Biochemical Properties of Big Renin Extracted from Human Plasma RICHARD P. DAY AND JOHN A. LUETSCHER Department of Medicine, Division of Endocrinology, Stanford University School of Medicine, Stanford, California 94305 renin or big renin previously activated. Using sheep substrate, the enzyme kinetics of normal renin and previously activated big renin were identical, while inactive big renin possessed a higher Michaelis constant. These data indicate that big renin is closely related biochemically to normal plasma renin. As the activation of big renin results in the formation of the substance even more similar to normal renin, the possibility exists that big renin may prove to be a precursor form of normal renin. (J Clin Endocrinol Metab 40: 1085, 1975)
ABSTRACT. The properties of big renin, a relatively inactive form of renin isolated from human plasma, were examined following partial purification by gel filtration. Exposure of big renin to pH 3.0-3.6, or brief incubation with trypsin or pepsin, resulted in a ten-fold increase in enzymatic activity. Activation was not effected by 4M NaCl, 6M urea, or incubation with neuraminidase. Both before and after activation, big renin eluted from Sephadex gel more rapidly than normal plasma renin. During polyacrylamide gel disc electrophoresis, inactive big renin migrated more slowly than either normal
T
HE biochemical heterogeneity of many circulating polypeptide hormones has recently been established (1). Likewise, biochemically distinct forms of the renal hormone renin have been demonstrated. A relatively inactive renin, which may be activated by exposure to acid pH, has been extracted from porcine (2) and rabbit (3) kidney tissue. This inactive renin elutes more rapidly from Sephadex gel than normal renin, indicating that it possesses a higher molecular weight. A similar form of renin has been isolated from plasma and kidney extracts of certain patients (4,5), as well as from human amniotic fluid (5-7). The present study further examines the biochemical characteristics of this "big renin" both before and after its activation. Materials and Methods Renin assay. Renin activity in all experiments was determined using a modification of the plasma renin concentration (PRC) method (8). Received January 13, 1975. This research was supported by grants from the National Aeronautics and Space Administration (NGR-05-020-456) and the National Institutes of Health (HL-13917) and (HL-17364). Dr. Luetscher is the recipient of a Research Career Award (K6 AM14176).
Samples were incubated at 37 C and pH 7.4 with (partially purified) sheep substrate (8) at a final concentration of 1600 ng angiotensin/ml, in the presence of 1 mM disodium EDTA, 1.6 mM dimercaprol, and 6.8 mM 8-hydroxyquinoline. In the initial gel filtration of plasma aliquots described in Fig. 1, pooled plasma from anephric patients was also used to supply substrate. Following incubation for 1 to 18 h samples were diluted with 2 vol 0.1M Tris-HCl buffer, pH 7.4, boiled for 10 min, and centrifuged. Angiotensin I in the supernate was measured by radioimmunoassay (9). Renin activity is expressed as ng of angiotensin I generated per ml of sample per hour of incubation. Gel filtration. One-half ml aliquots of plasma or eluate were applied to a Sephadex G-75 column, 2 cm in diameter by 90 cm in length, previously equilibrated with sodium phosphate buffer pH 7.4 (0.1M NaCl, 0.05M sodium phos-
phate, and 0.1% disodium EDTA). Two ml fractions were collected. The void volume of the column, measured with blue dextran, was 40 ml; the total volume, measured using 3 H 2 O, was 152 ml. Molecular weight estimates were made following gel filtration of multiple samples; values are expressed as the mean ± the standard error of the mean. The Sephadex column was calibrated with bovine serum albumin (KD = 0.16), ovalbumin (KD = 0.27) and chymotrypsinogen (KD = 0.46) (10). Protein concentration in the eluates was estimated spectrophotometrically at 280 nm.
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Activation
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studies. One-ml samples were
dialyzed for 24 h at 4 C against 40 vol buffer adjusted to selected pH values ranging from pH 2.0 to pH 10.0. Buffers of pH 3.6-10.0 were prepared using 10M NaOH to adjust a solution of 0.025M sodium citrate and 0.075M sodium
dihydrogen phosphate to the various pH. More acidic buffers were prepared using 0.05M glycine, adjusted to the correct pH using lM HC1. Following the return of the sample pH to 7.4 by dialysis at 4 C, renin activity was measured. The activity of big renin previously dialyzed to pH 3.3 was measured using both human and sheep substrate. Samples were incubated with trypsin, final concentration 50 fig/m\, at 37 C in sodium phosphate buffer, pH 7.4. Incubation was terminated by the addition of soybean trypsin inhibitor, 100 /ng/ml. Incubation of samples with pepsin, 50. fig/nd, and neuraminidase, 2 mg/ml, was performed at 37 C using 0.05M sodium acetate buffer containing 0.01M CaCl2 and adjusted to pH 4.5 and 5.0, respectively. Incubations with these two enzymes were terminated by the addition of an equal volume of 0.25M sodium phosphate buffer, pH 7.4, containing 1.0% disodium EDTA. Control values for enzyme incubations were obtained by exposing samples to incubation media containing no enzyme. Under the stated assay conditions, trypsin alone exhibited reninlike activity of less than 2 ng/ml/h, and pepsin alone showed less than 0.2 ng/ml/h renin activity. Samples exposed to urea were first incubated at either 4 C or 37 C for 2 h in the presence of 6M urea and 0.1% dimercaprol, and subsequently dialyzed at 4 C against sodium phosphate buffer, pH 7.4, containing 0.1% dimercaprol. Samples dialyzed against 4M NaCl were assayed for renin activity using sheep substrate previously dialyzed against 4M NaCl. Electrophoresis. Disc gel electrophoresis was performed in a 7.5% polyacrylamide resolving gel 6 cm in length placed below a 2.5% polyacrylamide gel 1 cm in length (11). Gels were polymerized in 0.3M Tris-HCl buffer, pH 8.9. Samples to be studied were concentrated by dialysis against 30% sucrose, and 20 ^il aliquots were applied to the gels. Electrophoresis was conducted at 20 C using 3 mA current per sample with 0.377M Tris-glycine buffer, pH 8.3, as the reservoir buffer. The migration during electrophoresis was visualized by the addition of bromophenol blue to the reservoir buffer. Electrophoresis was terminated upon the
JCE & M • 1975 Vol 40 • No 6
migration of the bromophenol blue front to within 1 cm of the end of the resolving gel, and the gels were subsequently sliced into 2 mm segments. Renin was allowed to diffuse from the gel segments following maceration in three volumes of sodium phosphate buffer, pH 7.4; renin activity was determined in the diffusate as described above. In these experiments, renin activity is expressed as ng of angiotensin I generated per mm of gel per hour of incubation. Solutions of bovine serum albumin and chymotrypsinogen, 1 mg/ml, were used as protein standards; following electrophoresis, standards were identified in the gels by staining with Coomassie Blue (11). Kinetic studies. The enzyme kinetics of normal and big renin were investigated using partially purified sheep substrate in concentrations ranging from 50 ng/ml to 1600 ng/ml. Samples, containing equal amounts of renin activity when saturated with substrate, were incubated from 1 to 18 h. The maximal rate of angiotensin I generation was used in the subsequent analysis. Michaelis-Menten constants were calculated by standard linear regression techniques (12); in all cases, the correlation coefficient exceeded 0.98. Materials. Normal renin was prepared from the plasma of normal patients undergoing salt restriction or furosemide diuresis. Big renin was extracted from plasma of patients with Wilms' tumor or diabetic nephropathy; clinical details of these patients are presented elsewhere (5). Both normal renin and big renin were partially purified by gel filtration before use in the other experiments. Bovine serum albumin and ovalbumin were obtained from Nutritional Biochemicals, Cleveland, Ohio. Bovine trypsin, procine pepsin, Type V neuraminidase, soybean trypsin inhibitor, and bovine achymotrypsinogen A were purchased from Sigma Chemical Co., St. Louis, Missouri.
Results Gel filtration of normal plasma revealed a single peak of renin activity which appeared after 68.7 ± 0.2 ml had eluted from the column (KD = 0.26) (Fig. 1, top). Dialysis of the active fractions to pH 3.3, followed by dialysis of the samples back to pH 7.4, did not appreciably alter the renin activity present in the fractions (Fig. 1, top, dashed lines). In contrast, gel filtration of
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BIOCHEMISTRY OF BIG RENIN 1OJ0
1087
I
•
I
i
HORMAL RENfi
75 "
If) 1
5.0 FIG. 1. Gel filtration of normal renin (top) and big renin (bottom). Renin activity was measured both before (A) and after (o) dialysis to pH 3.3. Ordinate: renin activity (ng/ml/h); abscissa: volume of buffer eluted (ml).
/ / .
10
I
"
tiff
60 70 VOLUME ELUTED (ml)
80
90
FIG. 2. Activation of big renin as a function of pH. Ordinate: renin activity (ng/ml/h); abscissa: pH of initial dialysate.
4 6 ACTIVATION pH
the plasma from the patient with Wilms' tumor revealed big renin activity. Maximum activity appeared after an elution volume of 59.8 ± 0.5 ml (KD = 0.18) (Fig.
K)
1, bottom). By Sephadex gel filtration, normal plasma renin exhibited an apparent molecular weight of 43,000 ± 1,000, while big renin possessed a molecular weight
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DAY AND LUETSCHER
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Incubation of big renin with trypsin or equal to 63,000 ± 2,000, calculated by pepsin resulted in greatly increased renin comparison with pure protein standards. Dialysis of the fractions containing big activity (Fig. 3). Incubation of big renin renin to pH 3.3 as described above in- with trypsin, 50 /ng/ml, for 10 s increased creased the renin activity present tenfold the renin activity sixfold; maximal activa(Fig. 1, bottom, dashed line). Following tion was observed after a 3-min incubation dialysis of big renin to pH values of 3.6 or (Fig. 3, top). Incubation with pepsin, 50 lower, increased renin activity was ob- /xg/ml, resulted in a similar time course of served (Fig. 2). Big renin dialyzed to pH activation (Fig. 3, bottom). Maximal activa4.0 to 10.0 showed no appreciable increase tion of big renin with pepsin occurred after in activity. Maximal activation occurred 30-100 s. The activity of normal renin did following dialysis to pH 3.3; below this not change following incubation with pH, denaturation of renin probably oc- either trypsin or pepsin. curred. When renin activity was measured Incubation of big renin with neurusing human substrate, dialysis to pH 3.3 aminidase for 3 h at 37 C did not inincreased its activity from 6.6 ng/ml/h to 54 crease renin activity appreciably above ng/ml/h. Using sheep substrate, dialysis of control levels (Table 1). The enzymatic big renin to pH 3.3 increased renin activity activity of big renin did not increase in response to high ionic strength media; 4M from 16.9 ng/ml/h to 225 ng/ml/h. 200
FIG. 3. Activation of big renin by trypsin (top) and pepsin (bottom). Ordinate: renin activity (ng/mlyh); abscissa: duration of incubation (min).
3 INCUBATION
4
TIME (min)
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BIOCHEMISTRY OF BIG RENIN
NaCl inhibited both normal renin and big renin to a similar extent (Table 1). Exposure of both big renin and normal renin to 6M urea, followed by dialysis against 0.1% dimercaprol, was an effective denaturing process for both renins (Table 1). Following activation, big renin eluted from Sephadex gel in a manner similar to that of unactivated big renin (Fig. 4). Whether previously exposed to pH 3.3 by dialysis (Fig. 4, top), or incubation with trypsin for three minutes (Fig. 4, bottom), big renin appeared at an elution volume of 60ml(KD = 0.18). Polyacrylamide gel disc electrophoresis, on the contrary, effected clear separation of unactivated and activated big renin (Fig. 5). The Rf value of big renin not previously activated was 0.36 (Fig. 5, upper right). Electrophoresis of normal plasma renin (Fig. 5, upper left), as well as big renin previously activated either with trypsin or by dialysis to pH 3.3 (Fig. 5, bottom)
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TABLE 1. Effect of neuraminidase, 4M NaCl, and 6M
urea upon normal and big renin activity
Treatment
Renin species
Control (ng/ml/h)
Experimental (ng/ml/h)
Neuraminidase
Big renin
12.8
15.7
4M NaCl
Normal renin Big renin
11.6 11.7
9.8 8.3
6M urea, 37 C
Normal Renin Big Renin
11.5 9.6
0.0 0.1
6M urea, 4 C
Normal Renin Big Renin
11.5 12.2
8.2 12.4
possessed Rf values of 0.47. Under equivalent conditions the Rf of bovine serum albumin and chymotrypsinogen was 0.38 and 0.56, respectively. Using partially purified sheep substrate, saturation kinetics for both normal renin and big renin could be demonstrated (Fig. 6). The Michaelis constant for normal renin was 185 ± 36 ng/ml. Big renin not
FIG. 4. Gel filtration of big renin previously activated by dialysis to pH 3.3 (top) or incubation with trypsin (bottom). Ordinate: renin activity (ng/ml/h); abscissa: volume of buffer eluted (ml).
TRYPSIN ACT1VKTED
50
60
70
80
90
VOLUME ELUTED (ml)
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DAY AND LUETSCHER
24
i
i
16
i
0\ \ >
6 !** Qb V
8
1.0
\ \ \
BIG BENIN
-
—
0
FlG. 5. Polyacrylamide gel disc electrophoresis of normal renin (upper left), unactivated big renin (upper right), and big renin previously activated by pH 3.3 dialysis (lower left) and tryptic digestion (lower right). Ordinate: renin activity (ng/mm/h); abscissa: distance from origin (Rf). The activity of unactivated big renin (upper right) was measured both before (A) and after (o) dialysis to pH 3.3.
previously activated possessed a Michaelis constant of 465 ± 150 ng/ml; after activation the Michaelis constant of big renin dropped to 151 ± 25 ng/ml. Discussion In the field of hormone research, examples of biochemically heterogeneous polypeptide agents abound (1). Previous investigators have also reported the existence of various forms of renal renin. Skeggs, using DEAE-cellulose chromatography, separated four different forms of hog renin (13) as well as four different forms of human renal renin (14). The molecular weights of the different forms of human renal renin isolated in the latter study were estimated to range from 38,000 to 39,000 (14). Gel filtration of partially purified human renin extracted from kidneys ob-
tained at autopsy yielded two peaks of activity, possessing apparent molecular weights of 42,000 and 38,000 (4). Similar results were obtained with renin extracted from rat kidney (2). Because of the similarity of molecular weight values obtained in these studies, the possible existence of renin isozymes has been suggested (2). In other studies, strikingly different results have been observed in regard to renin heterogeneity. Boyd (2,15) isolated two forms of porcine renin possessing markedly different properties. Renin A was stable to changes in pH; on the contrary, renin B showed increased activity after exposure to either acidic (pH 4.5) or basic (pH 10.0) media. Sephadex gel filtration established a molecular weight for renin B of 60,000; both renin A and the activated form of renin B exhibited a molecular weight of 38,000. Similar results were obtained by
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BIOCHEMISTRY OF BIG RENIN I
I
1091 I
i
i
r
NORMAL RENIN
FIG. 6. Enzyme kinetics of normal renin (top) and big renin (bottom), using partially purified sheep substrate. Big renin kinetics were studied both before (A) and after (o) activation by dialysis to pH 3.3. Ordinate: reciprocal of renin activity [(ng/ ml/h)"1 x 10]; abscissa: reciprocal of substrate concentration [(ng/ml)"1 x 102].
i
i
i
i
i
i
4 -
2 -
20
Leckie, who examined rabbit kidney extracts before and after dialysis to pH 2.5 (3). In man, the big renin found in plasma and tumor extracts of a patient with Wilms' tumor was similarly found to elute more rapidly than normal renin from Sephadex gel, possessing an apparent molecular weight of 60,000 (4). Big renin activity was subsequently found in plasma and kidney extracts from other patients, as well as in normal amniotic fluid (5). Lumbers (6) found that the activity of amniotic fluid
renin increased when exposed to pH 3.3-3.6; in the present study, activation of human big renin took place upon dialysis to pH 3.0-3.6 (Fig. 2). Activation of big renin was of similar magnitude whether sheep substrate or human substrate was used. The concentration of sheep substrate used to assay renin activity in these experiments was well above the Michaelis constant of either normal renin or big renin (Fig. 6). Thus, the activation of big renin was chiefly the result of an increase in
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DAY AND LUETSCHER
JCE & M • 1975 Vol 40 • No 6
maximal enzymatic velocity, and not the more sensitive to antirenin antibodies than result of only an increased affinity for normal renin (4). The present evidence would therefore indicate that big renin, substrate. In the present study, both trypsin and upon activation, resembles normal renin in pepsin were found to activate big renin its enzymatic and certain physical propertenfold during 1- to 3-min incubations (Fig. ties, but not in its behavior during gel 3). One hour incubations of human amni- filtration. Since gel filtration (16) and elecotic fluid with trypsin or pepsin also in- trophoresis (11) measure not only molecucreased the renin activity two- to three- lar weight, but also measure shape and fold (7). Incubation of big renin with charge, further work will be necessary to neuraminidase did not result in activation, define the physical changes which accomindicating that activation does not involve pany the activation of big renin. Renin-like enzymes have been isolated the liberation of sialic acid residues (Table 1). Likewise, reduction of disulfide bonds from a number of different tissues. The by dimercaprol in the presence of 6M urea renin in amniotic fluid (5-7) is very similar did not effect activation; instead, such to big renin, while renin activity in murine treatment denatured both normal and big salivary gland extracts is indistinguishable renin (Table 1). Boyd (15) has shown that from normal renin (17-18). Skeggs has exposure of porcine renin B to 4M NaCl described an enzyme named pseudorenin results in its disassociation into renin A and (14,19), which forms angiotensin I from the a renin binding protein. In the present synthetic tetradecapeptide substrate, but study, the activity of big renin did not which has no activity at pH 7.4 with normal increase when incubated with substrate in renin substrate. The enzyme tonin (20) can form angiotensin II directly from the presence of 4M NaCl (Table 1). In their studies, both Boyd (2) and Lec- the synthetic tetradecapeptide, with a kie (3) observed that acid activation of Michaelis constant possibly of the order of porcine or rabbit renal renin resulted in its 10,000 ng/ml. A renin-like enzyme named less rapid elution from Sephadex gel, and iso-renin has been isolated from brain and proposed that activation led to a decrease adrenals; it possesses optimal activity at in apparent molecular weight. Human big pH 5.0, is less sensitive to antirenin anrenin extracted from Wilms* tumor, follow- tibodies than normal renin, and does not ing acidification to pH 3.3, still eluted from exhibit saturable kinetics with homologous Sephadex gel more rapidly than normal substrate (21). In contrast, big renin isorenin (4). In the present study, acid or lated from human plasma releases antrypsin activation did not alter the elution giotensin I from human substrate at pH 7.4 behavior of big renin on Sephadex gel (Figs. and 37 C (4), and following activation pos1, 4). Thus, on the basis of gel filtration sesses enzymatic properties similar to studies, the activation of human big renin those of normal plasma renin (Fig. 6). does not involve changes in the molecular Thus, the biochemical similarities suggest weight. On the other hand, polyacrylamide a structural relationship only between gel electrophoresis of human big renin normal renin and big renin, and not besuggests that acid activation results in tween renin and the other renin-like ensignificant structural change. The migration zymes reported. of activated human big renin in polyaFor many polypeptide hormones, the crylamide gel was faster than inactive big synthesis of a precursor molecule has been renin, and similar to normal renin (Fig. 5). shown to precede the formation and reLikewise, activated big renin and normal lease of the active agent (22,23). The renin share similar kinetics (Fig. 6), and enzyme trypsin has been implicated in both have optimal activity to pH 5.5 (4). vitro in the formation of parathormone Big renin following activation is slightly from its prohormone (24); similar findings
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BIOCHEMISTRY OF BIG RENIN have been reported for insulin (25) and ACTH (26). Pancreatic proteases likewise are produced in inactive forms, and are subsequently activated in the intestinal lumen by trypsin (27). On the basis of their study of renin in amniotic fluid, Morris and Lumbers have suggested that cathepsins may be responsible for the activation of renin (7), although their data are consistent with the notion that renin, like pepsin, may be activated by autocatalysis at acid pH (27). The present study, on the basis of the physical and enzymatic parameters investigated, suggests that big renin may prove to be the precursor of normal renin. For this to be true, the method of activation in vivo of big renin would necessarily be by some means other than those used in vitro in this study. Furthermore, the isolation of big renin from normal kidney tissue has not yet been accomplished (5). To directly implicate big renin as the renin precursor, more direct evidence concerning its biosynthesis by normal renin tissue and its subsequent conversion to normal renin in vivo is necessary. Nevertheless, the comparison of the present evidence concerning big renin to the data available for other polypeptide hormones suggests a possible role of big renin in normal renin biosynthesis and release. Acknowledgments The authors wish to express their thanks to Mrs. Carol Gonzales for her expert technical assistance, and to Mrs. Brooke Pyatt for her aid in the preparation of the manuscript. References 1. Yalow, R. S., In Greer, R. O. (ed.), Recent Progress in Hormone Research, vol. 30, Academic Press, New York, 1974, p. 597. 2. Boyd, G. W., In Genest, J., and E. Koiw (ed.), Hypertension, Springer-Verlag, Berlin, 1972, p. 161.
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3. Leckie, B., Clin Sci 44: 301, 1973. 4. Day, R. P., and J. A. Luetscher,/ Clin Endocrinol Metab 38: 923, 1974. 5. , , and C. M. Gonzales, J Clin Endocrinol Metab 40: 1018, 1975. 6. Lumbers, E. R., Enzymologia 40: 329, 1971. 7. Morris, B. J., and E. R. Lumbers, Biochim Biophys Ada 289: 385, 1972. 8. Skinner, S. L., Circ Res 20: 391, 1967. 9. Beckerhoff, R., J. A. Luetscher, and D. Wilkinson, J Clin Endocrinol Metab 34: 1067, 1972. 10. Andrews, P., BiochemJ 91: 222, 1964. 11. Smith, I., In Smith, I. (ed.), Chromatographic and Electrophoretic Techniques, vol. 2, Wiley, New York, 1968, p. 365. 12. Goldstein, A., Biostatistics, MacMillan, New York, 1964, p. 129. 13. Skeggs, L. T., K. E. Lentz, J. R. Kahn, and H. Hochstrasser, Circ Res 21: Supp. II, 91, 1967. 14. , , , M. Levine, and F. E. Dorer, In Genest, J., and E. Koiw (eds.), Hypertension, Springer-Verlag, Berlin, 1972, p. 149. 15. Boyd, G. W., Circ Res 35: 426, 1974. 16. Siegel, L. M., and K. J. Monty, Biochim Biophys Ada 112: 346, 1966. 17. Cohen, S., J. M. Taylor, K. Murakami, A. M. Michelakis, and T. Inagami, Biochemistry 11: 4286, 1972. 18. Michelakis, A. M., H. Yoshida, J. Menzie, K. Murakami, and T. Inagami, Endocrinology 94: 1101, 1974. 19. Skeggs, L. T., K. E. Lentz, J. R. Kahn, F. E. Dorer, and M. Levine, Circ Res 75: 451, 1969. 20. Boucher, R., J. Asselin, and J. Genest, Circ Res 34: Supp. I, 1-203, 1974. 21. Ganten, D., P. Granger, V. Ganten, R. Boucher, and J. Genest, In Genest, J., and E. Koiw (eds.), Hypertension, Springer-Verlag, Berlin, 1972, p. 423. 22. Kemper, B., J. F. Habener, J. T. Potts, Jr., and A. Rich, Proc Natl Acad Sci 69: 643, 1972. 23. Steiner, D. F., D. Cunningham, L. Spigelman, and B. Arn, Science 157: 697, 1972. 24. Cohn, D. V., R. R. MacGregor, L. L. H. Chu, J. R. Kimmel, and J. W. Hamilton, Proc Natl Acad Sci 69: 1521, 1972. 25. Kemmer, W., J. D. Peterson, A. H. Rubenstein, and D. F. Steiner, Diabetes 21: Supp. 2, 572, 1972. 26. Gewirtz, G., B. Schneider, D. T. Krieger, and R. S. Yalow, J Clin Endocrinol Metab 38: 227, 1974. 27. Linderstr0m-Lang, K., In Luck, J. M. (ed.), Annual Review of Biochemistry, vol. 6, Annual Reviews, 1937, p. 60.
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ABSTRACT. The properties of big renin, a relatively inactive form of renin isolated from human plasma, were examined following partial purification by gel filtration. Exposure of big renin to pH 3.0-3.6, or brief incubation with trypsin or pepsin, resulted in a ten-fold increase in enzymatic activity. Activation was not effected by 4M NaCl, 6M urea, or incubation with neuraminidase. Both before and after activation, big renin eluted from Sephadex gel more rapidly than normal plasma renin. During polyacrylamide gel disc electrophoresis, inactive big renin migrated more slowly than either normal
T
HE biochemical heterogeneity of many circulating polypeptide hormones has recently been established (1). Likewise, biochemically distinct forms of the renal hormone renin have been demonstrated. A relatively inactive renin, which may be activated by exposure to acid pH, has been extracted from porcine (2) and rabbit (3) kidney tissue. This inactive renin elutes more rapidly from Sephadex gel than normal renin, indicating that it possesses a higher molecular weight. A similar form of renin has been isolated from plasma and kidney extracts of certain patients (4,5), as well as from human amniotic fluid (5-7). The present study further examines the biochemical characteristics of this "big renin" both before and after its activation. Materials and Methods Renin assay. Renin activity in all experiments was determined using a modification of the plasma renin concentration (PRC) method (8). Received January 13, 1975. This research was supported by grants from the National Aeronautics and Space Administration (NGR-05-020-456) and the National Institutes of Health (HL-13917) and (HL-17364). Dr. Luetscher is the recipient of a Research Career Award (K6 AM14176).
Samples were incubated at 37 C and pH 7.4 with (partially purified) sheep substrate (8) at a final concentration of 1600 ng angiotensin/ml, in the presence of 1 mM disodium EDTA, 1.6 mM dimercaprol, and 6.8 mM 8-hydroxyquinoline. In the initial gel filtration of plasma aliquots described in Fig. 1, pooled plasma from anephric patients was also used to supply substrate. Following incubation for 1 to 18 h samples were diluted with 2 vol 0.1M Tris-HCl buffer, pH 7.4, boiled for 10 min, and centrifuged. Angiotensin I in the supernate was measured by radioimmunoassay (9). Renin activity is expressed as ng of angiotensin I generated per ml of sample per hour of incubation. Gel filtration. One-half ml aliquots of plasma or eluate were applied to a Sephadex G-75 column, 2 cm in diameter by 90 cm in length, previously equilibrated with sodium phosphate buffer pH 7.4 (0.1M NaCl, 0.05M sodium phos-
phate, and 0.1% disodium EDTA). Two ml fractions were collected. The void volume of the column, measured with blue dextran, was 40 ml; the total volume, measured using 3 H 2 O, was 152 ml. Molecular weight estimates were made following gel filtration of multiple samples; values are expressed as the mean ± the standard error of the mean. The Sephadex column was calibrated with bovine serum albumin (KD = 0.16), ovalbumin (KD = 0.27) and chymotrypsinogen (KD = 0.46) (10). Protein concentration in the eluates was estimated spectrophotometrically at 280 nm.
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Activation
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studies. One-ml samples were
dialyzed for 24 h at 4 C against 40 vol buffer adjusted to selected pH values ranging from pH 2.0 to pH 10.0. Buffers of pH 3.6-10.0 were prepared using 10M NaOH to adjust a solution of 0.025M sodium citrate and 0.075M sodium
dihydrogen phosphate to the various pH. More acidic buffers were prepared using 0.05M glycine, adjusted to the correct pH using lM HC1. Following the return of the sample pH to 7.4 by dialysis at 4 C, renin activity was measured. The activity of big renin previously dialyzed to pH 3.3 was measured using both human and sheep substrate. Samples were incubated with trypsin, final concentration 50 fig/m\, at 37 C in sodium phosphate buffer, pH 7.4. Incubation was terminated by the addition of soybean trypsin inhibitor, 100 /ng/ml. Incubation of samples with pepsin, 50. fig/nd, and neuraminidase, 2 mg/ml, was performed at 37 C using 0.05M sodium acetate buffer containing 0.01M CaCl2 and adjusted to pH 4.5 and 5.0, respectively. Incubations with these two enzymes were terminated by the addition of an equal volume of 0.25M sodium phosphate buffer, pH 7.4, containing 1.0% disodium EDTA. Control values for enzyme incubations were obtained by exposing samples to incubation media containing no enzyme. Under the stated assay conditions, trypsin alone exhibited reninlike activity of less than 2 ng/ml/h, and pepsin alone showed less than 0.2 ng/ml/h renin activity. Samples exposed to urea were first incubated at either 4 C or 37 C for 2 h in the presence of 6M urea and 0.1% dimercaprol, and subsequently dialyzed at 4 C against sodium phosphate buffer, pH 7.4, containing 0.1% dimercaprol. Samples dialyzed against 4M NaCl were assayed for renin activity using sheep substrate previously dialyzed against 4M NaCl. Electrophoresis. Disc gel electrophoresis was performed in a 7.5% polyacrylamide resolving gel 6 cm in length placed below a 2.5% polyacrylamide gel 1 cm in length (11). Gels were polymerized in 0.3M Tris-HCl buffer, pH 8.9. Samples to be studied were concentrated by dialysis against 30% sucrose, and 20 ^il aliquots were applied to the gels. Electrophoresis was conducted at 20 C using 3 mA current per sample with 0.377M Tris-glycine buffer, pH 8.3, as the reservoir buffer. The migration during electrophoresis was visualized by the addition of bromophenol blue to the reservoir buffer. Electrophoresis was terminated upon the
JCE & M • 1975 Vol 40 • No 6
migration of the bromophenol blue front to within 1 cm of the end of the resolving gel, and the gels were subsequently sliced into 2 mm segments. Renin was allowed to diffuse from the gel segments following maceration in three volumes of sodium phosphate buffer, pH 7.4; renin activity was determined in the diffusate as described above. In these experiments, renin activity is expressed as ng of angiotensin I generated per mm of gel per hour of incubation. Solutions of bovine serum albumin and chymotrypsinogen, 1 mg/ml, were used as protein standards; following electrophoresis, standards were identified in the gels by staining with Coomassie Blue (11). Kinetic studies. The enzyme kinetics of normal and big renin were investigated using partially purified sheep substrate in concentrations ranging from 50 ng/ml to 1600 ng/ml. Samples, containing equal amounts of renin activity when saturated with substrate, were incubated from 1 to 18 h. The maximal rate of angiotensin I generation was used in the subsequent analysis. Michaelis-Menten constants were calculated by standard linear regression techniques (12); in all cases, the correlation coefficient exceeded 0.98. Materials. Normal renin was prepared from the plasma of normal patients undergoing salt restriction or furosemide diuresis. Big renin was extracted from plasma of patients with Wilms' tumor or diabetic nephropathy; clinical details of these patients are presented elsewhere (5). Both normal renin and big renin were partially purified by gel filtration before use in the other experiments. Bovine serum albumin and ovalbumin were obtained from Nutritional Biochemicals, Cleveland, Ohio. Bovine trypsin, procine pepsin, Type V neuraminidase, soybean trypsin inhibitor, and bovine achymotrypsinogen A were purchased from Sigma Chemical Co., St. Louis, Missouri.
Results Gel filtration of normal plasma revealed a single peak of renin activity which appeared after 68.7 ± 0.2 ml had eluted from the column (KD = 0.26) (Fig. 1, top). Dialysis of the active fractions to pH 3.3, followed by dialysis of the samples back to pH 7.4, did not appreciably alter the renin activity present in the fractions (Fig. 1, top, dashed lines). In contrast, gel filtration of
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BIOCHEMISTRY OF BIG RENIN 1OJ0
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I
•
I
i
HORMAL RENfi
75 "
If) 1
5.0 FIG. 1. Gel filtration of normal renin (top) and big renin (bottom). Renin activity was measured both before (A) and after (o) dialysis to pH 3.3. Ordinate: renin activity (ng/ml/h); abscissa: volume of buffer eluted (ml).
/ / .
10
I
"
tiff
60 70 VOLUME ELUTED (ml)
80
90
FIG. 2. Activation of big renin as a function of pH. Ordinate: renin activity (ng/ml/h); abscissa: pH of initial dialysate.
4 6 ACTIVATION pH
the plasma from the patient with Wilms' tumor revealed big renin activity. Maximum activity appeared after an elution volume of 59.8 ± 0.5 ml (KD = 0.18) (Fig.
K)
1, bottom). By Sephadex gel filtration, normal plasma renin exhibited an apparent molecular weight of 43,000 ± 1,000, while big renin possessed a molecular weight
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DAY AND LUETSCHER
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Incubation of big renin with trypsin or equal to 63,000 ± 2,000, calculated by pepsin resulted in greatly increased renin comparison with pure protein standards. Dialysis of the fractions containing big activity (Fig. 3). Incubation of big renin renin to pH 3.3 as described above in- with trypsin, 50 /ng/ml, for 10 s increased creased the renin activity present tenfold the renin activity sixfold; maximal activa(Fig. 1, bottom, dashed line). Following tion was observed after a 3-min incubation dialysis of big renin to pH values of 3.6 or (Fig. 3, top). Incubation with pepsin, 50 lower, increased renin activity was ob- /xg/ml, resulted in a similar time course of served (Fig. 2). Big renin dialyzed to pH activation (Fig. 3, bottom). Maximal activa4.0 to 10.0 showed no appreciable increase tion of big renin with pepsin occurred after in activity. Maximal activation occurred 30-100 s. The activity of normal renin did following dialysis to pH 3.3; below this not change following incubation with pH, denaturation of renin probably oc- either trypsin or pepsin. curred. When renin activity was measured Incubation of big renin with neurusing human substrate, dialysis to pH 3.3 aminidase for 3 h at 37 C did not inincreased its activity from 6.6 ng/ml/h to 54 crease renin activity appreciably above ng/ml/h. Using sheep substrate, dialysis of control levels (Table 1). The enzymatic big renin to pH 3.3 increased renin activity activity of big renin did not increase in response to high ionic strength media; 4M from 16.9 ng/ml/h to 225 ng/ml/h. 200
FIG. 3. Activation of big renin by trypsin (top) and pepsin (bottom). Ordinate: renin activity (ng/mlyh); abscissa: duration of incubation (min).
3 INCUBATION
4
TIME (min)
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BIOCHEMISTRY OF BIG RENIN
NaCl inhibited both normal renin and big renin to a similar extent (Table 1). Exposure of both big renin and normal renin to 6M urea, followed by dialysis against 0.1% dimercaprol, was an effective denaturing process for both renins (Table 1). Following activation, big renin eluted from Sephadex gel in a manner similar to that of unactivated big renin (Fig. 4). Whether previously exposed to pH 3.3 by dialysis (Fig. 4, top), or incubation with trypsin for three minutes (Fig. 4, bottom), big renin appeared at an elution volume of 60ml(KD = 0.18). Polyacrylamide gel disc electrophoresis, on the contrary, effected clear separation of unactivated and activated big renin (Fig. 5). The Rf value of big renin not previously activated was 0.36 (Fig. 5, upper right). Electrophoresis of normal plasma renin (Fig. 5, upper left), as well as big renin previously activated either with trypsin or by dialysis to pH 3.3 (Fig. 5, bottom)
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TABLE 1. Effect of neuraminidase, 4M NaCl, and 6M
urea upon normal and big renin activity
Treatment
Renin species
Control (ng/ml/h)
Experimental (ng/ml/h)
Neuraminidase
Big renin
12.8
15.7
4M NaCl
Normal renin Big renin
11.6 11.7
9.8 8.3
6M urea, 37 C
Normal Renin Big Renin
11.5 9.6
0.0 0.1
6M urea, 4 C
Normal Renin Big Renin
11.5 12.2
8.2 12.4
possessed Rf values of 0.47. Under equivalent conditions the Rf of bovine serum albumin and chymotrypsinogen was 0.38 and 0.56, respectively. Using partially purified sheep substrate, saturation kinetics for both normal renin and big renin could be demonstrated (Fig. 6). The Michaelis constant for normal renin was 185 ± 36 ng/ml. Big renin not
FIG. 4. Gel filtration of big renin previously activated by dialysis to pH 3.3 (top) or incubation with trypsin (bottom). Ordinate: renin activity (ng/ml/h); abscissa: volume of buffer eluted (ml).
TRYPSIN ACT1VKTED
50
60
70
80
90
VOLUME ELUTED (ml)
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DAY AND LUETSCHER
24
i
i
16
i
0\ \ >
6 !** Qb V
8
1.0
\ \ \
BIG BENIN
-
—
0
FlG. 5. Polyacrylamide gel disc electrophoresis of normal renin (upper left), unactivated big renin (upper right), and big renin previously activated by pH 3.3 dialysis (lower left) and tryptic digestion (lower right). Ordinate: renin activity (ng/mm/h); abscissa: distance from origin (Rf). The activity of unactivated big renin (upper right) was measured both before (A) and after (o) dialysis to pH 3.3.
previously activated possessed a Michaelis constant of 465 ± 150 ng/ml; after activation the Michaelis constant of big renin dropped to 151 ± 25 ng/ml. Discussion In the field of hormone research, examples of biochemically heterogeneous polypeptide agents abound (1). Previous investigators have also reported the existence of various forms of renal renin. Skeggs, using DEAE-cellulose chromatography, separated four different forms of hog renin (13) as well as four different forms of human renal renin (14). The molecular weights of the different forms of human renal renin isolated in the latter study were estimated to range from 38,000 to 39,000 (14). Gel filtration of partially purified human renin extracted from kidneys ob-
tained at autopsy yielded two peaks of activity, possessing apparent molecular weights of 42,000 and 38,000 (4). Similar results were obtained with renin extracted from rat kidney (2). Because of the similarity of molecular weight values obtained in these studies, the possible existence of renin isozymes has been suggested (2). In other studies, strikingly different results have been observed in regard to renin heterogeneity. Boyd (2,15) isolated two forms of porcine renin possessing markedly different properties. Renin A was stable to changes in pH; on the contrary, renin B showed increased activity after exposure to either acidic (pH 4.5) or basic (pH 10.0) media. Sephadex gel filtration established a molecular weight for renin B of 60,000; both renin A and the activated form of renin B exhibited a molecular weight of 38,000. Similar results were obtained by
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BIOCHEMISTRY OF BIG RENIN I
I
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i
i
r
NORMAL RENIN
FIG. 6. Enzyme kinetics of normal renin (top) and big renin (bottom), using partially purified sheep substrate. Big renin kinetics were studied both before (A) and after (o) activation by dialysis to pH 3.3. Ordinate: reciprocal of renin activity [(ng/ ml/h)"1 x 10]; abscissa: reciprocal of substrate concentration [(ng/ml)"1 x 102].
i
i
i
i
i
i
4 -
2 -
20
Leckie, who examined rabbit kidney extracts before and after dialysis to pH 2.5 (3). In man, the big renin found in plasma and tumor extracts of a patient with Wilms' tumor was similarly found to elute more rapidly than normal renin from Sephadex gel, possessing an apparent molecular weight of 60,000 (4). Big renin activity was subsequently found in plasma and kidney extracts from other patients, as well as in normal amniotic fluid (5). Lumbers (6) found that the activity of amniotic fluid
renin increased when exposed to pH 3.3-3.6; in the present study, activation of human big renin took place upon dialysis to pH 3.0-3.6 (Fig. 2). Activation of big renin was of similar magnitude whether sheep substrate or human substrate was used. The concentration of sheep substrate used to assay renin activity in these experiments was well above the Michaelis constant of either normal renin or big renin (Fig. 6). Thus, the activation of big renin was chiefly the result of an increase in
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DAY AND LUETSCHER
JCE & M • 1975 Vol 40 • No 6
maximal enzymatic velocity, and not the more sensitive to antirenin antibodies than result of only an increased affinity for normal renin (4). The present evidence would therefore indicate that big renin, substrate. In the present study, both trypsin and upon activation, resembles normal renin in pepsin were found to activate big renin its enzymatic and certain physical propertenfold during 1- to 3-min incubations (Fig. ties, but not in its behavior during gel 3). One hour incubations of human amni- filtration. Since gel filtration (16) and elecotic fluid with trypsin or pepsin also in- trophoresis (11) measure not only molecucreased the renin activity two- to three- lar weight, but also measure shape and fold (7). Incubation of big renin with charge, further work will be necessary to neuraminidase did not result in activation, define the physical changes which accomindicating that activation does not involve pany the activation of big renin. Renin-like enzymes have been isolated the liberation of sialic acid residues (Table 1). Likewise, reduction of disulfide bonds from a number of different tissues. The by dimercaprol in the presence of 6M urea renin in amniotic fluid (5-7) is very similar did not effect activation; instead, such to big renin, while renin activity in murine treatment denatured both normal and big salivary gland extracts is indistinguishable renin (Table 1). Boyd (15) has shown that from normal renin (17-18). Skeggs has exposure of porcine renin B to 4M NaCl described an enzyme named pseudorenin results in its disassociation into renin A and (14,19), which forms angiotensin I from the a renin binding protein. In the present synthetic tetradecapeptide substrate, but study, the activity of big renin did not which has no activity at pH 7.4 with normal increase when incubated with substrate in renin substrate. The enzyme tonin (20) can form angiotensin II directly from the presence of 4M NaCl (Table 1). In their studies, both Boyd (2) and Lec- the synthetic tetradecapeptide, with a kie (3) observed that acid activation of Michaelis constant possibly of the order of porcine or rabbit renal renin resulted in its 10,000 ng/ml. A renin-like enzyme named less rapid elution from Sephadex gel, and iso-renin has been isolated from brain and proposed that activation led to a decrease adrenals; it possesses optimal activity at in apparent molecular weight. Human big pH 5.0, is less sensitive to antirenin anrenin extracted from Wilms* tumor, follow- tibodies than normal renin, and does not ing acidification to pH 3.3, still eluted from exhibit saturable kinetics with homologous Sephadex gel more rapidly than normal substrate (21). In contrast, big renin isorenin (4). In the present study, acid or lated from human plasma releases antrypsin activation did not alter the elution giotensin I from human substrate at pH 7.4 behavior of big renin on Sephadex gel (Figs. and 37 C (4), and following activation pos1, 4). Thus, on the basis of gel filtration sesses enzymatic properties similar to studies, the activation of human big renin those of normal plasma renin (Fig. 6). does not involve changes in the molecular Thus, the biochemical similarities suggest weight. On the other hand, polyacrylamide a structural relationship only between gel electrophoresis of human big renin normal renin and big renin, and not besuggests that acid activation results in tween renin and the other renin-like ensignificant structural change. The migration zymes reported. of activated human big renin in polyaFor many polypeptide hormones, the crylamide gel was faster than inactive big synthesis of a precursor molecule has been renin, and similar to normal renin (Fig. 5). shown to precede the formation and reLikewise, activated big renin and normal lease of the active agent (22,23). The renin share similar kinetics (Fig. 6), and enzyme trypsin has been implicated in both have optimal activity to pH 5.5 (4). vitro in the formation of parathormone Big renin following activation is slightly from its prohormone (24); similar findings
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BIOCHEMISTRY OF BIG RENIN have been reported for insulin (25) and ACTH (26). Pancreatic proteases likewise are produced in inactive forms, and are subsequently activated in the intestinal lumen by trypsin (27). On the basis of their study of renin in amniotic fluid, Morris and Lumbers have suggested that cathepsins may be responsible for the activation of renin (7), although their data are consistent with the notion that renin, like pepsin, may be activated by autocatalysis at acid pH (27). The present study, on the basis of the physical and enzymatic parameters investigated, suggests that big renin may prove to be the precursor of normal renin. For this to be true, the method of activation in vivo of big renin would necessarily be by some means other than those used in vitro in this study. Furthermore, the isolation of big renin from normal kidney tissue has not yet been accomplished (5). To directly implicate big renin as the renin precursor, more direct evidence concerning its biosynthesis by normal renin tissue and its subsequent conversion to normal renin in vivo is necessary. Nevertheless, the comparison of the present evidence concerning big renin to the data available for other polypeptide hormones suggests a possible role of big renin in normal renin biosynthesis and release. Acknowledgments The authors wish to express their thanks to Mrs. Carol Gonzales for her expert technical assistance, and to Mrs. Brooke Pyatt for her aid in the preparation of the manuscript. References 1. Yalow, R. S., In Greer, R. O. (ed.), Recent Progress in Hormone Research, vol. 30, Academic Press, New York, 1974, p. 597. 2. Boyd, G. W., In Genest, J., and E. Koiw (ed.), Hypertension, Springer-Verlag, Berlin, 1972, p. 161.
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3. Leckie, B., Clin Sci 44: 301, 1973. 4. Day, R. P., and J. A. Luetscher,/ Clin Endocrinol Metab 38: 923, 1974. 5. , , and C. M. Gonzales, J Clin Endocrinol Metab 40: 1018, 1975. 6. Lumbers, E. R., Enzymologia 40: 329, 1971. 7. Morris, B. J., and E. R. Lumbers, Biochim Biophys Ada 289: 385, 1972. 8. Skinner, S. L., Circ Res 20: 391, 1967. 9. Beckerhoff, R., J. A. Luetscher, and D. Wilkinson, J Clin Endocrinol Metab 34: 1067, 1972. 10. Andrews, P., BiochemJ 91: 222, 1964. 11. Smith, I., In Smith, I. (ed.), Chromatographic and Electrophoretic Techniques, vol. 2, Wiley, New York, 1968, p. 365. 12. Goldstein, A., Biostatistics, MacMillan, New York, 1964, p. 129. 13. Skeggs, L. T., K. E. Lentz, J. R. Kahn, and H. Hochstrasser, Circ Res 21: Supp. II, 91, 1967. 14. , , , M. Levine, and F. E. Dorer, In Genest, J., and E. Koiw (eds.), Hypertension, Springer-Verlag, Berlin, 1972, p. 149. 15. Boyd, G. W., Circ Res 35: 426, 1974. 16. Siegel, L. M., and K. J. Monty, Biochim Biophys Ada 112: 346, 1966. 17. Cohen, S., J. M. Taylor, K. Murakami, A. M. Michelakis, and T. Inagami, Biochemistry 11: 4286, 1972. 18. Michelakis, A. M., H. Yoshida, J. Menzie, K. Murakami, and T. Inagami, Endocrinology 94: 1101, 1974. 19. Skeggs, L. T., K. E. Lentz, J. R. Kahn, F. E. Dorer, and M. Levine, Circ Res 75: 451, 1969. 20. Boucher, R., J. Asselin, and J. Genest, Circ Res 34: Supp. I, 1-203, 1974. 21. Ganten, D., P. Granger, V. Ganten, R. Boucher, and J. Genest, In Genest, J., and E. Koiw (eds.), Hypertension, Springer-Verlag, Berlin, 1972, p. 423. 22. Kemper, B., J. F. Habener, J. T. Potts, Jr., and A. Rich, Proc Natl Acad Sci 69: 643, 1972. 23. Steiner, D. F., D. Cunningham, L. Spigelman, and B. Arn, Science 157: 697, 1972. 24. Cohn, D. V., R. R. MacGregor, L. L. H. Chu, J. R. Kimmel, and J. W. Hamilton, Proc Natl Acad Sci 69: 1521, 1972. 25. Kemmer, W., J. D. Peterson, A. H. Rubenstein, and D. F. Steiner, Diabetes 21: Supp. 2, 572, 1972. 26. Gewirtz, G., B. Schneider, D. T. Krieger, and R. S. Yalow, J Clin Endocrinol Metab 38: 227, 1974. 27. Linderstr0m-Lang, K., In Luck, J. M. (ed.), Annual Review of Biochemistry, vol. 6, Annual Reviews, 1937, p. 60.
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