamide gel electrophoresis; amino acid analysis of the human substrate differs from that reported for hog, especially in the Asp, Glu and Gly composition.

INTRODUCTION The role of the renin angiotensin system in the physiology of normotensive man as well as in the genesis and maintenance of hypertensive disease continues to be an area of active investigation. The lack of full characterization, both physical and chemical, of the two principle protein molecules involved in the system, the enzyme renin and its globulin substrate angiotensinogen, remains a prominent limitation. These proteins are known to possess species differences [1], yet the current concepts on the nature of renin substrate in man are largely derived from lower animal sources [2-9]. The renal enzyme renin cleaves its substrate between position 10 and 11 (LeuLeu) to generate the decapeptide, angiotensin I [10, 11]. Whether the proteolytic activity of renin is specific solely for the leucyl-leucine bond of angiotensinogen remains inconclusive [12]. The C-terminal dipeptide histidyl-leucine of angiotensin I is cleaved by angiotensin converting enzyme [5] to produce the vasopressor octapeptide angiotensin I I [ 13]. Quantitation of generated angiotensin is at present the only method available for estimation of renin and renin substrate concentrations. One mol of angiotensin is assumed to be generated, per mol of substrate. Definitive studies of the

209 system require an alternate direct method for quantitation of these proteins. Purification of renin substrate beside physical chemical characterization offers the opportunity to develop a direct radioimmunoassay for this protein. In addition, this purified protein is essential in determining the kinetic parameters of the renin reaction in man, since a homologous system is required. In the present study, we have purified and partially characterized human renin substrate from pooled plasma of normotensive subjects. MATERIALS AND METHODS

Ammonium sulfate fractionation Pooled plasma containing 0 . 3 8 ~ EDTA or serum was adjusted to 1.5 M (NH4)2SO 4 by the addition of either solid ammonium sulfate or a saturated solution, stirred overnight at 4 °C and centrifuged at 5000 × g in a refrigerated centrifuge at 4 °C for 30 min. The precipitate was washed twice with a 1.5 M (NH4)2SO4 solution. The combined supernatant liquids were adjusted to 2.3 M (NH4)2SO4 and stirred for 4 h at 4 °C. After centrifugation as above, the precipitates were disolved and exhaustively dialyzed against distilled deionized water and subsequently freeze dried.

Gel exclusion chromatography Gel filtration was performed with Sephadex G-150 (Pharmacia Fine Chemicals) equilibrated in 0.05 M Tris.HC1 buffer (pH 8.4) on a 5 cm × 100 cm chromatographic column.

DEAE cellulose chromatography Ion exchange chromatography on DEAE-Cellulose (Whatman) equilibrated in 0.05 M Tris.HC1 buffer, pH 8.4, containing 0.03 M NaC1 was performed in a 2.5 cm × 45 cm column. Bound renin substrate was eluted in a NaC1 gradient (0.030.15 M).

Chromatography on calcium phosphate gel Calcium phosphate gel (Bio-Rad) equilibrated in 0.001 M sodium phosphate buffer, pH 6.8, +0.5 M NaC1 was used in 2.5 cm x 45 cm chromatographic column Bound renin substrate was eluted in a phosphate gradient (0.001-0.2 M) with 0.5 M NaCI at pH 6.8.

Isoelectric focusing Separation by isoelectric point was performed on a LKB 8121 column with ampholines (pH 3-6 or 3.5-10, LKB-Produkter AB) and a sucrose gradient following the method of Vestberg and Svensson [14].

Preparative polyacrylamide gel electrophoresis A Shandon preparative polyacrylamide gel electrophoresis unit was employed using a 7.5 ~o acrylamide (Eastman) and an electrolyte buffer of 0.05 M Tris containing 0.0383 M glycine, pH 8.3. Elution buffer was 0.43 M Tris containing 9.244 M acetic acid.

210

Miscellaneous methods Immuno-diffusion and immunoelectrophoresis against human serum antiserum as well as analytical polyacrylamide gel electrophoresis were performed as previously described [15, 16]. Protein concentrations were determined by the method of Lowry et al. [17]. Amino acid analysis Amino acid analysis was performed by the method of Spackman et al. [18] modified by the micro-technique of Bohlen et al. [19]. Renin substrate quantitation Renin substrate concentration was determined by incubation of the isolated protein fraction with homologous angiotensinase free renin [7] at 37 °C and pH 5.5 in the presence of 0.015 M EDTA and 1 • 10 -4 M diisopropylfluorophosphate (DFP) for 30 min. The generated angiotensin I was quantitated either by bioassay in a nephrectomized ganglionic blocked rat by the method of Sambhi and Barrett [20], or by radioimmunoassay employing the method of Eggena et al. [21]. RESULTS

Ammonium sulfate fractionation The initial fractionation of plasma with (NH4)2SO 4 (1.5-2.3 M) led to a 7-fold purification with recoveries ranging from 70-90 ~. No angiotensinogen was detected in either the 1.5 M precipitate or the 2.3 M supernatant fractions. Initially a DEAE batch procedure following the (NH4)2SO4 fractionation was employed (0.04-0.11 M sodium phosphate, pH 7.4). This procedure was found to be unsatisfactory since only a marginal increase in purity was obtained and recoveries were only 4 0 - 6 0 ~ of the applied angiotensinogen. Gel exclusion chromatography On column chromatography with Sephadex G-150 of the ammonium sulfate fraction a 17-fold increase in purity was obtained with 9 5 - 1 0 0 ~ recoveries of the applied protein (Fig. 1). Angiotensinogen was located in the descending shoulder of the main protein peak (Fig. 1). The major contaminant of the isolated protein was found by immunoelectrophoresis (Fig. 2) and analytical polyacrylamide gel electrophoresis, (Fig. 3) to be albumin. The single peak of the angiotensinogen activity appeared in an elution volume smaller than that of the albumin. DEAE cellulose chromatography The results of ion exchange chromatography in this medium are shown in Fig. 4. Angiotensinogen was eluted in a NaC1 gradient between 0.03 and 0.15 M leading to a 4-fold purification and recoveries of 90-100 ~ . The immunoelectrophoretic patterns and analytical polyacrylamide after this step are shown in Figs. 2 and 3, respectively. When DEAE chromatography was performed at a lower pH (7.4) or in phosphate or acetate buffers as low as pH 5.5, greater contamination of the substrate with albumin was observed. Under all conditions of chromatography on DEAE cellulose, only a single peak with angiotensinogen activity was observed.

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Biochimica et Biophysica Acta, 427 (1976) 208-217 © Elsevier Scientific Publishing Company, Amsterdam --Printed in The Netherlands BBA 37255 P U R I...
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