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Renal renin extraction and biochemical characterization
Comp. Biochem. Physiol. Vol. 68B, pp. 329 to 332
0305-0491/81/0201-0329$02.00/0
© Pergamon Press Ltd 1981. Printed in Great Britain
RENAL RENIN EXTRACTION AND BIOCHEMICAL CHARACTERIZATION CHUN SIK PARZ Division of Nephrology, Department of Medicine, School of Mcdicinc, M-023, Univcrsity of California, San Diego, La Jolla, CA 92093, U.S.A.
(Received 15 May 1980) A h s t r a c t - - l . A method for extraction of renal renin with high and reproducible yield was described.
2. About 90~o of the total renal renin activity was found in the supernatant at 105,000 g for 60 min. 3. The molecular weight of extracted renin of 3 different species was about 45,000 dalton. 4. The isoelectric point of renal renin of pig, dog and mouse was 5.10, 5.46 and 5.94, respectively. 5. Acid treatment of renal renin resulted in the change of its isoelectric point and inactivation of enzymatic activity.
INTROD UCTION A remarkable correlation between the granulation of juxtaglomerular (JG) cells, the amount of extractable renal renin and plasma renin activity was observed in a variety of experimental conditions (Gross et al., 1964; Hartroft, 1963; Miksche et al., 1970; Tobian et al., 1959). It was reported that a quantitative correlation exists between renal activity and the rate of renin secretion (Fray, 1978; Park et al., 1978). All these observations indicate that the renal renin activity is one among the important determinants of renin secretion. In spite of the importance of renin in fluid and water balance, there is no standard procedure for the determination of renal renin activity. As an index of this, the reported values of renal renin activity of rats fed normal salt diet varies from 4 to 4000 ng Ang I/rag kidney.hr, depending upon the different procedures (De Senarclens et al., 1977; Kotchen et al., 1974; Miiller-Suur et al., 1975; Silverman et al., 1974). The purpose of this investigation was threefold. The first was to develop a method for the quantitative extraction and determination of dog renal renin. The second was to study some biochemical characteristics of canine renal renin, which have not yet been studied. The biochemical characteristics of renal renin of other species were also determined and compared to those of dog renin. The third was to examine whether the extracted dog renin is all active form or not. MATERIALS AND METHODS
Extraction of renal renin The detailed procedures for the preparation of renal cortical slices were described previously (Park et al., 1978). Briefly, slices of renal cortex (about 300 mg) was homogenized in ice-cold 0.25 M sucrose using an all glass tissue homogenizer. After homogenation, subcellular fractions were obtained by centrifuging the homogenate at 700 g for 10 min (Nuclear Fraction as well as cell debris), at 10,000g for 10 min (Mitrochondrial Fraction), and at 105,000g for 60 min (Microsomal Fraction and its supernatant). All subcellular fractions and the supernatant (105,000 g) as well as the remaining homogenate were frozen at -20°C until 329
assay of renin activity. The distribution of renal renin in each subcellular fraction was determined in 16 renal cortical samples from two dogs.
Molecular weight of renin (dog, pig and rat) The molecular weight of renin was estimated by gel filtration of 2ml of the 105,000g supernatant using a Sephadex G-100 column (2.5 x 45 cm). The column was pre-equilibrated with 50mM sodium phosphate buffer, pH 7.4 and calibrated by determining the averaged elution volume (Kay) of each of the following molecular weight standards (Pharmacia Kit); ribonuclease A (13,700mol. wt), chymotrypsinogen A (25,000mol. wt), ovalbumin (45,000 mol. wt), bovine serum albumin (68,000 mol. wt), aldolase (158,000 mol. wt)and blue dextran 2000. The relative concentration of blue dextran in the column eluate was determined spectrophotometrically at 620 nm and the molecular maker proteins were assayed by the method of Lowry et al. (1951). pH-stability of dog renal renin Fifty microliters of the supernatant of dog renal cortical homogenate from 4 dogs was treated at 4°C with 1 ml of maleate buffer (0.2 M) in which the pH ranged from 1 to 12, (adjusted either with HC1 or NaOH). After 1, 2, 3 (each 1 sample) and 24 hr (3 samPles ) following the treatment at each pH, 25 and 50/~l of each sample was incubated with l ml renin substrate (pH 6.0) for the determination of renin activity. Gel electrophoresis and isoelectric focusing of renin Discontinuous polyacrylamide gel electrophoresis was carried out in glass tubes (5 x 65 mm) containing separating gel (7.5~ acrylamide at pH 8.8) and stacking gel (2.5~ acrylamide at pH 6.8) by the method of Gabriel (1971). The supernatant (105,000 g) was overlayered on the top of the stacking gel and electrophoresis was conducted at 4°C at 3 mA per tube until the tracking dye, bromophenol blue, moved to within 5 mm of the top of the separating gel. The isoelectric point of renin was determined by the method of Wrigley (1971) on a 7.5~o polyacrylamide containing ampholyte, p H 4 ~ or 3-10 (Ampholine, LKB Instruments, Inc.). Electrophoresis was carried out at a maximum current of 2 mA per tube for 90 min. The gels were cut at 3 mm thickness, which were then macerated in 0.5 ml of double distilled water. Renin activity and pH (at 25°C) were determined in the eluates of each gel slice. Renin activity of renal homogenate and column eluates was determined by incubating renin samples with dog
330
CHUN SIK PARK
renin substrate prepared from 48 hr nephrectomized dogs as described previously (Park et al., 1978). The recovery of angiotensin I (0.5, 1.0, 2, 3, 4 and 5 ng in duplicate) added to 1 ml of renin substrate was determined after incubation at 37°C for 60 min. Adequacy of renin substrate was examined by incubating renin samples for 15, 30 and 60 min (n = 8). Angiotensin I (Ang I) generated after the incubation at 37°C, pH6.0 was measured by the method of Haber et al. (1969), using New England Nuclear Angiotensin I Radioimmunoassay Kit.
Recovery and generation of angiotensin The recovery of Ang I added to the renin substrate in the incubating mixture was 92.1 _+ 6.1~o. The generation of Ang I by incubation of the superoatant of renal homogenate at 105,000g with renin substrate was linear up to 60 min. The incubation conditions were made to consume no more than 20~ of renin substrate by diluting renin samples.
Table 1. Percentage distribution of renal renin in the subcellular fractions of dog renal cortical homogenate
Fraction
% of total* (Mean + SEM)
I Nuclear
2.4 + 0.3
2 Mitochondrial
1.8 + 0.2
3 Microsomal
1.5 + 0.i
4 Supernatant
87.9 + 2.2
5 Sum (Recovery)
93.5 + 2.0
* Distribution expressed as percentage of total renin activity measured in the homogenate (n = 16 experiments).
RESULTS
Cellular distribution Distribution of renin in the subcellular fractions are shown in Table 1. The supematant contained the highest renin activity, accounting for 87.9 + 2.2~ of the total. The overall recovery of renin from the three particulate fractions and the soluble supernatant was 93.5 + 2.0% of the total activity measured in the cortical homogenate.
Molecular weight of renin From Fig. 1 it can be seen that molecular weight of renal renin of dog, pig and rat are about the size of ovalbumin. The estimated molecular weight of renin for each species (n = 3) are as follows: dog, 43,500 + 400; pig, 46,000 + 500 and rat, 48,500 + 700.
lsoelectric point of renal renin A typical experiment of renin activity and pH of each gel slice on gel electrofocusing of dog renin is shown in Fig. 2. A sharp peak of renin activity was observed in one gel slice, the pH of which was taken as the isoelectric point of renin. In the same way the isoelectric point of mouse and pig renin were determined. The isoelectric points of dog, pig and mouse
renin was 5.46 + 0.02, 5.19 + 0.02 and 5.94 + 0.03, respectively (n = i0). The isoelectric point of dog renin was significantly different from that of pig and mouse renin (P < 0.001). The isoelectric point of pig renin purified by the method of Haas et al. (1954) up to step 5 (Haas et al., 1954), was 4.65 _ 0.05 (n = 5). The difference in the isoelectric points of fresh and commercial pig renin is statistically significant (P < 0.001).
Electrophoretic mobility of renin Only a single peak of renin activity was revealed on polyacrylamide gel at mobility of about 0.5 relative to tracking dye bromophenol blue. A single peal of renin activity was also observed with pig and mouse renin (results are not shown).
The pH stability of dog renin Renin activity following preincubation at different pHs is shown in Fig. 3. In the pH range of 3-10, renin activity was stable up to 24 hr. However at pH values above and below this range, renin was unstable and rapidly inactivated after 3 hr of incubation at pH 2 or 11. Renin activity was decreased to approximately
0.6_-~ease A 0.4-
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DOG~
0.2
P I o;
v°lburnin
PIG~ " ~ J
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;
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Fig. 1. The molecular weight of dog, pig and rat renin (n = 3 for each species) was estimated using SephadexG-100.
Biochemistry of renin pH
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o Renin Activity
331
following three grounds. First, values of SEM were very small indicating a consistent degree of renin extraction. For instance, renal renin activity deterII • mined on 8 batches of renal cortex from 1 dog was 76.8 + 3.9 ng Ang I/mg tissue.hr. Second, over 90% of the total renal renin activity was found in the soluble fraction, which indicates that the degree of 5.( homogenation was sufficient to extract most, if not all of the renin. Third, the extraction was sensitive Ienough to detect changes in the renal renin activity as small as 12% in dogs (Park et al., 1978). z It has been reported that renin activity is mainly localized in the mitochondrial (Morimoto et al., 1972; O 4.0 4 7 I0 13 Nustad et al., 1970), lysosomal (Ogino et al., 1967) or GEL SLICE NUMBER between two subcellular fractions (Grass & Barajas, Fig. 2. A representative experiment showing the isoelectric 1975). Upon increasing the degree of homogenation, point of dog renal renin. however, most renin activity was shifted from the particulate fractions to the supematant (Morimoto et al., 1972; Nustad & Rubin, 1970). Thus, the extensive homogenation used in our studies may account for the fact that most of renin activity was found in the 3000 supematant. This result is consistent with a recent report by Inagami et al. (1977). They observed that more than 90% of hog and rat renal renin activity was in the supernatant fraction. The molecular weight of dog renin, as well as rat g and pig renin, was about 43,500 daltons (see Fig. 1). ~2ooo This value is in close agreement with the reported molecular weight of renin of human (Murakami et al., 1977; Slater & Haber, 1978; Rubin, 1972) and rat (Inagami et al., 1977; Lauritzen et al., 1976). Thus, it seems uniform in terms of the molecular weight of )oco renin throughout various mammalian species. The isoelectric point of dog renal renin was 5.46 + 0.02, indicating that renin is an acidic protein. However, significant species difference in the isoelectric point of renin, about I pH unit was noticed. Consistent with I I our observations, different isoelectric points of renin 3 6 9 in different species have been reported: human, 5.25 pH (Murakami et al., 1977); pig, 5.2 (Inagami & MuraFig. 3. pH-dependence of dog renal renin activity. Renin kami, 1977); rat, 5.4 (Lauritzen et al., 1976). samples were treated with Tris or maleate buffer pH There are several recent reports indicating the existranged 1.0 to 12.0 at 4°C. Each point represents mean ence of inactive renin, which can be activated by acid + SEM of six experiments. (Boyd, 1974; Day et al., 1974; De Senarclens et al., 1977; Inagami et al., 1977). However, no activation was observed at pH 3 in our studies. Rather, a signifi33% of that at pH 3-10 after 3 hr and completely cant inactivation of renin was observed at pH 3, in inactivated by 24 hr. At pH values lower than 2 or contrast with other recent reports (Levine et al., 1978; higher than 11, renin was completely inactivated only Rubin, 1972). Judged from the molecular weight, iso1 hr after the preincubation. At no pH was there any electric point and electrophoretic mobility of renin, evidence of acid activation of an inactive form of only one form of renin, all of which is enzymatically active, was present in the extracts. Consequently, it renin. appears that renin extracted by this procedure is a quantitative measure of its activity in the kidney. DISCUSSION In summary, a method of renal renin extraction A standard procedure for the extraction of renal was described, which seems adequate to determine renin is not yet available, and little information was renal renin activity under various physiological conprovided about the degree of extraction of renin. For ditions. The extracted renin was uniform in terms of example, variable g forces are used in centrifugation. molecular weight, electrophoretic mobility and isoHowever, it is now evident that extraction procedures electric point. Most importantly, the extracted renin can greatly affect the amount of renin obtained from a was enzymatically all active. The molecular weight of kidney. Renal renin activity is reported to vary over three species was about the same, but there was 10-fold by different authors (Haas et al., 1954; Mori- notable difference in the isoelectric point among moto et al., 1972; Waldh~iusl et al., 1970). The pro- species. The exposure of renin to acid caused a cedure used in this study appears to be an appro- marked change in its enzymatic activity and isoelecpriate one for determining renal renin activity on the tric point.
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332
CHUN SIK PARK
Acknowledoements--The author thanks the China Medical Board of New York and National Kidney Foundation for the fellowship awarded to C. S. Park. This work was supported by NIH grant AM05077 and HL 18575.
REFERENCES BOYD G. W. (1974) A protein-bound form of Porcine renal renin. Circulation. Res. 35, 426-438. DAY R. P., LEUTSCHER J. A. & GONZALES C. M. (1975) Occurrence of big renin in human plasma, amniotic fluid and kidney extracts. J. clin. Endocr. Metab. 40, 1078-1084. DE SENARCLENS C. F., PRICAM C. E., BANICHAHI F. D. & VALLOTTON M. B. (1977) Renin synthesis, storage, and release in the rat: A morphological and biochemical study. Kidney Int. 11, 161-169. FRAY J. C. S. (1978) Mechanism of increased renin release during sodium depletion. 234, F376-F380. GABRIEL O. (1971) Analytical disc gel electrophoresis. In Methods in Enzymology. Vol. 22, pp. 565-578. Academic Press, New York. GRASS D. M. & BARAJASL. (1975) The large-scale isolation of renin-containing granules from rabbit renal cortex by zonal centrifugation. J. lab. clin. Med. 85, 467-477. GRASS F., SCHAECHTEL1N G., BRUNNER H. & PETERS G. (1964) The role of the renin-angiotensin system in blood pressure regulation and kidney function. Can. Med. Ass. J. 90, 258-262. HAAS E., LAMFORMH. V. & GOLDBLATTH. (1954) A simple method for the extraction and partial purification of renin. Archs Biochem. Biophys. 48, 256-260. HABER E., KOERNER T., PAGE L. B., KLIMAN B. & PURNODE A. (1969) Application of a radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects. J. clin. Endocr. Metab. 29, 1349-1955. HARTROFT P. M. (1963) Juxtaglomerular cells. Circulation Res. 12, 525-534. INAGAMIT., HIRASE S., MURAKAMIK. & MATOBAT. (1977) Native form of renin in the kidney. J. biol. Chem. 252, 7733-7737. INAGAMI T. & MURAKAMIK. (1977) Pure renin. Isolation from hog kidney and characterization. J. biol. Chem. 252, 2978-2983. KOTCHEN T. A., MAULL K. I., LUKE R., REES D. & FLAMENBAUMW. (1974) Effect of acute and chronic calcium administration on plasma renin. J. clin. Invest. 54, 1279-1286.
LAURITZEN M., DANISGARRDJ. J., RUBIN I. & LAURITZEN E. (1976) A comparison of the properties of renin isolated from pig and rat kidney. Biochem. J. 155, 317-323. LEVINE M., LENTZ K. E., KAHN J. R., DORER F. E. & SKEGGS L. T. (1978) Studies on high molecular weight renin from hog kidney. Circulation. Res. 42, 368-375. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-277. MIKSCHE L. W., MIKSCHE U. & GROSS F. (1970) Effect of sodium restriction on renal hypertension and on renin activity in the rat. Circulation Res. 27, 973 984. MORIMOTO S., YAMAMOTOK. & VEDA J. (1972) Isolation of renin granules from the dog kidney cortex. J. appl. Physiol. 33, 306-331. MURAKAM~K., INAGAMIT. & HAAS E. (1977) Partial purification of human renin. Circulation Res. (Suppl II) 41, 11-4-I1-7. MULLER-SUUR R., GUTSCHE H.-U., SAMWER K. F.. OELKERS W. & HIERHOLZER K. (1975) Tubuloglomerular feedback in rat kidneys of different renin contents. Pfliigers Arch. 9es Physiol. 359, 33-56. NUSTAD K. & RUBIN I. (1970) Subcellular localization of renin and kininogenase in the rat kidney. Br. J. Pharmac. 40, 326-333. OGINO K., MATSUNAGAM., SAITO N., KIRA J. & TAKAYASU M. Renin and acid adenosine triphosphatase as lysosomal enzymes. Japan Circ. J. 31, 1-8. PARK C. S., MALVIN R. L., MURRAY R. D. & CHO K. W. (1978) Renin secretion as a function of renal renin content in dogs. Am. J. Physiol. 234, F506 F509. RUmN I. (1972) Purification of hog renin. Properties of purified hog renin. Scand. J. clin. lab. Invest. 29, 51 58. SILVERMANA.-J. & BARAJASL. (1974) Effect of reserpine on the juxtaglomerular granular cells and renal nerves. Lab. Invest. 30, 723-731. SLATER E. E. & HABERE. (1978) A large form of renin from normal human kidney. J. clin. Endocr. Metab. 47, 105-109. TOBIAN L., JANECK J. & TOMBOULIANA. (1959) Correlation between granulation of juxtaglomerular cells and extractable renin in rats with experimental hypertension. Proc. Soc. exp. Biol. Med. 100, 9446. WALDH,~USL W., LUCAS C. P., CONN J. W., LUTZ J. H. & COHEN E. L. (1970) Studies on the partial isolation of human renin. Biochim. biophys. Acta 221,536-548. WRIGLEY C. W. (1971) Gel electrofocusing. In Methods in Enzymology Vol. 22, pp. 559-564. Academic Press, New York.
0305-0491/81/0201-0329$02.00/0
© Pergamon Press Ltd 1981. Printed in Great Britain
RENAL RENIN EXTRACTION AND BIOCHEMICAL CHARACTERIZATION CHUN SIK PARZ Division of Nephrology, Department of Medicine, School of Mcdicinc, M-023, Univcrsity of California, San Diego, La Jolla, CA 92093, U.S.A.
(Received 15 May 1980) A h s t r a c t - - l . A method for extraction of renal renin with high and reproducible yield was described.
2. About 90~o of the total renal renin activity was found in the supernatant at 105,000 g for 60 min. 3. The molecular weight of extracted renin of 3 different species was about 45,000 dalton. 4. The isoelectric point of renal renin of pig, dog and mouse was 5.10, 5.46 and 5.94, respectively. 5. Acid treatment of renal renin resulted in the change of its isoelectric point and inactivation of enzymatic activity.
INTROD UCTION A remarkable correlation between the granulation of juxtaglomerular (JG) cells, the amount of extractable renal renin and plasma renin activity was observed in a variety of experimental conditions (Gross et al., 1964; Hartroft, 1963; Miksche et al., 1970; Tobian et al., 1959). It was reported that a quantitative correlation exists between renal activity and the rate of renin secretion (Fray, 1978; Park et al., 1978). All these observations indicate that the renal renin activity is one among the important determinants of renin secretion. In spite of the importance of renin in fluid and water balance, there is no standard procedure for the determination of renal renin activity. As an index of this, the reported values of renal renin activity of rats fed normal salt diet varies from 4 to 4000 ng Ang I/rag kidney.hr, depending upon the different procedures (De Senarclens et al., 1977; Kotchen et al., 1974; Miiller-Suur et al., 1975; Silverman et al., 1974). The purpose of this investigation was threefold. The first was to develop a method for the quantitative extraction and determination of dog renal renin. The second was to study some biochemical characteristics of canine renal renin, which have not yet been studied. The biochemical characteristics of renal renin of other species were also determined and compared to those of dog renin. The third was to examine whether the extracted dog renin is all active form or not. MATERIALS AND METHODS
Extraction of renal renin The detailed procedures for the preparation of renal cortical slices were described previously (Park et al., 1978). Briefly, slices of renal cortex (about 300 mg) was homogenized in ice-cold 0.25 M sucrose using an all glass tissue homogenizer. After homogenation, subcellular fractions were obtained by centrifuging the homogenate at 700 g for 10 min (Nuclear Fraction as well as cell debris), at 10,000g for 10 min (Mitrochondrial Fraction), and at 105,000g for 60 min (Microsomal Fraction and its supernatant). All subcellular fractions and the supernatant (105,000 g) as well as the remaining homogenate were frozen at -20°C until 329
assay of renin activity. The distribution of renal renin in each subcellular fraction was determined in 16 renal cortical samples from two dogs.
Molecular weight of renin (dog, pig and rat) The molecular weight of renin was estimated by gel filtration of 2ml of the 105,000g supernatant using a Sephadex G-100 column (2.5 x 45 cm). The column was pre-equilibrated with 50mM sodium phosphate buffer, pH 7.4 and calibrated by determining the averaged elution volume (Kay) of each of the following molecular weight standards (Pharmacia Kit); ribonuclease A (13,700mol. wt), chymotrypsinogen A (25,000mol. wt), ovalbumin (45,000 mol. wt), bovine serum albumin (68,000 mol. wt), aldolase (158,000 mol. wt)and blue dextran 2000. The relative concentration of blue dextran in the column eluate was determined spectrophotometrically at 620 nm and the molecular maker proteins were assayed by the method of Lowry et al. (1951). pH-stability of dog renal renin Fifty microliters of the supernatant of dog renal cortical homogenate from 4 dogs was treated at 4°C with 1 ml of maleate buffer (0.2 M) in which the pH ranged from 1 to 12, (adjusted either with HC1 or NaOH). After 1, 2, 3 (each 1 sample) and 24 hr (3 samPles ) following the treatment at each pH, 25 and 50/~l of each sample was incubated with l ml renin substrate (pH 6.0) for the determination of renin activity. Gel electrophoresis and isoelectric focusing of renin Discontinuous polyacrylamide gel electrophoresis was carried out in glass tubes (5 x 65 mm) containing separating gel (7.5~ acrylamide at pH 8.8) and stacking gel (2.5~ acrylamide at pH 6.8) by the method of Gabriel (1971). The supernatant (105,000 g) was overlayered on the top of the stacking gel and electrophoresis was conducted at 4°C at 3 mA per tube until the tracking dye, bromophenol blue, moved to within 5 mm of the top of the separating gel. The isoelectric point of renin was determined by the method of Wrigley (1971) on a 7.5~o polyacrylamide containing ampholyte, p H 4 ~ or 3-10 (Ampholine, LKB Instruments, Inc.). Electrophoresis was carried out at a maximum current of 2 mA per tube for 90 min. The gels were cut at 3 mm thickness, which were then macerated in 0.5 ml of double distilled water. Renin activity and pH (at 25°C) were determined in the eluates of each gel slice. Renin activity of renal homogenate and column eluates was determined by incubating renin samples with dog
330
CHUN SIK PARK
renin substrate prepared from 48 hr nephrectomized dogs as described previously (Park et al., 1978). The recovery of angiotensin I (0.5, 1.0, 2, 3, 4 and 5 ng in duplicate) added to 1 ml of renin substrate was determined after incubation at 37°C for 60 min. Adequacy of renin substrate was examined by incubating renin samples for 15, 30 and 60 min (n = 8). Angiotensin I (Ang I) generated after the incubation at 37°C, pH6.0 was measured by the method of Haber et al. (1969), using New England Nuclear Angiotensin I Radioimmunoassay Kit.
Recovery and generation of angiotensin The recovery of Ang I added to the renin substrate in the incubating mixture was 92.1 _+ 6.1~o. The generation of Ang I by incubation of the superoatant of renal homogenate at 105,000g with renin substrate was linear up to 60 min. The incubation conditions were made to consume no more than 20~ of renin substrate by diluting renin samples.
Table 1. Percentage distribution of renal renin in the subcellular fractions of dog renal cortical homogenate
Fraction
% of total* (Mean + SEM)
I Nuclear
2.4 + 0.3
2 Mitochondrial
1.8 + 0.2
3 Microsomal
1.5 + 0.i
4 Supernatant
87.9 + 2.2
5 Sum (Recovery)
93.5 + 2.0
* Distribution expressed as percentage of total renin activity measured in the homogenate (n = 16 experiments).
RESULTS
Cellular distribution Distribution of renin in the subcellular fractions are shown in Table 1. The supematant contained the highest renin activity, accounting for 87.9 + 2.2~ of the total. The overall recovery of renin from the three particulate fractions and the soluble supernatant was 93.5 + 2.0% of the total activity measured in the cortical homogenate.
Molecular weight of renin From Fig. 1 it can be seen that molecular weight of renal renin of dog, pig and rat are about the size of ovalbumin. The estimated molecular weight of renin for each species (n = 3) are as follows: dog, 43,500 + 400; pig, 46,000 + 500 and rat, 48,500 + 700.
lsoelectric point of renal renin A typical experiment of renin activity and pH of each gel slice on gel electrofocusing of dog renin is shown in Fig. 2. A sharp peak of renin activity was observed in one gel slice, the pH of which was taken as the isoelectric point of renin. In the same way the isoelectric point of mouse and pig renin were determined. The isoelectric points of dog, pig and mouse
renin was 5.46 + 0.02, 5.19 + 0.02 and 5.94 + 0.03, respectively (n = i0). The isoelectric point of dog renin was significantly different from that of pig and mouse renin (P < 0.001). The isoelectric point of pig renin purified by the method of Haas et al. (1954) up to step 5 (Haas et al., 1954), was 4.65 _ 0.05 (n = 5). The difference in the isoelectric points of fresh and commercial pig renin is statistically significant (P < 0.001).
Electrophoretic mobility of renin Only a single peak of renin activity was revealed on polyacrylamide gel at mobility of about 0.5 relative to tracking dye bromophenol blue. A single peal of renin activity was also observed with pig and mouse renin (results are not shown).
The pH stability of dog renin Renin activity following preincubation at different pHs is shown in Fig. 3. In the pH range of 3-10, renin activity was stable up to 24 hr. However at pH values above and below this range, renin was unstable and rapidly inactivated after 3 hr of incubation at pH 2 or 11. Renin activity was decreased to approximately
0.6_-~ease A 0.4-
~ ChymotrypsinogenA
[
DOG~
0.2
P I o;
v°lburnin
PIG~ " ~ J
%./\ '
;
' '
,'o
Fig. 1. The molecular weight of dog, pig and rat renin (n = 3 for each species) was estimated using SephadexG-100.
Biochemistry of renin pH
OoH
6.C
o Renin Activity
331
following three grounds. First, values of SEM were very small indicating a consistent degree of renin extraction. For instance, renal renin activity deterII • mined on 8 batches of renal cortex from 1 dog was 76.8 + 3.9 ng Ang I/mg tissue.hr. Second, over 90% of the total renal renin activity was found in the soluble fraction, which indicates that the degree of 5.( homogenation was sufficient to extract most, if not all of the renin. Third, the extraction was sensitive Ienough to detect changes in the renal renin activity as small as 12% in dogs (Park et al., 1978). z It has been reported that renin activity is mainly localized in the mitochondrial (Morimoto et al., 1972; O 4.0 4 7 I0 13 Nustad et al., 1970), lysosomal (Ogino et al., 1967) or GEL SLICE NUMBER between two subcellular fractions (Grass & Barajas, Fig. 2. A representative experiment showing the isoelectric 1975). Upon increasing the degree of homogenation, point of dog renal renin. however, most renin activity was shifted from the particulate fractions to the supematant (Morimoto et al., 1972; Nustad & Rubin, 1970). Thus, the extensive homogenation used in our studies may account for the fact that most of renin activity was found in the 3000 supematant. This result is consistent with a recent report by Inagami et al. (1977). They observed that more than 90% of hog and rat renal renin activity was in the supernatant fraction. The molecular weight of dog renin, as well as rat g and pig renin, was about 43,500 daltons (see Fig. 1). ~2ooo This value is in close agreement with the reported molecular weight of renin of human (Murakami et al., 1977; Slater & Haber, 1978; Rubin, 1972) and rat (Inagami et al., 1977; Lauritzen et al., 1976). Thus, it seems uniform in terms of the molecular weight of )oco renin throughout various mammalian species. The isoelectric point of dog renal renin was 5.46 + 0.02, indicating that renin is an acidic protein. However, significant species difference in the isoelectric point of renin, about I pH unit was noticed. Consistent with I I our observations, different isoelectric points of renin 3 6 9 in different species have been reported: human, 5.25 pH (Murakami et al., 1977); pig, 5.2 (Inagami & MuraFig. 3. pH-dependence of dog renal renin activity. Renin kami, 1977); rat, 5.4 (Lauritzen et al., 1976). samples were treated with Tris or maleate buffer pH There are several recent reports indicating the existranged 1.0 to 12.0 at 4°C. Each point represents mean ence of inactive renin, which can be activated by acid + SEM of six experiments. (Boyd, 1974; Day et al., 1974; De Senarclens et al., 1977; Inagami et al., 1977). However, no activation was observed at pH 3 in our studies. Rather, a signifi33% of that at pH 3-10 after 3 hr and completely cant inactivation of renin was observed at pH 3, in inactivated by 24 hr. At pH values lower than 2 or contrast with other recent reports (Levine et al., 1978; higher than 11, renin was completely inactivated only Rubin, 1972). Judged from the molecular weight, iso1 hr after the preincubation. At no pH was there any electric point and electrophoretic mobility of renin, evidence of acid activation of an inactive form of only one form of renin, all of which is enzymatically active, was present in the extracts. Consequently, it renin. appears that renin extracted by this procedure is a quantitative measure of its activity in the kidney. DISCUSSION In summary, a method of renal renin extraction A standard procedure for the extraction of renal was described, which seems adequate to determine renin is not yet available, and little information was renal renin activity under various physiological conprovided about the degree of extraction of renin. For ditions. The extracted renin was uniform in terms of example, variable g forces are used in centrifugation. molecular weight, electrophoretic mobility and isoHowever, it is now evident that extraction procedures electric point. Most importantly, the extracted renin can greatly affect the amount of renin obtained from a was enzymatically all active. The molecular weight of kidney. Renal renin activity is reported to vary over three species was about the same, but there was 10-fold by different authors (Haas et al., 1954; Mori- notable difference in the isoelectric point among moto et al., 1972; Waldh~iusl et al., 1970). The pro- species. The exposure of renin to acid caused a cedure used in this study appears to be an appro- marked change in its enzymatic activity and isoelecpriate one for determining renal renin activity on the tric point.
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CHUN SIK PARK
Acknowledoements--The author thanks the China Medical Board of New York and National Kidney Foundation for the fellowship awarded to C. S. Park. This work was supported by NIH grant AM05077 and HL 18575.
REFERENCES BOYD G. W. (1974) A protein-bound form of Porcine renal renin. Circulation. Res. 35, 426-438. DAY R. P., LEUTSCHER J. A. & GONZALES C. M. (1975) Occurrence of big renin in human plasma, amniotic fluid and kidney extracts. J. clin. Endocr. Metab. 40, 1078-1084. DE SENARCLENS C. F., PRICAM C. E., BANICHAHI F. D. & VALLOTTON M. B. (1977) Renin synthesis, storage, and release in the rat: A morphological and biochemical study. Kidney Int. 11, 161-169. FRAY J. C. S. (1978) Mechanism of increased renin release during sodium depletion. 234, F376-F380. GABRIEL O. (1971) Analytical disc gel electrophoresis. In Methods in Enzymology. Vol. 22, pp. 565-578. Academic Press, New York. GRASS D. M. & BARAJASL. (1975) The large-scale isolation of renin-containing granules from rabbit renal cortex by zonal centrifugation. J. lab. clin. Med. 85, 467-477. GRASS F., SCHAECHTEL1N G., BRUNNER H. & PETERS G. (1964) The role of the renin-angiotensin system in blood pressure regulation and kidney function. Can. Med. Ass. J. 90, 258-262. HAAS E., LAMFORMH. V. & GOLDBLATTH. (1954) A simple method for the extraction and partial purification of renin. Archs Biochem. Biophys. 48, 256-260. HABER E., KOERNER T., PAGE L. B., KLIMAN B. & PURNODE A. (1969) Application of a radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects. J. clin. Endocr. Metab. 29, 1349-1955. HARTROFT P. M. (1963) Juxtaglomerular cells. Circulation Res. 12, 525-534. INAGAMIT., HIRASE S., MURAKAMIK. & MATOBAT. (1977) Native form of renin in the kidney. J. biol. Chem. 252, 7733-7737. INAGAMI T. & MURAKAMIK. (1977) Pure renin. Isolation from hog kidney and characterization. J. biol. Chem. 252, 2978-2983. KOTCHEN T. A., MAULL K. I., LUKE R., REES D. & FLAMENBAUMW. (1974) Effect of acute and chronic calcium administration on plasma renin. J. clin. Invest. 54, 1279-1286.
LAURITZEN M., DANISGARRDJ. J., RUBIN I. & LAURITZEN E. (1976) A comparison of the properties of renin isolated from pig and rat kidney. Biochem. J. 155, 317-323. LEVINE M., LENTZ K. E., KAHN J. R., DORER F. E. & SKEGGS L. T. (1978) Studies on high molecular weight renin from hog kidney. Circulation. Res. 42, 368-375. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-277. MIKSCHE L. W., MIKSCHE U. & GROSS F. (1970) Effect of sodium restriction on renal hypertension and on renin activity in the rat. Circulation Res. 27, 973 984. MORIMOTO S., YAMAMOTOK. & VEDA J. (1972) Isolation of renin granules from the dog kidney cortex. J. appl. Physiol. 33, 306-331. MURAKAM~K., INAGAMIT. & HAAS E. (1977) Partial purification of human renin. Circulation Res. (Suppl II) 41, 11-4-I1-7. MULLER-SUUR R., GUTSCHE H.-U., SAMWER K. F.. OELKERS W. & HIERHOLZER K. (1975) Tubuloglomerular feedback in rat kidneys of different renin contents. Pfliigers Arch. 9es Physiol. 359, 33-56. NUSTAD K. & RUBIN I. (1970) Subcellular localization of renin and kininogenase in the rat kidney. Br. J. Pharmac. 40, 326-333. OGINO K., MATSUNAGAM., SAITO N., KIRA J. & TAKAYASU M. Renin and acid adenosine triphosphatase as lysosomal enzymes. Japan Circ. J. 31, 1-8. PARK C. S., MALVIN R. L., MURRAY R. D. & CHO K. W. (1978) Renin secretion as a function of renal renin content in dogs. Am. J. Physiol. 234, F506 F509. RUmN I. (1972) Purification of hog renin. Properties of purified hog renin. Scand. J. clin. lab. Invest. 29, 51 58. SILVERMANA.-J. & BARAJASL. (1974) Effect of reserpine on the juxtaglomerular granular cells and renal nerves. Lab. Invest. 30, 723-731. SLATER E. E. & HABERE. (1978) A large form of renin from normal human kidney. J. clin. Endocr. Metab. 47, 105-109. TOBIAN L., JANECK J. & TOMBOULIANA. (1959) Correlation between granulation of juxtaglomerular cells and extractable renin in rats with experimental hypertension. Proc. Soc. exp. Biol. Med. 100, 9446. WALDH,~USL W., LUCAS C. P., CONN J. W., LUTZ J. H. & COHEN E. L. (1970) Studies on the partial isolation of human renin. Biochim. biophys. Acta 221,536-548. WRIGLEY C. W. (1971) Gel electrofocusing. In Methods in Enzymology Vol. 22, pp. 559-564. Academic Press, New York.