- Email: [email protected]
Electrophoretic characterization of active renin from human kidney and inactive renin from a human chorionic cell culture
Moiefular and Cefhdar End~rinoZo~,
42 (1985) 175-183
175
Elsevier Scientific Publishers Ireland, Ltd. MCE 01364
Electrophoretic characterization of active renin from human kidney and inactive renin from a human chorionic cell culture C. Auzan, C. Devaux, A.M. Houot, I. Laboulandine, P. Corvol. J. Menard and A. Chr~bach 1 INSERM, Unite’36, f 7 rue du Fer - ci-Moulin, 75005 Paris (France), and ’ Section on Macromolecular Analysis, Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20205 (U.S.A.)
(Received 6 February 1985; accepted 14 June 1985)
Keywords: renin, human kidney, active; renin, human chorionic culture, inactive; gel electrophoresis;
gel electrofocusing; molecular
size; molecular net charge.
Summary Enzymatically inactive human renin from chorionic cells in culture is significantly distinct in polyacrylamide gel electrophoresis (pH 8.17, O*C) from active human kidney renin. The inactive renin is larger and more basic than the active renin; their molecular weights derived from gel electrophoretic retardation coefficients relate as 47.5/35.3 kDa, their valences (net protons/molecule) as 2.14/1.85. In gel electrofocusing conducted in a mixture of simple buffers, both inactive and active renins exhibit 2 components at the steady-state. The molecular size and basicity of inactive renin are consistent with the hypothesis that it may be a precursor (prorenin), although the possibility that it is an inhibitor complex cannot be ruled out.
Immunoactive plasma renin consists of up to 80% of an inactive form (Sealey and Laragh, 1975) of the enzyme, while kidney renin is mostly in an active form. It is not as yet known whether inactive plasma renin is a precursor, possibly related to prorenin found in biosynthetic studies (Re et al., 1982; Catanzara et al., 1983; Galen et al., 1984) and what functions it may have. To investigate these questions it appeared important to separate active and inactive renins completely, and to physically characterize inactive renin, i.e., to determine its size and charge properties. The chain structure of active renin has been fully elucidated (Imai et al., 1983). Correspondence: A. Chrambach, Building 10, Room 8C413, NIH, Bethesda, MD 20205 (U.S.A.). 0303-7207/85/$03.30
Previous separations between active and inactive renins have been reported using (a) DEAE chromatography (Shalkes et al,, 1978); (b) reverse-phase partition chromatography on octylSepharose (Chang et al., 1981); (c) affinity chromatography on Affigei blue (Atlas et al., 19?I), on a column of mono~lonal anti-renin antibodies (Galen et al., 1984), or on Sepharose-pepstatin (Atlas et al., 1982). Pepstatin being a competitive inhibitor of renin (Guyenne et al., 1976), a pepstatin column retains renin and passes inactive renin. Both active and inactive renin are adsorbed onto an antibody cdumn but elute at different rates in view of the much reduced affinity of the antibody for inactive renin. This study aims at physically characterizing active and inactive forms of renin in order to determine their difference. It
0 1985 Elsevier Scientific Publishers Ireland, Ltd.
176
employed native ’ inactive renin produced by human chorionic cells in culture, and native ’ renin from infarcted human kidney. Since the RIA for their detection does not discriminate between active and inactive renins, it could be used for the detection of both species (Guyenne et al., 1980). Prior determinations of the molecular size ’ of inactive renin have not been made. However, the molecular weight (M,) of active renin has been calculated as 37200 by DNA sequencing (Imai et al., 1983). The M, of prorenin has also been established by the same method: The profragment consists of renin extended on the N-terminal side by 46 amino acid residues, i.e., by 5410 (Imai et al., 1983). Of course, these IV, values do not necessarily reflect the post-translational states of the molecule as it exists in plasma, nor do they take into account the known glycosylation at 2 sites of the molecule (Imai et al., 1983). Materials and methods (i) Active and inactive renins Inactive renin was obtained from human chorionic cells in culture which produce the inactive form of the enzyme (Acker et al., 1982). The culture medium contained 100 ng of immunoactive renin/ml. The medium was stored in aliquots at -20°C for several months. It was thawed prior to use and used without dilution. Active human renin was obtained from infarcted kidneys removed at surgery. The tissue was homogenized just prior to use as needed during a 6 week period. Homogenization was carried out in 0.1 M phosphate buffer, pH 7.5, containing protease inhibitors as described (Marukami and Inagami, 1975). After centrifugation for 30 min at 20000 x g the supernatant was collected and stored in aliquots at -20°C. The specific activity
The term ‘native’ is used here to underline the fact that the proteins under study were active rather than denatured. Obviously, any species removed from its biological milieu ceases to be native in the strict sense of the term and is likely to undergo structural and/or conformational alterations. The term ‘molecular size’ is used to stress the fact that the methods used to measure it are sensitive to molecular geometry, not to mass (see Discussion).
was 0.1 Goldblatt units (GU)/mg protein and 120 ng immunoreactive renin/mg of protein, using the direct RIA (Guyenne et al., 1980). The supernatant was diluted 20-fold just prior to use. (2) Polyacrylamide gel eiectrophoresis (A) Selection of pH. The pH of PAGE was optimized, using a systematic decrease of the pH of stacking gels (Chrambach, 1980) of negative polarity, 0°C and measuring the recovery of immunoactive renin at each of the pHs. Discontinuous buffer systems with trailing ion net mobilities (TINM) of approximately 0.050 (Chrambach and Jovin, 1983) operative at pH 10.33, 9.41, 8.40 and 7.83 were found to yield a recovery of the immunoactivities exceeding the standard curve while the stack in a system operative at pH 7.05 yieided only a 10% recovery. Thus, to optimize separation based on net charge differences (Chrambach, 1980), the buffer system stacking at pH 7.83 (TINM 0.046) was selected. To unstack the renins in the same buffer system at moderate gel concentrations, the TINM in the resolving gel was raised to 0.087, with a corresponding increase in operative pH to 8.17. The composition of the buffer system is given in Table 1. Recovery of immunoactivity was tested on stacking gels. The position of the stack was excised and the sliced diffusate was analyzed for immunoactivity as described in section C. (B) Ferguson plot analysis. PAGE was carried out at 0-2°C in resolving gels at 5, 6, 7, 8, 11, 12 and 13 ST, 2 %CBi, as described (Chrambach and Rodbard, 1981). Protein loads per cylindrical gel of 6 mm diameter were lo-50 ~1 renin and 17Q-400 ~1 inactive renin. Resolving gels were sliced into 1 mm slices, using an electrovibrator type gel cutter (Hoefer Scientific). Alternate gel slices were suspended in RIA buffer. Rr’s were calculated by dividing the slice numbers corresponding to peaks by the slice number of the section carrying the tracking dye, bromphenolblue. Ferguson plots (log R, vs. %T) were computed and evaluated statistically using the PAGE-PACK programs of D. Rodbard (Chrambach and Rodbard, 1981). In particular, these computations yielded values and confidence limits for the slope (KK) and y-intercept (Y,) of the Ferguson plot which are measures of molecular size and net charge respectively, as
177
TABLE
1
COMPOSITION ACTIVE AND
OF THE INACTIVE
DISCONTINUOUS RENINS Prepared
BUFFER
SYSTEM
OPTIMIZED
buffer
FOR
THE
GEL
ELECTROPHORESIS
Operative PH,Y
buffer a
1-00
TINM
Catholyte (upper buffer)
Trick 1NKOH
3.58 g/l 10.0 ml
4 x concentrated Stacking gel buffer
1 M HaSO, Bistris
23.68 ml/100 12.47 g
ml
5.91
7.83
0.046
1 M H,S04
23.68 mI/lOO ml
6.94
8.17
0.087
Bistris
36.90 g
1NHCI Bistris
10.0 ml/l 4.18 g
4 x concentrated Resolving gel buffer Anolyte (lower buffer)
b
8.05
6.50
a ‘Operative’ conditions are those in the gel after passage of the sulfate/fricinate boundary. ’ The 4-fold concentration allows for polymerization of a gel by a 7 : 2.5 : 0.5 volume ratio of acrylamide lyst mixture.
well as the ellipsoidal joint 95% confidence envelopes of K, and Y,. (C) Radioimmunoassays. Both active and inactive renins were assayed in the diffusate of gel slices using the RIA described previously (Guyenne et al., 1980). The diffusate was prepared by suspending gel slices of 1 mm width in 0.5 ml RIA buffer and allowing them to diffuse overnight at 4°C. (D) Molecular weight determination. K, and Y, values translated to the conventional parameters descriptive of molecular size and net charge, molecular weight and valence (net protons/molecule), using a standard curve of 6 vs. the geometric mean radius, E (nm). K, values of the standards were obtained by experimental verification of stacking, followed by Ferguson plot analysis, using 6 to 25 gel concentrations (section B). E was calculated from the known masses (in parentheses) of the following standards, assuming sphericity, partial specific volume, v, of 0.74 and 0 hydration: ~-1actaIbumin (14 200; Sigma No. L4385); soybean trypsin inhibitor (22 700; Boehringer No. 109886); /3-lactoglobulin (35 000; Sigma No. L-7880); pepsinogen (40400; Sigma No. PO258); ovalbumin (43500; Pharmacia No. 170441-01); bovine serum albumin (67000; Phar-
OF
stock solution/buffer/cata-
macia No. 17-0441-01); transferrin (90000; Sigma No. T-2252); bovine serum albumin dimer (134 000); lactic dehydrogenase I (135 000; Boehringer No. 153378); cataiase (232000; Pharmacia No. 17-0~1-01); ferritin (450000; Pharmacia No. 17-0441-01); /&galactosidase (520000; Sigma No. G-8511). (3) Electrofocusing (A) Selection of a BEF system and determination ofits stability. A natural pH gradient was formed by buffer electrofocusing (BEF) at 20 V/cm, using a 5 %T, 15 %C uATD gel as described (An der Lan and Chrambach, 1981) which contained a 0.04 M concentration of the following buffers: MES (Sigma No. M8210), ACES (Sigma No. A9758), MOPS (Aldrich No. 16377-5), HEPES (Sigma No. H3375), EPPS (Aldrich No. 16374-O), TAPS (Aldrich No. 21-993.2), tricine (Aldrich No. 16-378-2) bicine (Sigma No. B 3876), threonine (Aldrich No. T 3420-7), hydroxyproline (Aldrich No. H 5440-9) glycine (Aldrich No. G 620-l), @-alanine (Aldrich No. 14.604-4), GABA (Sigma No. A 2129) and histidine (Sigma No. H 8000). Catholyte was 0.1 M histidine, anolyte 0.1 M acetic acid. Stability of the pH gradient between pH 4 and 6 was ascertained for 70 h of BEF.
178 ACTIVE
RENIN
0
INACTIVE
RENIN
6%T
1
I
IQ
09
0.7 0.6 06 04 08
0 603
h ,i$-g”
20
30 31.32
NUMBER
Fig. 1. PAGE patterns of immunoactive, enzymatically active renin from human kidney and inactive renin from chorionic cells in culture. PAGE at 0-4°C; pH 8.17; TINM 0.087; ionic strength 0.01; negative polarity. Arrows designate the position of the moving boundary front.
179
(B) BEF ojrenins. lo-50 ~1 of active renin and 170-400 ~1 of inactive renin were applied after 16-24 h of BEF. Gels were sliced and assayed for pH and subsequently for immunoactivity (see sections 2B and C). Results (I) Inactive renin is larger and less negatively charged at pH 8 than act&e renin Active and inactive renins were analyzed by polyacrylamide gel electrophoresis at pH 817,O”C, at gel concentrations varying from 5 to 13 %T, 2 SC,,. Alternate gel slices of 1 mm thickness were suspended in RIA buffer, stored at 4°C overnight and analyzed by specific RIA (Fig. 1). The migra-
rA
tion distances corresponding to the peak positions of immunoactivity were measured (in terms of slice number) and divided by the slice number corresponding to the moving boundary front to yield the characteristic R, values at each of 6 gel concentrations. These were used to construct the Ferguson plots (Fig. 2A). The Ferguson plot of inactive renin exhibits the lesser y-intercept, Y, (net charge), and the larger slope, K, (size), on this plot. This result is statistically significant within 95% confidence limits, as shown by its negative vertical and positive horizontal displacement of the joint 95% confidence envelopes of K, and Y,, along the Y, and K, axes respectively, compared to active renin (Fig. 2B). (21 Molecular weights and vaiences at pH 8 of inactive and active renins Experimental K, and Y, values were translated to conventional parameters descriptive of molecular size and net charge, using a standard curve of F,vs. z (nm) under the assumptions of sphericity, partial specific volume v= 0.74 and 0 hydration for both renins and standard proteins. Table 2 shows the resulting values of molecular weight and valence (net protons/molecule). (31 Optimal gel concentration at 0 %T for the separation of inactive and active renins Computation of the optimal gel concentration for the resolution of active and inactive renins shows a single optimum at 0 ST, as well as a synergism between charge and size separation. This result directed the separation to electrofocusing (Fig. 19 of Chrambach, 1980).
Fig. 2. Panel A: Ferguson plots (pH 8.17, negative polarity, ionic strength 0.01, O”C, TINM 0.087) for active (solid line) and inactive (dashed line) renins. Correlation coefficients, in that order, are 0.993 and 0.980. Panel B: joint 95% confidence envelopes of K, and YO.
(4) Inactive and active renins separate and exhibit 2 components each upon e~e~trojoc~sing To minimize the prospect for an artifactual interaction between renins and carrier ampholytes, electrofocusing was carried out in a mixture of simple buffers (Cuono et al., 1983). Stability for 70 h of the gradient between pH 4 and 6 was ascertained. Inactive and active renins applied onto this buffer pH gradient give rise to peaks of immunoactivity (Fig. 3) which position at declining pHs as a function of electrofocusing time (Fig. 4). Two peaks each of immunoactive renin, and of immunoactive inactive renin, are observed in elec-
180
INACTIVE RENIN
ACTIVE RENIN
0 8h
100
6.C
100
6.C
80
80 60
60
5.c
40
40 20
5
10
15
20
25
30
35
40
45
40
50
5
10
15
20
25
30
35
40
45
50
0
24h
5 45
I I
i%
5
2
53*525
6.0
5 -100
I
g I
a
-60
a.0 5
10
15
20
25
30
35
40
45
Q
80
-
5.0
s 7
- 40
z
-20
T i
50
01 48h
loo
100
80
80
60
60
1
40 20 5
10
15
20
25
30
35
40
45
50
SLICE NUMBER Fig. 3. BEF of immunoactive enzymatically active renin from kidney and enzymatically The buffer mixture generating the pH gradient is described under Methods. 20 V/cm.
inactive O-4”C,
renin from chorionic 5 %T, 15 SC,,,.,.
cells in culture.
181
TABLE
2
PHYSICAL
CHARACTERISTICS
Property
Molecular
OF ACTIVE
AND
INACTIVE
RENINS
Active renin
weight (kDa)
Inactive
renin
Value
95% confidence limits
Value
95% confidence limits
35.3
8.8-90.8
41.5
14.2-112.3
Valence a
2.14
1.85
PI’
5.46, 5.18
5.59, 5.35
a Net negative
charge
at pH 8.17, 0°C (net protons/molecule).
trofocusing at the steady-state. Viewed as a set, the 2 inactive renins exhibit higher pZ’ values than the 2 active renins. Their characteristic pZ’ values are listed in Table 2. Discussion This study shows that inactive renin from human chorionic cells in culture is significantly larger and less negatively charged at pH 8 than active renin from human kidney. This conclusion rests on the displacement of the joint 95% confidence envelopes of K, and Y, toward larger K, values (the abscissa in Fig. 2B) and lower Y, values (the ordinate in Fig. 2B). It is therefore entirely based on experimental parameters descriptive of molecular size and net charge which are not subject to any assumptions. By contrast, the molecular size and net charge parameters derived from K, and Y, respectively,
Fig. 4. Positions in the pH gradient of enzymatically inactive renin from chorionic cells in culture (closed symbols) and active renin from kidney (open symbols) as a function of electrofocusing time in BEF. Conditions as in Fig. 3.
molecular weight, M,, and valence, I’, are burdened (a) by the assumptions that unknown and standard proteins are spherical, possess a partial specific volume of a ‘normal protein’, 0.74, and are completely non-hydrated (Chrambach et al., 1976). Since these 3 assumptions are a priori erroneous, one can only hope to average out differences in those 3 parameters by using as many standard proteins as possible, preferably 20 or more. However, a severe limit to the number of available standard proteins is set by the requirement that they stack in the buffer system optimized for the unknown. This condition was met by 12 proteins in this study. (b) In addition, the ‘translation’ of K, to M, assumes that standards and unknown are chemically homogeneous in the sense that they contain similar proportions of protein, carbohydrate and lipid moieties. This assumption already does not hold for the standards alone - the selected ones contain, for instance, different proportions of carbohydrate. Even more so, this assumption fails when the standards and unknown contain disproportionate fractions of non-proteinaceous moieties. (c) A third reason to base the conclusion that the inactive renin is larger than the active renin on the differences in K, rather than M, relates to the fact that the sequence analysis provides a measure of mass (daltons) while gel electrophoresis (as well as any other separation method except sedimentation equilibrium) provides a measure of geometry, such as molecular surface area or equivalent radius (nm) (Chrambach et al., 1976). Thus, the molecular size found in gel electrophoresis changes with such parameters as pH and ionic strength, to the degree
182
that these parameters have different effects on the conformation of standards and unknown (Rodbard and Chrambach, 1971). These considerations undoubtedly account for the difference between the M, values given in Table 2 for the active renin and the known value of its mass derived from sequence analysis. The ?t4, value does, however, provide evidence that both the active and inactive renins are monomeric. The isoelectric points for the active and inactive renins given in Table 2 are within the ranges previously reported for renins from various sources (5.35-5.55 (Galen et al., 1979) and 5.40-5.73 (Chang et al., 1981) for kidney active renin; 5.25-5.68 for plasma active renin (Chang et al., 1981); 5.40-5.61 for kidney inactive renin (Chang et al., 1981); 5.25-6.37 for plasma inactive renin). All previous data agree with the present report showing that inactive renin is more basic a protein than active renin. However, while all of the previous ‘PI’ data refer to pHs at arbitrary electrofocusing times and are therefore variable, the values reported here refer to the constant value which the pH assumes after a certain electrofocusing time (see Fig. 4) and therefore represent true (although still only apparent 3, pZ values. The rough agreement with previous values also suggests that binding of renins to synthetic carrier ampholytes (e.g., Ampholine) is negligible or, at least, limited to ionic interactions similar to those with simple buffers (Cuono et al., 1983). However, previously reported heterogeneity of renins in electrofocusing beyond 2 species (Galen et al., 1979; Chang et al., 1981) appears suspect in the light of the finding (Fig. 3) that 3 species coalesce to 2 with indistinguishable pl values, as the electrofocusing time is increased. The gel electrophoresis of active renin from kidney under the conditions of this study does not give rise to an immunoactive form with modified size and/or charge akin to the inactive renin from chorionic cell culture. Equally, the inactive renin from a tissue culture source does not give rise to
3
The pls are apparent (pl’ values) since the ionic strength at the site of pH measurement is not 0, the temperature of pH measurement is not that of electrofocusing, the viscosity of the medium is not that of water, and the buffer milieu is not free of non-ionic constituents.
an immunoactive form modified in size and/or net charge akin to the active renin from kidney. Thus, under the conditions used for their separation, there appears to be no interconversion between active and inactive species of the enzyme. The gel electrophoretic conditions, described in this work may possibly provide a method capable of resolving plasma renin from inactive renin in conjunction with a recently described (Menard et al., 1984) ELBA more sensitive than the RIA which was available at the time of this study. Finally, the results obtained in the present study are consistent with the hypothesis (Acker et al., 1982) of inactive renin being the precursor of renin (prorenin) as (a) inactive renin has a higher M, than active renin; (b) inactive renin is a more basic protein than active renin as expected for prorenin, the profragment being very rich in basic amino acids. However, the possibility that inactive renin from amniotic cells in culture may be an inhibitor complex (Inagami et al., 1983) cannot be ruled out by the present data. Since the inactive renin is more basic than the active renin and not more acidic, the difference between them cannot be due to glucosylation of inactive renin (Johnson et al., 1979). References Acker, G.M., Galen, F.X., Devaux, C., Foote, S., Papernik, A., Pesty, A., Menard, J. and Corvol, P. (1982) J. Clin. Endocrinol. Metab. 55, 902-908. An der Lan, B. and Chrambach, A. (1981) In: Gel Electrophoresis of Proteins: A Practical Approach, Eds.: B.D. Hames and D. Rickwood (Information Retrieval Press, London and Washington, DC) pp. 157-187. Atlas, S.A., Sealey, J.E., Dharmgrongartama, E., Hesson, T.E. and Laragh, J.H. (1981) Hypertension 3 (Suppl. l), 30-40. Atlas, S.A., Sealey, J.E., Hesson, T.E., Kaplan, A.P., Menard, J., Corvol, P. and Laragh, J.H. (1982) Hypertension 4 (Suppl. 2), 85-95. Catanzara, D.F., Mulins, J.J. and Morris, J.B. (1983) J. Biol. Chem. 258, 7354-7368. Chang, Jin-Jyc M., Kisaragi, M.. Okamoto, H. and Inagami, T. (1981) Hypertension 3, 509-515. Chrambach, A. (1980) J. Mol. Cell. Biochem. 29, 3-22. Chrambach, A. and Jovin, T.M. (1983) Electrophoresis 4. 190-204. Chrambach, A. and Rodbard, D. (1981) In: Electrophoresis of Proteins: A Practical Approach, Eds.: B.D. Hames and D. Rickwood (Information Retrieval Press, London and Washington, DC) pp. 157-187. Chrambach, A., Jovin, T.M., Svendsen, P.J. and Rodbard, D.
183
(1976) In: Methods of Protein Separation, Vol. 2, Ed.: N. Catsimpoolas (Plenum Press, New York) pp. 27-144. Cuono, C.B., Chapo, G.A., Chrambach, A. and Hjelmeland, L.M. (1983) Electrophoresis 4, 404407. Galen, F.X., Devaux, C., Guyenne, T., Menard, J. and Corvol, P. (1979) J. Biol. Chem. 254, 4848-4854. Galen, F.X., Devaux, C., Houot, A.M., Menard, J., Corvol, P., Corvol, M.T., Gubler, M.C., Mounier, F. and Camilleri, J.P. (1984) J. Clin. Invest. 73, 11441156. Galen, F.X., Devaux, C., Atlas, S., Guyenne, T., Menard, J., Corvol, P., Simon, D., Cazaubon, C., Richer, P., Badouaille, G., Richaud, J.P., Gros, P. and Pau, E. (1984) J. Clin. Invest. 74, 723-736. Guyenne, T., Devaux, C., Menard, J. and Corvol, P. (1976) J. Clin. Endocrinol. Metab. 43, 1301-1306. Guyenne, T.T., Galen, F.X., Devaux, C., Corvol, P. and Menard, J. (1980) Hypertension 2, 465-470. Imai, T., Miyazaki, H., Hirose, S., Hori, H., Hayashi, L., Kageyama, R., Ohkubo, H., Nakanishi, S. and Murakami,
K. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7405-7409. Inagami, T., Misono, K., Inagaki, T. and Takii, Y. (1983) In: Protease Inhibitors: Medical and Biological Aspects, Eds.: N. Katanuma, H. Umezawa and H. Holzer (Jap. Sci. Sot. Press, Tokyo) pp. 251-259. Johnson, R.L., Poisner, A.M. and Crist, R.D. (1979) B&hem. Pharmacol. 28, 1791-1799. Maruakami, K. and Inagami, T. (1975) Biochem. Biophys. Res. Commun. 62, 757-763. Menard J., Bews, J. and Heusser, J. (1984) J. Hypertension 2, 245-278. Re, R., Fallon, J.L., Dzau, V., Ouay, S.C. and Haber, E. (1982) Life Sci. 30, 99-106. Rodbard, D. and Chrambach, A. (1971) Anal. Biochem. 40, 95-134. Sealey, J.E. and Laragh, J.H. (1975) Circ. Res. (Suppl. 1) 36, 10-16. Shalkes, A.A., Gibson, R.R. and Skinner, S.L. (1978) Clin. Sci. Mol. Med. 55, 41-50.
42 (1985) 175-183
175
Elsevier Scientific Publishers Ireland, Ltd. MCE 01364
Electrophoretic characterization of active renin from human kidney and inactive renin from a human chorionic cell culture C. Auzan, C. Devaux, A.M. Houot, I. Laboulandine, P. Corvol. J. Menard and A. Chr~bach 1 INSERM, Unite’36, f 7 rue du Fer - ci-Moulin, 75005 Paris (France), and ’ Section on Macromolecular Analysis, Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20205 (U.S.A.)
(Received 6 February 1985; accepted 14 June 1985)
Keywords: renin, human kidney, active; renin, human chorionic culture, inactive; gel electrophoresis;
gel electrofocusing; molecular
size; molecular net charge.
Summary Enzymatically inactive human renin from chorionic cells in culture is significantly distinct in polyacrylamide gel electrophoresis (pH 8.17, O*C) from active human kidney renin. The inactive renin is larger and more basic than the active renin; their molecular weights derived from gel electrophoretic retardation coefficients relate as 47.5/35.3 kDa, their valences (net protons/molecule) as 2.14/1.85. In gel electrofocusing conducted in a mixture of simple buffers, both inactive and active renins exhibit 2 components at the steady-state. The molecular size and basicity of inactive renin are consistent with the hypothesis that it may be a precursor (prorenin), although the possibility that it is an inhibitor complex cannot be ruled out.
Immunoactive plasma renin consists of up to 80% of an inactive form (Sealey and Laragh, 1975) of the enzyme, while kidney renin is mostly in an active form. It is not as yet known whether inactive plasma renin is a precursor, possibly related to prorenin found in biosynthetic studies (Re et al., 1982; Catanzara et al., 1983; Galen et al., 1984) and what functions it may have. To investigate these questions it appeared important to separate active and inactive renins completely, and to physically characterize inactive renin, i.e., to determine its size and charge properties. The chain structure of active renin has been fully elucidated (Imai et al., 1983). Correspondence: A. Chrambach, Building 10, Room 8C413, NIH, Bethesda, MD 20205 (U.S.A.). 0303-7207/85/$03.30
Previous separations between active and inactive renins have been reported using (a) DEAE chromatography (Shalkes et al,, 1978); (b) reverse-phase partition chromatography on octylSepharose (Chang et al., 1981); (c) affinity chromatography on Affigei blue (Atlas et al., 19?I), on a column of mono~lonal anti-renin antibodies (Galen et al., 1984), or on Sepharose-pepstatin (Atlas et al., 1982). Pepstatin being a competitive inhibitor of renin (Guyenne et al., 1976), a pepstatin column retains renin and passes inactive renin. Both active and inactive renin are adsorbed onto an antibody cdumn but elute at different rates in view of the much reduced affinity of the antibody for inactive renin. This study aims at physically characterizing active and inactive forms of renin in order to determine their difference. It
0 1985 Elsevier Scientific Publishers Ireland, Ltd.
176
employed native ’ inactive renin produced by human chorionic cells in culture, and native ’ renin from infarcted human kidney. Since the RIA for their detection does not discriminate between active and inactive renins, it could be used for the detection of both species (Guyenne et al., 1980). Prior determinations of the molecular size ’ of inactive renin have not been made. However, the molecular weight (M,) of active renin has been calculated as 37200 by DNA sequencing (Imai et al., 1983). The M, of prorenin has also been established by the same method: The profragment consists of renin extended on the N-terminal side by 46 amino acid residues, i.e., by 5410 (Imai et al., 1983). Of course, these IV, values do not necessarily reflect the post-translational states of the molecule as it exists in plasma, nor do they take into account the known glycosylation at 2 sites of the molecule (Imai et al., 1983). Materials and methods (i) Active and inactive renins Inactive renin was obtained from human chorionic cells in culture which produce the inactive form of the enzyme (Acker et al., 1982). The culture medium contained 100 ng of immunoactive renin/ml. The medium was stored in aliquots at -20°C for several months. It was thawed prior to use and used without dilution. Active human renin was obtained from infarcted kidneys removed at surgery. The tissue was homogenized just prior to use as needed during a 6 week period. Homogenization was carried out in 0.1 M phosphate buffer, pH 7.5, containing protease inhibitors as described (Marukami and Inagami, 1975). After centrifugation for 30 min at 20000 x g the supernatant was collected and stored in aliquots at -20°C. The specific activity
The term ‘native’ is used here to underline the fact that the proteins under study were active rather than denatured. Obviously, any species removed from its biological milieu ceases to be native in the strict sense of the term and is likely to undergo structural and/or conformational alterations. The term ‘molecular size’ is used to stress the fact that the methods used to measure it are sensitive to molecular geometry, not to mass (see Discussion).
was 0.1 Goldblatt units (GU)/mg protein and 120 ng immunoreactive renin/mg of protein, using the direct RIA (Guyenne et al., 1980). The supernatant was diluted 20-fold just prior to use. (2) Polyacrylamide gel eiectrophoresis (A) Selection of pH. The pH of PAGE was optimized, using a systematic decrease of the pH of stacking gels (Chrambach, 1980) of negative polarity, 0°C and measuring the recovery of immunoactive renin at each of the pHs. Discontinuous buffer systems with trailing ion net mobilities (TINM) of approximately 0.050 (Chrambach and Jovin, 1983) operative at pH 10.33, 9.41, 8.40 and 7.83 were found to yield a recovery of the immunoactivities exceeding the standard curve while the stack in a system operative at pH 7.05 yieided only a 10% recovery. Thus, to optimize separation based on net charge differences (Chrambach, 1980), the buffer system stacking at pH 7.83 (TINM 0.046) was selected. To unstack the renins in the same buffer system at moderate gel concentrations, the TINM in the resolving gel was raised to 0.087, with a corresponding increase in operative pH to 8.17. The composition of the buffer system is given in Table 1. Recovery of immunoactivity was tested on stacking gels. The position of the stack was excised and the sliced diffusate was analyzed for immunoactivity as described in section C. (B) Ferguson plot analysis. PAGE was carried out at 0-2°C in resolving gels at 5, 6, 7, 8, 11, 12 and 13 ST, 2 %CBi, as described (Chrambach and Rodbard, 1981). Protein loads per cylindrical gel of 6 mm diameter were lo-50 ~1 renin and 17Q-400 ~1 inactive renin. Resolving gels were sliced into 1 mm slices, using an electrovibrator type gel cutter (Hoefer Scientific). Alternate gel slices were suspended in RIA buffer. Rr’s were calculated by dividing the slice numbers corresponding to peaks by the slice number of the section carrying the tracking dye, bromphenolblue. Ferguson plots (log R, vs. %T) were computed and evaluated statistically using the PAGE-PACK programs of D. Rodbard (Chrambach and Rodbard, 1981). In particular, these computations yielded values and confidence limits for the slope (KK) and y-intercept (Y,) of the Ferguson plot which are measures of molecular size and net charge respectively, as
177
TABLE
1
COMPOSITION ACTIVE AND
OF THE INACTIVE
DISCONTINUOUS RENINS Prepared
BUFFER
SYSTEM
OPTIMIZED
buffer
FOR
THE
GEL
ELECTROPHORESIS
Operative PH,Y
buffer a
1-00
TINM
Catholyte (upper buffer)
Trick 1NKOH
3.58 g/l 10.0 ml
4 x concentrated Stacking gel buffer
1 M HaSO, Bistris
23.68 ml/100 12.47 g
ml
5.91
7.83
0.046
1 M H,S04
23.68 mI/lOO ml
6.94
8.17
0.087
Bistris
36.90 g
1NHCI Bistris
10.0 ml/l 4.18 g
4 x concentrated Resolving gel buffer Anolyte (lower buffer)
b
8.05
6.50
a ‘Operative’ conditions are those in the gel after passage of the sulfate/fricinate boundary. ’ The 4-fold concentration allows for polymerization of a gel by a 7 : 2.5 : 0.5 volume ratio of acrylamide lyst mixture.
well as the ellipsoidal joint 95% confidence envelopes of K, and Y,. (C) Radioimmunoassays. Both active and inactive renins were assayed in the diffusate of gel slices using the RIA described previously (Guyenne et al., 1980). The diffusate was prepared by suspending gel slices of 1 mm width in 0.5 ml RIA buffer and allowing them to diffuse overnight at 4°C. (D) Molecular weight determination. K, and Y, values translated to the conventional parameters descriptive of molecular size and net charge, molecular weight and valence (net protons/molecule), using a standard curve of 6 vs. the geometric mean radius, E (nm). K, values of the standards were obtained by experimental verification of stacking, followed by Ferguson plot analysis, using 6 to 25 gel concentrations (section B). E was calculated from the known masses (in parentheses) of the following standards, assuming sphericity, partial specific volume, v, of 0.74 and 0 hydration: ~-1actaIbumin (14 200; Sigma No. L4385); soybean trypsin inhibitor (22 700; Boehringer No. 109886); /3-lactoglobulin (35 000; Sigma No. L-7880); pepsinogen (40400; Sigma No. PO258); ovalbumin (43500; Pharmacia No. 170441-01); bovine serum albumin (67000; Phar-
OF
stock solution/buffer/cata-
macia No. 17-0441-01); transferrin (90000; Sigma No. T-2252); bovine serum albumin dimer (134 000); lactic dehydrogenase I (135 000; Boehringer No. 153378); cataiase (232000; Pharmacia No. 17-0~1-01); ferritin (450000; Pharmacia No. 17-0441-01); /&galactosidase (520000; Sigma No. G-8511). (3) Electrofocusing (A) Selection of a BEF system and determination ofits stability. A natural pH gradient was formed by buffer electrofocusing (BEF) at 20 V/cm, using a 5 %T, 15 %C uATD gel as described (An der Lan and Chrambach, 1981) which contained a 0.04 M concentration of the following buffers: MES (Sigma No. M8210), ACES (Sigma No. A9758), MOPS (Aldrich No. 16377-5), HEPES (Sigma No. H3375), EPPS (Aldrich No. 16374-O), TAPS (Aldrich No. 21-993.2), tricine (Aldrich No. 16-378-2) bicine (Sigma No. B 3876), threonine (Aldrich No. T 3420-7), hydroxyproline (Aldrich No. H 5440-9) glycine (Aldrich No. G 620-l), @-alanine (Aldrich No. 14.604-4), GABA (Sigma No. A 2129) and histidine (Sigma No. H 8000). Catholyte was 0.1 M histidine, anolyte 0.1 M acetic acid. Stability of the pH gradient between pH 4 and 6 was ascertained for 70 h of BEF.
178 ACTIVE
RENIN
0
INACTIVE
RENIN
6%T
1
I
IQ
09
0.7 0.6 06 04 08
0 603
h ,i$-g”
20
30 31.32
NUMBER
Fig. 1. PAGE patterns of immunoactive, enzymatically active renin from human kidney and inactive renin from chorionic cells in culture. PAGE at 0-4°C; pH 8.17; TINM 0.087; ionic strength 0.01; negative polarity. Arrows designate the position of the moving boundary front.
179
(B) BEF ojrenins. lo-50 ~1 of active renin and 170-400 ~1 of inactive renin were applied after 16-24 h of BEF. Gels were sliced and assayed for pH and subsequently for immunoactivity (see sections 2B and C). Results (I) Inactive renin is larger and less negatively charged at pH 8 than act&e renin Active and inactive renins were analyzed by polyacrylamide gel electrophoresis at pH 817,O”C, at gel concentrations varying from 5 to 13 %T, 2 SC,,. Alternate gel slices of 1 mm thickness were suspended in RIA buffer, stored at 4°C overnight and analyzed by specific RIA (Fig. 1). The migra-
rA
tion distances corresponding to the peak positions of immunoactivity were measured (in terms of slice number) and divided by the slice number corresponding to the moving boundary front to yield the characteristic R, values at each of 6 gel concentrations. These were used to construct the Ferguson plots (Fig. 2A). The Ferguson plot of inactive renin exhibits the lesser y-intercept, Y, (net charge), and the larger slope, K, (size), on this plot. This result is statistically significant within 95% confidence limits, as shown by its negative vertical and positive horizontal displacement of the joint 95% confidence envelopes of K, and Y,, along the Y, and K, axes respectively, compared to active renin (Fig. 2B). (21 Molecular weights and vaiences at pH 8 of inactive and active renins Experimental K, and Y, values were translated to conventional parameters descriptive of molecular size and net charge, using a standard curve of F,vs. z (nm) under the assumptions of sphericity, partial specific volume v= 0.74 and 0 hydration for both renins and standard proteins. Table 2 shows the resulting values of molecular weight and valence (net protons/molecule). (31 Optimal gel concentration at 0 %T for the separation of inactive and active renins Computation of the optimal gel concentration for the resolution of active and inactive renins shows a single optimum at 0 ST, as well as a synergism between charge and size separation. This result directed the separation to electrofocusing (Fig. 19 of Chrambach, 1980).
Fig. 2. Panel A: Ferguson plots (pH 8.17, negative polarity, ionic strength 0.01, O”C, TINM 0.087) for active (solid line) and inactive (dashed line) renins. Correlation coefficients, in that order, are 0.993 and 0.980. Panel B: joint 95% confidence envelopes of K, and YO.
(4) Inactive and active renins separate and exhibit 2 components each upon e~e~trojoc~sing To minimize the prospect for an artifactual interaction between renins and carrier ampholytes, electrofocusing was carried out in a mixture of simple buffers (Cuono et al., 1983). Stability for 70 h of the gradient between pH 4 and 6 was ascertained. Inactive and active renins applied onto this buffer pH gradient give rise to peaks of immunoactivity (Fig. 3) which position at declining pHs as a function of electrofocusing time (Fig. 4). Two peaks each of immunoactive renin, and of immunoactive inactive renin, are observed in elec-
180
INACTIVE RENIN
ACTIVE RENIN
0 8h
100
6.C
100
6.C
80
80 60
60
5.c
40
40 20
5
10
15
20
25
30
35
40
45
40
50
5
10
15
20
25
30
35
40
45
50
0
24h
5 45
I I
i%
5
2
53*525
6.0
5 -100
I
g I
a
-60
a.0 5
10
15
20
25
30
35
40
45
Q
80
-
5.0
s 7
- 40
z
-20
T i
50
01 48h
loo
100
80
80
60
60
1
40 20 5
10
15
20
25
30
35
40
45
50
SLICE NUMBER Fig. 3. BEF of immunoactive enzymatically active renin from kidney and enzymatically The buffer mixture generating the pH gradient is described under Methods. 20 V/cm.
inactive O-4”C,
renin from chorionic 5 %T, 15 SC,,,.,.
cells in culture.
181
TABLE
2
PHYSICAL
CHARACTERISTICS
Property
Molecular
OF ACTIVE
AND
INACTIVE
RENINS
Active renin
weight (kDa)
Inactive
renin
Value
95% confidence limits
Value
95% confidence limits
35.3
8.8-90.8
41.5
14.2-112.3
Valence a
2.14
1.85
PI’
5.46, 5.18
5.59, 5.35
a Net negative
charge
at pH 8.17, 0°C (net protons/molecule).
trofocusing at the steady-state. Viewed as a set, the 2 inactive renins exhibit higher pZ’ values than the 2 active renins. Their characteristic pZ’ values are listed in Table 2. Discussion This study shows that inactive renin from human chorionic cells in culture is significantly larger and less negatively charged at pH 8 than active renin from human kidney. This conclusion rests on the displacement of the joint 95% confidence envelopes of K, and Y, toward larger K, values (the abscissa in Fig. 2B) and lower Y, values (the ordinate in Fig. 2B). It is therefore entirely based on experimental parameters descriptive of molecular size and net charge which are not subject to any assumptions. By contrast, the molecular size and net charge parameters derived from K, and Y, respectively,
Fig. 4. Positions in the pH gradient of enzymatically inactive renin from chorionic cells in culture (closed symbols) and active renin from kidney (open symbols) as a function of electrofocusing time in BEF. Conditions as in Fig. 3.
molecular weight, M,, and valence, I’, are burdened (a) by the assumptions that unknown and standard proteins are spherical, possess a partial specific volume of a ‘normal protein’, 0.74, and are completely non-hydrated (Chrambach et al., 1976). Since these 3 assumptions are a priori erroneous, one can only hope to average out differences in those 3 parameters by using as many standard proteins as possible, preferably 20 or more. However, a severe limit to the number of available standard proteins is set by the requirement that they stack in the buffer system optimized for the unknown. This condition was met by 12 proteins in this study. (b) In addition, the ‘translation’ of K, to M, assumes that standards and unknown are chemically homogeneous in the sense that they contain similar proportions of protein, carbohydrate and lipid moieties. This assumption already does not hold for the standards alone - the selected ones contain, for instance, different proportions of carbohydrate. Even more so, this assumption fails when the standards and unknown contain disproportionate fractions of non-proteinaceous moieties. (c) A third reason to base the conclusion that the inactive renin is larger than the active renin on the differences in K, rather than M, relates to the fact that the sequence analysis provides a measure of mass (daltons) while gel electrophoresis (as well as any other separation method except sedimentation equilibrium) provides a measure of geometry, such as molecular surface area or equivalent radius (nm) (Chrambach et al., 1976). Thus, the molecular size found in gel electrophoresis changes with such parameters as pH and ionic strength, to the degree
182
that these parameters have different effects on the conformation of standards and unknown (Rodbard and Chrambach, 1971). These considerations undoubtedly account for the difference between the M, values given in Table 2 for the active renin and the known value of its mass derived from sequence analysis. The ?t4, value does, however, provide evidence that both the active and inactive renins are monomeric. The isoelectric points for the active and inactive renins given in Table 2 are within the ranges previously reported for renins from various sources (5.35-5.55 (Galen et al., 1979) and 5.40-5.73 (Chang et al., 1981) for kidney active renin; 5.25-5.68 for plasma active renin (Chang et al., 1981); 5.40-5.61 for kidney inactive renin (Chang et al., 1981); 5.25-6.37 for plasma inactive renin). All previous data agree with the present report showing that inactive renin is more basic a protein than active renin. However, while all of the previous ‘PI’ data refer to pHs at arbitrary electrofocusing times and are therefore variable, the values reported here refer to the constant value which the pH assumes after a certain electrofocusing time (see Fig. 4) and therefore represent true (although still only apparent 3, pZ values. The rough agreement with previous values also suggests that binding of renins to synthetic carrier ampholytes (e.g., Ampholine) is negligible or, at least, limited to ionic interactions similar to those with simple buffers (Cuono et al., 1983). However, previously reported heterogeneity of renins in electrofocusing beyond 2 species (Galen et al., 1979; Chang et al., 1981) appears suspect in the light of the finding (Fig. 3) that 3 species coalesce to 2 with indistinguishable pl values, as the electrofocusing time is increased. The gel electrophoresis of active renin from kidney under the conditions of this study does not give rise to an immunoactive form with modified size and/or charge akin to the inactive renin from chorionic cell culture. Equally, the inactive renin from a tissue culture source does not give rise to
3
The pls are apparent (pl’ values) since the ionic strength at the site of pH measurement is not 0, the temperature of pH measurement is not that of electrofocusing, the viscosity of the medium is not that of water, and the buffer milieu is not free of non-ionic constituents.
an immunoactive form modified in size and/or net charge akin to the active renin from kidney. Thus, under the conditions used for their separation, there appears to be no interconversion between active and inactive species of the enzyme. The gel electrophoretic conditions, described in this work may possibly provide a method capable of resolving plasma renin from inactive renin in conjunction with a recently described (Menard et al., 1984) ELBA more sensitive than the RIA which was available at the time of this study. Finally, the results obtained in the present study are consistent with the hypothesis (Acker et al., 1982) of inactive renin being the precursor of renin (prorenin) as (a) inactive renin has a higher M, than active renin; (b) inactive renin is a more basic protein than active renin as expected for prorenin, the profragment being very rich in basic amino acids. However, the possibility that inactive renin from amniotic cells in culture may be an inhibitor complex (Inagami et al., 1983) cannot be ruled out by the present data. Since the inactive renin is more basic than the active renin and not more acidic, the difference between them cannot be due to glucosylation of inactive renin (Johnson et al., 1979). References Acker, G.M., Galen, F.X., Devaux, C., Foote, S., Papernik, A., Pesty, A., Menard, J. and Corvol, P. (1982) J. Clin. Endocrinol. Metab. 55, 902-908. An der Lan, B. and Chrambach, A. (1981) In: Gel Electrophoresis of Proteins: A Practical Approach, Eds.: B.D. Hames and D. Rickwood (Information Retrieval Press, London and Washington, DC) pp. 157-187. Atlas, S.A., Sealey, J.E., Dharmgrongartama, E., Hesson, T.E. and Laragh, J.H. (1981) Hypertension 3 (Suppl. l), 30-40. Atlas, S.A., Sealey, J.E., Hesson, T.E., Kaplan, A.P., Menard, J., Corvol, P. and Laragh, J.H. (1982) Hypertension 4 (Suppl. 2), 85-95. Catanzara, D.F., Mulins, J.J. and Morris, J.B. (1983) J. Biol. Chem. 258, 7354-7368. Chang, Jin-Jyc M., Kisaragi, M.. Okamoto, H. and Inagami, T. (1981) Hypertension 3, 509-515. Chrambach, A. (1980) J. Mol. Cell. Biochem. 29, 3-22. Chrambach, A. and Jovin, T.M. (1983) Electrophoresis 4. 190-204. Chrambach, A. and Rodbard, D. (1981) In: Electrophoresis of Proteins: A Practical Approach, Eds.: B.D. Hames and D. Rickwood (Information Retrieval Press, London and Washington, DC) pp. 157-187. Chrambach, A., Jovin, T.M., Svendsen, P.J. and Rodbard, D.
183
(1976) In: Methods of Protein Separation, Vol. 2, Ed.: N. Catsimpoolas (Plenum Press, New York) pp. 27-144. Cuono, C.B., Chapo, G.A., Chrambach, A. and Hjelmeland, L.M. (1983) Electrophoresis 4, 404407. Galen, F.X., Devaux, C., Guyenne, T., Menard, J. and Corvol, P. (1979) J. Biol. Chem. 254, 4848-4854. Galen, F.X., Devaux, C., Houot, A.M., Menard, J., Corvol, P., Corvol, M.T., Gubler, M.C., Mounier, F. and Camilleri, J.P. (1984) J. Clin. Invest. 73, 11441156. Galen, F.X., Devaux, C., Atlas, S., Guyenne, T., Menard, J., Corvol, P., Simon, D., Cazaubon, C., Richer, P., Badouaille, G., Richaud, J.P., Gros, P. and Pau, E. (1984) J. Clin. Invest. 74, 723-736. Guyenne, T., Devaux, C., Menard, J. and Corvol, P. (1976) J. Clin. Endocrinol. Metab. 43, 1301-1306. Guyenne, T.T., Galen, F.X., Devaux, C., Corvol, P. and Menard, J. (1980) Hypertension 2, 465-470. Imai, T., Miyazaki, H., Hirose, S., Hori, H., Hayashi, L., Kageyama, R., Ohkubo, H., Nakanishi, S. and Murakami,
K. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7405-7409. Inagami, T., Misono, K., Inagaki, T. and Takii, Y. (1983) In: Protease Inhibitors: Medical and Biological Aspects, Eds.: N. Katanuma, H. Umezawa and H. Holzer (Jap. Sci. Sot. Press, Tokyo) pp. 251-259. Johnson, R.L., Poisner, A.M. and Crist, R.D. (1979) B&hem. Pharmacol. 28, 1791-1799. Maruakami, K. and Inagami, T. (1975) Biochem. Biophys. Res. Commun. 62, 757-763. Menard J., Bews, J. and Heusser, J. (1984) J. Hypertension 2, 245-278. Re, R., Fallon, J.L., Dzau, V., Ouay, S.C. and Haber, E. (1982) Life Sci. 30, 99-106. Rodbard, D. and Chrambach, A. (1971) Anal. Biochem. 40, 95-134. Sealey, J.E. and Laragh, J.H. (1975) Circ. Res. (Suppl. 1) 36, 10-16. Shalkes, A.A., Gibson, R.R. and Skinner, S.L. (1978) Clin. Sci. Mol. Med. 55, 41-50.