/ . Biochem., 80, 1277-1285 (1976)
Dog Renal Kallikrein: Purification and Some Properties Chiaki MORIWAKI,* Kyosuke MIYAZAKI,* Yoshifumi MATSUDA,* Hiroshi MORIYA,* Yukio FUJIMOTO,*-1 and Hiroshi UEKI** •Laboratory of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Shinjuku-ku, Tokyo 162, and "Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862 Received for publication, April 3, 1976
The contents of kallikrein [EC 3.4.21.8] in the kidneys of various animals were estimated and the activity was found to be most potent in dogs. The dog renal kallikrein (DRK) was located mainly in the kidney cortex. Following the activation of a dog kidney cortex homogenate with acetone, kallikrein was purified about 2,000-fold with an overall yield of 18 % by diethylaminoethyl (DEAE)-cellulose adsorption, acetone fractionation, and chromatography on Sephadex G-75 and DEAE-Sephadex A-50. The final purified preparation of dog renal kallikrein had a vasodilator activity of 65.5 KU per AIM, and appeared to be homogeneous both in disc electrophoresis and ultracentrifugal analysis. Its molecular weight was estimated to be approximately 3.8x10* from the sedimentation coefficient obtained by ultracentrifugation, and by Sephadex gel filtration. However, isoelectric fractionation of the purified DRK preparation gave three isoelectric point, 3.9, 4.1, and 4.3. The DRK had an optimum pH of about 8.6 and was stable at pH 8. This enzyme was hardly inhibited by Trasylol, soybean trypsin inhibitor, ovomucoid trypsin inhibitor or potato kallikrein inhibitors. These properties were compared with those of kallikrein from other sources; DRK appeared to be similar to urinary kallikrein.
It has been suggested by recent investigations that the kallikrein [EC 3.4.21.8]-kinin system seems to relate to the renal function (7-7). The infusion of kinin into the renal artery of dog {1-4) and man (5) causes an increase of renal sodium excretion, , . , Present address: Department of Biochemistry, Hok, .. , . . „. • io• un kaido Institute of Pharmaceutical Sciences, Otaru-shi, Hokkaido 047-0? Abbreviations: *BAEE, N-a-benzoyl-L-arginine ethyl ester; TAME, N-a-tosyl-L-arginine methyl ester; SBTI, soybean trypsin inhibitor; OTI, ovomucoid trypsin inhibitor. Vol. 80, No. 6, 1976 1277 1
and an increase in the excretion of urinary kallikrein on administration on mineralocorticoid(s) (6, 7) or saline load (7) has been also reported. Not much attention has been paid to renal kallikrein itself, although many investigators have studied urinary kallikrein. The origin of urinary kallikrein was once as, , , , sumed to be the pancreas, but that was later con„ ' . .. sidered unlikely (8). Since small kallikrein activity was demonstrated in kidney extract (9), the kidney was considered as a possible origin of urinary kallikrein (70-72). However, another report (73) is incompatible with this view and the relationship
1278
C. MORIWAKI, K. MIYAZAKI, Y. MATSUDA, H. MORIYA, Y. FUJIMOTO, and H. UEKI
between renal and urinary kallikrein is not yet clear. This confusion may be due to the use of relatively crude preparations of kallikrein in these investigations and the lack of strict comparability among these kallikreins. The purpose of the present study is the purification of kallikrein from dog kidney (DRK) and comparison of the properties of DRK with those of other purified kallikreins.
thawing procedure was repeated several times. Deionized water was added to the gruel (2 ml per 1 g of original wet tissue weight) and it was shaken vigorously, the allowed to stand for 18-24 hr at 4°. In the case of the acetone activation experiment, deionized water was replaced by various concentrations of acetone solution; the procedure was as above. The homogenate was centrifuged at 7,000 x g for 30 min, and the supernatant was dialyzed for 2 days against deionized water. The EXPERIMENTS AND RESULTS precipitate formed during dialysis was removed by Assay of Kallikrein Activities—The vasodilator centrifugation. The vasodilator activity of the activity was determined as described previously supernatant was assayed and the activities in the {14,15) by measuring the blood flow increase at the whole, the medulla and the cortex of the kidneys of femoral artery of a dog. The activity is expressed various animals are shown in Table I. In general, in terms of kallikrein units: KU. The esterolytic the kidney cortex contained more kallikrein than the activity was assayed by colorimetric measurement medulla, and dog kidney was especially active. with chromotropic acid using TAME (Protein The perfused kidney did not show a marked differResearch Foundation, Osaka) as a substrate (75). ence in activity compared with the unperfused one, The activity is expressed in terms of esterolytic units so the following experiments were performed using (EU) equal to /*moles of TAME hydrolyzed per unperfused kidneys. On activation with acetone min at 30° in 0.05 M Tris-HCl buffer, pH 8.0. The (10, 20, 30, and 60%, V/V), the maximum activity kinin-forming activity was estimated with bovine was found at 20%, amounting to approximately 3 kininogen as a substrate, and the contractile re- times that of the water extract, so the acetone consponse of an isolated guinea pig ileum was recorded. centration for activation was fixed at 20%. As a standard, synthetic bradykinin (BRS 640, Purification of DRK—All steps were carried Sandoz Pharmaceuticals, Switzerland) was em- out at 0-4°. ployed ; the activity is expressed in terms of bradyStep 1. Activation by acetone: From about kinin equivalent, as described previously (75). 30 kidneys, 280 g of kidney cortex was obtained, Determination of Protein Concentration—Pro- and the homogenate was activated with acetone as tein concentration was determined spectrophoto- described above. The activity was 0.03 KU and metrically by measuring the absorbance at 280 nm, 0.006 EU per / W using a Hitachi spectrophotometer, model 124 with a cell of 1 cm light path, assuming tentatively that TABLE I. Kallikrein contents in the kidneys of various animals. The vasodilator activity was determined withthe value of £ t ^* = 10.0. out any activation treatment of the tissue homogenates. Chemicals—All chemicals used in this investigation were of analytical or reagent grade. Activity (KU per gram wet weight) Preliminary Experiments for the Purification of Species — Cortex Medulla Whole DRK—Healthy adult dogs of either sex, weighing 8-15 kg, were killed by bleeding and the kidneys — Dog 1.4-2.8 0.06-0.16 were removed immediately. Subsequently, the — Cow 0.25-0.32 1.1-1.7 kidneys were washed thoroughly or one kidney of — Rabbit 1.4 0.9 each pair was psrfused with ice-cold saline for 3-4 Rat 0.4 0.5-0.8 0.3 hr to remove as much blood as possible. The Hog 0.2-0.4 0.3 — kidneys of cows, horses, and hogs were obtained from a slaughterhouse, and those of rabbits, guinea Guinea pig — — 0.3 pigs, and rats (Donryu strain) were also employed. Horse — 0.1 0.08 The cortex and medulla were separated with scisHuman 0.1 0.12 0.07 sors. The tissues were weighed, crushed with sea sand, frozen overnight, and thawed. This freeze- —, not examined. J. Biochem.
1279
DOG RENAL KALLIKREIN
Step 2. DEAE-cellulose adsorption: Four liters of deionized water was added to the homogenate. After adjusting the pH to 7.0-7.5, 20 g of DEAE-cellulosc (0.89 meq/g, Brown Co., U.S.A.) was added to the solution as described previously (16) and packed in a column (3x35 cm) after adsorption for 2 hr. The initial eluate with 0.05 M Tris-HCl buffer had no vasodilator activity. On the other hand, the eluate with 0.5 M buffer possessed the greatest vasodilator activity (Table IT). However, this eluate gave only 10% recovery of the esterolytic activity. This suggested that there was considerable contamination by TAME esterases other than kaUikrein in the kidney tissue. Step 3. Fractionation with acetone: The active fraction eluted with 0.5 M buffer was dialyzed for 1 day against tap water and for 1 more day against deionized water. The contents of the dialysis sac were diluted with deionized water to make a final protein concentration of approximately 10/lj80 per ml, and the diluted solution was adjusted to pH 7.0. Acetone was added to make a final concentration of 50% (V/V) and the precipitate formed was centrifuged off at 15,000 xg for 30 min. Cold acetone was again added to the supernatant to 80% (V/V) and the precipitate was collected by centrifugation. About 9-fold purification was achieved by this procedure with 84% recovery of the vasodilator activity.
Step 4. Sephadex G-75 gel filtration: The acetone powder of the above precipitate was dissolved and filtrated through a Sephadex G-75 column (Pharmacia AB, Sweden), as shown in Fig. 1. Though the esterolytic activity was distributed in several fractions, the vasodilator activity formed a single peak (fractions 27-36) with 58 % recovery. Step 5. DEAE-Sephadex A-50 chromatography: The pooled fraction of DRK was dialyzed overnight against deionized water and concentrated to approximately 5 ml as described previously (16). This concentrated solution was applied to a DEAE-Sephadex A-50 column (3.5 ±0.5 meq/g, Pharmacia AB, Sweden). As shown in Fig. 2, most of the activity was eluted and these fractions (tubes 52-59) were combined. Step 6. Sephadex G-100 gel filtration: The active fraction was dialyzed and concentrated as mentioned in step 5, and final purification was performed on a Sephadex G-100 column, as shown in Fig. 3. The overall results of these purification steps are summarized in Table II. The purified DRK preparation showed an activity of 65.5 KU per Ats0. This value is about 2,200 times higher than that of this initial homogenate, but the purification factor for the esterolytic activity was only 190. Most of the non-kallikrein TAME esterase(s) was 0.1.
1.5
0.1
/V/
JL \ r \ ''
1.0
20 •
15
i °'2
10
§ 0,1
5
0 0
20
10
60
80
3
/
',
0,05
0.05
i
0,5
0,3
S
I
0.1
_
20
/
.'
10
60
80
100
100 FRACTION MffBER (3.7 ML/TUBE)
FRACTION HS1BER (I.ZHL/TUBE)
Fig. 1. Gel filtration of the acetone (50-80%) precipitate on Sephadex G-75. About 500 mg of the dried powder was dissolved in 12 ml of 0.02 M acetic acidammonium acetate buffer (pH 7.4), and applied to a Sephadex G-75 column (2.5x90 cm). Elution was carried out with the same buffer at a flow rate of 10 ml per hr, and fractions of 4.2 ml were collected. • , vasodilator activity; O, esterolytic activity; , absorbancy at 280 nm. Vol. 80, No. 6, 1976
Fig. 2. DEAE-Sephadex A-50 column chromatography following gel filtration on Sephadex G-75. The concentrated active fraction (total Aua = 306) was applied to a DEAE-Sephadex column (1.5x30 cm) previously equilibrated with 0.02 M Tris-HCl buffer (pH. 7.7) containing 0.05 M NaCl. The column was eluated with a linear gradient of 0.05 to 0.4 M NaCl in the above buffer (400 ml), and fractions of 3.7 ml were collected. • , vasodilator activity; o , esterolytic activity; , absorbancy at 280 nm; , concentration of NaCl.
C. MORIWAKI, K. MIYAZAKI, Y. MATSUDA, H. MORIYA, Y. FUJIMOTO, and H. UEKI
1280
0.15 .
.0.15
A n
0.10 •
0,05 -
•0,05
X 0
10
.0.10
15 •
5
20
30
•
10
FRACTION NUWER (3,9 ML/TUBE) Fig. 3. Sephadex G-100 gel filtration following D E A E - S e p h a d e x A-50 column c h r o m a t o g r a p h y . A Sephadex G-100 column ( 1 . 5 x 9 0 c m ) was equilibrated with 0.05 M Tris-HCl buffer ( p H 7.4) containing 0.05 M NaCl. T h e flow rate was 10 ml per hr a n d fractions of 3.9 ml were
collected. • , vasodilator activity; O , esterolytic activity; sorbancy at 280 nm. TABLE II.
, ab-
Summary of the purification of D R K . Recovery
Steps
Homogenate and acetone-activated
Protein
100
(%)
Activity Vasodilator
Activity KU
EU
100
100
0.03
KU/.4m
Esterolytic
(P.F.)«
(1)
EU/,418O
0.006
(P.F.)i
(1)
DEAE-celluIose 0.05 M 0.5 M
5.7
0
12
0.0
13.5
86
10
0.19
0.013 (6)
0.004
(0
Acetone fractionation 0-50%
4.6
2
3
0.02
50-80%
1.5
84
7
1.7
(60)
0.030
(5)
0.1
58
2.3
17.7
(600)
0.139
(20)
Sephadex G-75
0.005
DEAE-Sephadex A-50
0.02
34
1.6
56.6
(1900)
0.477
(80)
Sephadex G-100
0.01
18
1.4
65.5
(2200)
1.124
(190)
* P.F. : purification factor.
removed by the DEAE-cellulose absorption procedure. The kinin-releasing activity of the purified DRK preparation was determined as 2.5 ft% bradykinin equivalents per min psr At30. Its caseinolytic activity (17) was negligible. Criteria for the Homogeneity of Purified DRK—
Disc electrophoresis of DRK was carried out by the
method of Orstein (18) and Davis (19) in 7.5% (W/V) polyacrylamide gel with 0.05 M Tris-glycinate buffer at pH 8.6 and the gel was stained with Coomassie Brilliant Blue. The preparation gave a single band, with a very faint sub-band. Sedimentation equilibrium analysis (Fig. 5-B) gave a linear function between the logarithm of the fringe disJ. Biochem.
DOG RENAL KALLIKREIN
1281
o
0.6 -
I
1 oo°°° o o
0,2
pi t
1 /
/
1 ' ^•"'t
°
10
5o
1-
o^A°
1
{
.1
1 oO°°°°°°°
I
0.1 .
6-
o
Pi 3.9
20
pi 1.3 1
I
5 3
A 2
u1 \ 30
•
1
1-
N 10
50
FRACTION NUMBER ( 2 HL/TUBE )
Fig. 4. Isoelectric fractionation of DRK. Isoelectric focusing was performed with carrier ampholyte (pH 3-5) on LKB 8100 equipment. Electrophoresis was carried out at 500 volts and 6-8° for 40 hr. O, pH; , absorbancy at 280 nm; • , vasodilator activity. placement of DRK and the distance from the center of the rotor (R), suggesting the homogeneity of this preparation. However, isoelectric fractionation gave three active peaks with pi's of 3.9, 4.1, and 4.3. This result indicates that DRK may consist of multiple form. Amino Acid Analysis—The purified DRK preparation (158 fig) was hydrolyzed at 110° for 32 hr in an evacuated, sealed tube with redistilled 6 N HC1, and subsequently analyzed in a Hitachi KLA3B amino acid analyser, according to the modified method of Spackman et al. (20). The recovery of amino acids was 66 %. The results of the analysis are shown in Table III. Estimation of the Molecular Weight—The molecular weight of DRK and dog urinary kallikrein were estimated to be 3.8 xlO4 and 2.6 xlO4, respectively, by Sephadex G-100 gel filtration (Fig. 5-A). Furthermore, an estimate of the molecular weight by ultracentrifugation (sedimentation equilibrium method) was made using a Spinco model E ultracentrifuge as described previously (27). The molecular weight calculated from Fig. 5-B was 3.8x10*. Some Properties of DRK in Comparison with Those of Kallikreins from Other Sources—Kallikreins: Dog urinary kallikrein was prepared by the method of Matsuda et al. (unpublished). Its vasodilator and esterolytic activities per AIM were Vol. 80, No. 6, 1976
TABLE III. Amino acid composition of the purifisd DRK. Aminr> npiri
Lys His Arg Asp Thrb Serb GIu Pro Gly Ala Cys Val Met lie Leu Tyr Phe Trp c
Mole (%)
Residues1 per molecule
Nearest integer
4.17 2.57 2.89 10.37 4.97 6.42 11.23 5.78 9.52 6.31 5.67 6.63 1.50 4.28 6.42 3.42 3.00 4.92
13.75 8.46 9.51 33.80 16.22 21.16 37.03 19.05 33.01 21.15 18.70 21.86 5.00 14.62 21.16 11.28 9.87 16.22
14 8- 9 9-10 34 16 21 37 19 33 21 19 22 5 15 21 11 10 16
» Calculated on the basis of a molecular weight of 3.8x10*. b The hydrolytic losses of threonine and serine were corrected using factors of 5.0% and 14.8%, respectively. « Tryptophan content was determined spectrophotometrically according to Goodwin and Morton (22).
C. MOR1WAKI, K. MIYAZAKI, Y. MATSUDA, H. MORIYA, Y. FUJ1MOTO, and H. UEKI
1282
QA)
2.0
\
V)B)
urinary k 1.5
-
k. C)\
D)
i
n
1
2 3 4 5 10 Molecular We1ght( xlO-4) Fig. 5A: Estimation of the approximate molecular weight of DRK by Sephadex G-100 gel filtration. One mg each of cytochrome c, SBTI, ovalbumin, bovine serum albumin and blue dextran and 0.5 mg each of DRK and partially purified dog urinary kallikrein was dissolved in 1 ml of Tris-HCl buffer (0.1 M, pH 7.7), and applied to a Sephadex G-100 column (1.5x90 cm). The flow rate was maintained at 11 ml per hr. Eluates of each sample were checked for absorbancy at 280 nm for SBTI, ovalbumin and bovine serum albumin, at 410 nm for cytochrome c and at 620 nm for blue dextran. The maximum activity was recorded for the kallikreins. A, Cytochrome c; B, SBTI; C, Ovalbumin; D, Bovine serum albumin.
168 KU and 4.15 EU, respectively. Dog pancreatic killikrein was partially purified by the method of Yamashita et al. (23). Human urinary and hog pancreatic kallikreins were partially purified by the modified methods of Matsuda et al. (24) and Moriya et al. (25); the vasodilator activities of these preparations were 67.7 and 23.25 KU per ^ u o , respectively. Heat stability: D R K and dog urinary kallikrein were heated at 50, 60, and 75° for 10, 30, and 60 min in 0.05 M Tris-HCl buffer at pH 8.0, and the TAME esterolytic activity was determined. The initial activities of DRK and dog urinary kallikrein were 4 and 6.3 x 10"* EU, respectively. DRK was stable to heat treatment, i.e. more than 80 % of the activity was retained after heating at 50° for 60 min, 60° for 30 min or 75° for 10 min and 60% of the activity was retained even after treatment at 75° for 60 min. Dog urinary kallikrein showed similar heat stability, i.e., about 50% of the activity was retained after heating at 75° for 60 min. Human urinary kallikrein showed almost the same heat stability as D R K and dog urinary kallikrein (24).
2.0 •
3
10
l.o
50
51
52
Fig. 5B: Sedimentation equilibrium centrifugation of DRK. The initial protein concentration was 2 mg per 100 ml of 0.1 M phosphate buffer (pH 7.0). The plot was read from an interference photograph of the equilibliu-n distribution at 20,410 rpm and 11° for 192 min.
Fig. 6. The pH profiles of the activities of dog renal, urinary and pancreatic kallikreins. TAME-esterolytic activity was determined at various pH's in 0.04 M Britton-Robinson wide-range buffer. The dog renal, urinary and pancreatic kallikreins had esterolytic activities of 4, 6.3 and 7 x 10"' EU (estimated in 0.05 M TrisHCl buffer, pH 8.0, at 30°), respectively. Each enzyme was incubated with TAME at the indicated pH for 30 min at 30°. Maximum activity was taken as 100 in the figure. O, dog urinary kallikrein; • , dog renal kallikrein; A , dog pancreatic kallikrein.
J. Biochem.
DOG RENAL KALLIKREIN
1283
TABLE IV. Inhibitory effects of various proteinase inhibitors on dog kallikreins. The enzyme solutions were preincubated with Trasylol, trypsin inhibitors of soybean (SBTI) and ovomucoid (OTI), and potato kallikrein inhibitors (PKJ-56 and PKI-64) as described in the text, in a total volume of 0.4 ml. The incubation mixtures (0.1 ml) were assayed on the arterial blood flow in dogs. Inhibition % Inhibitors
Trasylol
SBTI OTI PKJ-56 PKI-64
10 40 200 400 2000 500 1000 500 1000 2 10
KIU
/ig /ig /ig
2 pg 10
Renal (0.8KU)
Urinary (0.8KU)
Pancreatic (2.0KU)
N.D. N.D. N.D. N.D. 22 N.D. N.D. N.D. 20 N.D. N.D. N.D. N.D.
N.D. N.D. N.D. 12 36 N.D. 12 N.D. 15 N.D. N.D. N.D. N.D.
N.D. N.D. 18 56 60 N.D. 20 N.D. N.D. 30 95 10 85
N.D. : not detectable.
Effect of pH on activity: The pH profiles of the activity of a purified preparation of DRK, partially purified dog urinary, and pancreatic kallikreins were compared. The results are shown in Fig. 6. Both DRK and urinary kallikrein showed maximum activity at pH 8.6, but the optimum pH of pancreatic kallikrein was 8.0. Effect of various inhibitors:
The effect of
Trasylol (Bayer AG, West Germany) on the vasodilator activity of DRK and other kallikrein was determined after preincubation at 37° for 20 min in 0.05 M Tris-HCl buffer, pH 8.0. As shown in Table IV, dog urinary and renal kallikreins were hardly inhibited by Trasylol, but pancreatic kallikrein was sensitive to Trasylol. Hog pancreatic kallikrein was so sensitive to the inhibitor that 0.5 KIU of Trasylol caused 50% inhibition, and the activity was inhibited completely by 10 KIU. Human urinary kallikrein was similar to dog urinary kallikrein in this respect. Other proteinase inhibitors, such as SBTI, OTI (Sigma Chemical Co., U.S.A.) and potato kallikrein inhibitors (26) were also examined. As shown in Table IV, DRK and Vol. 80, No. 6, 1976
urinary kallikrein were not inhibited by these inhibitors. On the other hand, dog pancreatic kallikrein was markedly inhibited by potato kallikrein inhibitors. DISCUSSION In the present investigation, renal kallikrein was found mainly in the kidney cortex; this is in complete agreement with other reports (10,11, 27). However, the kallikrein content (per gram wet weight) was relatively low in comparison with those of other tissues and the value of god kidney was calculated to be about one-twentieth that of the pancreas (8). One possible explanation for this is that the kallikrein might be located in subcellular particles, existing in an inactive form (8). We made many attempts to destroy the subcellular particles, e.g. by dilution, freezing and thawing, acetone treatment, acidic pH, enzymic treatment and drastic homogenation in waring blenders (10, 11, 27, 28). In this investigation, repeated freezing and thawing and acetone treatment were used at
1284
C. MORIWAKI, K. MIYAZAKI, Y. MATSUDA, H. MORIYA, Y. FUJIMOTO, and H. UEKI
neutral pH for the activation of DRK for convenience in the purification procedure. As there was no difference in the kallikrein contents of kidneys with and without perfusion kidneys which were simple rinsed were employed for the purification of DRK. Possible contamination with plasma kallikrein was excluded by adsorption on DEAE-cellulose, which is known to adsorb the glandular kallikreins but not plasma kallikrein (29, 30). Although the purified DRK seemed almost pure on disc electrophoresis, electrofccusing of the preparation using carrier ampholyte gave three different active fractions with pi's of 3.9, 4.1, and 4.3. The occurrence of kallikreins multiple forms has been reported by many investigations, e.g. in hog pancreas (31), rat pancreas (32), bovine serum (55), and hog submaxillary gland (34). Werle et al. obtained five electrophoretically distinguishable kallikreins from hog pancreas (35) and eight from the submaxillary gland (34), and they considered that the difference was due to different contents of sialic acid (35). More detailed experiments are required on the multiple forms of DRK. On analysis of the amino acid composition, a low recovery of amino acids was found (66%). Although this suggests the possible presence of components other than amino acids, further experiments are required to confirm this. The apparent molecular weight of DRK was calculated to be 3.8x10* both by gel filtration on Sephadex G-100 and by ultracentrifugal analysis. The molecular weight of rat renal kallikrein was estimated to be about 3.8 x 104 by Nustad (28) and 4x10* by Porcelli et al. (36). The latter authors reported that the molecular weight of rat urinary kallikrein was 3.2xlO4 (36), but they suggested that the urinary kallikrein might be a partial degradation product of renal kallikrein. In our investigation, DRK was found to be a larger molecular than dog urinary kallikrein, and this suggests the above possibility. Nustad reported that the rat kidney homogenate had two pH optima at 6.5 and 8.5, using BAEE as a substrate (27). In our experiment, only one pH optimum, 8.6, was found, and this was in agreement with the results of Webster and Pierce (37). If an acidic and unstable BAEE esterase was present, as reported Nustad for dog kidney, it might have been removed during the purification procedures.
In addition to optimum pH, many similarities were found between dog renal and urinary kallikreins in the present investigation, e.g., heat stability and behaviour towards various inhibitors. Trasylol inhibited dog pancreatic kallikrein, but not urinary and renal kallikreins. SBTI had not inhibitory action on DRK. The results of SBTI inhibition are in agreement with the report of Nustad (28), but not with that of Werle and Vogel (13). Other proteinase inhibitors extracted from ovomucoid and potato inhibited neither DRK nor dog urinary kallikrein. However, dog pancreatic kallikrein was quite sensitive to the potato kallikrein inhibitors. DRK appeared to be different from the pancreatic and plasma kallikreins based on this inhibition experiment, together with the results on molecular weight, isoelectric point and heat stability. The overall results suggest that the origin of urinary kallikrein is the kidney. The inactive renal kallikrein might be activated at the functional stage of the kidney, e.g. as a result of ion balance, hypertension and water excretion; the urinary kallikrein seems to be a degradation product of activated renal kallikrein. A recent investigation by Kaizu and Margolius (38) utilizing a heterogeneous population of rat renal cortical cells in suspension seems to support this view. Our investigations on the purification of dog urinary kallikrein are continuing. The authors would like to acknowledge the helpful suggestions and observations of Prof. Dr. M. Schachter, Department of Physiology, University of Alberta. We are indebted to Dr. N. Murata, Faculty of Science, University of Tokyo, for carrying out ultracentrifugation and amino acid analysis. REFERENCES 1. Webster, M. & Gilmore, J.P. (1964) .-inter. J. Physiol. 206, 714-719 2. Barraclough, M.A. & Mills, I.H. (1965) Clin. Sci. 28, 67-74 3. Willis, L.R., Ludeus, J.H., Hook, J.B., & Williamson, H.E. (1969) Amer. J. Physiol. 217, 1-5 4. Stein, J.H., Congbalay, R.C., Karsh, D.L., Osgood, R.W., & Ferris, T.F. (1972) J. Clin. Invest. 51, 1709-1721 5. Gill, J.R., Ludeus, J.H., Hook, J.B., & Williamson, H.E. (1969) Amer. J. Physiol. 209, 844-849 / . Biochem.
DOG RENAL KALLIKREIN 6. Geller, R.C., Margolius, H.S., Pisano, J.J., & Keiser, H.R. (1972) Ore. Res. 31, 857-861 7. Marin-Grez, M. & Carretero, O.A. (1973) in Kininogenases (Haberland, G.L. & Rohen, J.W., eds.) pp. 113-121, F.K. Schattauer Stuttgart, New York 8. Webster, M.E. (1970) in Handbook of Experimental Pharmacology Vol. XXV, Bradykinin, Kallidin and Kallikrein (Eichler, O., Farah, H., Herken, R , & Werch, A.D., eds.) pp. 131-155, Springer Verlag, New York 9. Kraut, R , Frey, E.K., & Werle, E. (1930) Z. Physiol. Chem. 189, 97-L06 10. De Calvalho, I.F. & Diniz, C.R. (1964) Ann. New York Acad. Sci. 116, 912-917 11. De Calvalho, I.F. & Diniz, C.R. (1966) Biochim. Biophys. Ada 128, 136-148 12. Nustad, K. (1970) Br. J. Pharmac. 39, 73-86 13. Werle, E. & Vogel, R. (1961) Archs int. Pharmacodyn. Ther. 131, 257-261 14. Moriya, H., Yamazaki, K., Fukushima, H., & Moriwaki, C. (1965) /. Biochem. 58, 208-213 15. Moriwaki, C , Hojima, Y., & Moriya, H. (1974) Chemical & Pharmaceutical Bulletin 22(5), 975-983 16. Fujimoto, Y., Moriya, R , & Moriwaki, C. (1973) J. Biochem. 74, 239-246 37. Kunitz, M. (1947) /. Gen. Physiol. 30, 291-310 18. Orstein, L. (1964) .inn. New York Acad. Sci. 121, 321-349 19. Davis, B.J. (1964) Ann. New York Acad. Sci. 121, 404-427 20. Spackman, D.H., Stein, W.H., & Moore, S. (1958) Anal. Chem. 30, 1190-1206 21. Fujimoto, Y., Moriwaki, C , & Moriya, H. (1973) /. Biochem. 74, 247-252
Vol. 80, No. 6, 1976
1285 22. Goodwin, T.W. & Morton, R.A. (1946) Biochem. J. 40, 628-632 23. Yamashita, M., Hojima, Y., Moriwaki, C , & Moriya, H. (1974) Seikagaku (in Japanese) 46, 531 24. Matsuda, Y., Moriya, H., Fujimoto, Y., Hojima, Y., & Moriwaki, C. (1976) /. Biochem. 80, 671-679 25. Moriya, H., Kato, A., & Fukushima, H. (1969) Biochem. Pharmacol. 18, 549-552 26. Hojima, Y., Moriwaki, C , & Moriya, R (1973) /. Biochem. 73, 923-932 27. Nustad, K. (1970) Br. J. Pharmac. 39, 87-98 28. Nustad, K. (1970) Br. J. Pharmac. 39, 73-86 29. Webster, M.E. & Pierce, J.V. (1963) Ann. New York Acad. Sci. 104, 91-107 30. Peart, W.S., Loyd, A.M., & Thatcher, G.N. (1966) Biochem. J. 99, 708-716 31. Habermann, H. (1962) Z. Physiol. Chem. 328, 15-23 32. Yano, M., Nagasawa, S., & Suzuki, T. (1970) /. Biochem. 67, 713-725 33. Nustad, K. (1969) Scad. J. Clin. Lab. Invest. 24, 97-99 34. Fiedler, F., Muller, B., & Werle, E. (1970) Z. Physiol. Chem. 351, 1002-1006 35. Fritz, H., Eckert, I., & Werle, E. (1967) Z. Physiol. Chem. MS, 1120-1132 36. Porcelli, G., Croxatto, G.B., Marini-Bettolo, G., & Di Iorio, M. (1975) Life Sciences 16, 788 37. Webster, M.E. & Pierce, J.V. (1961) Proc. Soc. Exptl. Biol. Med. 107, 186-191 38. Kaizu, T. & Margolius, H.S. (1975) Biochim. Biophys. Ada 411, 305-315
Dog Renal Kallikrein: Purification and Some Properties Chiaki MORIWAKI,* Kyosuke MIYAZAKI,* Yoshifumi MATSUDA,* Hiroshi MORIYA,* Yukio FUJIMOTO,*-1 and Hiroshi UEKI** •Laboratory of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Shinjuku-ku, Tokyo 162, and "Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862 Received for publication, April 3, 1976
The contents of kallikrein [EC 3.4.21.8] in the kidneys of various animals were estimated and the activity was found to be most potent in dogs. The dog renal kallikrein (DRK) was located mainly in the kidney cortex. Following the activation of a dog kidney cortex homogenate with acetone, kallikrein was purified about 2,000-fold with an overall yield of 18 % by diethylaminoethyl (DEAE)-cellulose adsorption, acetone fractionation, and chromatography on Sephadex G-75 and DEAE-Sephadex A-50. The final purified preparation of dog renal kallikrein had a vasodilator activity of 65.5 KU per AIM, and appeared to be homogeneous both in disc electrophoresis and ultracentrifugal analysis. Its molecular weight was estimated to be approximately 3.8x10* from the sedimentation coefficient obtained by ultracentrifugation, and by Sephadex gel filtration. However, isoelectric fractionation of the purified DRK preparation gave three isoelectric point, 3.9, 4.1, and 4.3. The DRK had an optimum pH of about 8.6 and was stable at pH 8. This enzyme was hardly inhibited by Trasylol, soybean trypsin inhibitor, ovomucoid trypsin inhibitor or potato kallikrein inhibitors. These properties were compared with those of kallikrein from other sources; DRK appeared to be similar to urinary kallikrein.
It has been suggested by recent investigations that the kallikrein [EC 3.4.21.8]-kinin system seems to relate to the renal function (7-7). The infusion of kinin into the renal artery of dog {1-4) and man (5) causes an increase of renal sodium excretion, , . , Present address: Department of Biochemistry, Hok, .. , . . „. • io• un kaido Institute of Pharmaceutical Sciences, Otaru-shi, Hokkaido 047-0? Abbreviations: *BAEE, N-a-benzoyl-L-arginine ethyl ester; TAME, N-a-tosyl-L-arginine methyl ester; SBTI, soybean trypsin inhibitor; OTI, ovomucoid trypsin inhibitor. Vol. 80, No. 6, 1976 1277 1
and an increase in the excretion of urinary kallikrein on administration on mineralocorticoid(s) (6, 7) or saline load (7) has been also reported. Not much attention has been paid to renal kallikrein itself, although many investigators have studied urinary kallikrein. The origin of urinary kallikrein was once as, , , , sumed to be the pancreas, but that was later con„ ' . .. sidered unlikely (8). Since small kallikrein activity was demonstrated in kidney extract (9), the kidney was considered as a possible origin of urinary kallikrein (70-72). However, another report (73) is incompatible with this view and the relationship
1278
C. MORIWAKI, K. MIYAZAKI, Y. MATSUDA, H. MORIYA, Y. FUJIMOTO, and H. UEKI
between renal and urinary kallikrein is not yet clear. This confusion may be due to the use of relatively crude preparations of kallikrein in these investigations and the lack of strict comparability among these kallikreins. The purpose of the present study is the purification of kallikrein from dog kidney (DRK) and comparison of the properties of DRK with those of other purified kallikreins.
thawing procedure was repeated several times. Deionized water was added to the gruel (2 ml per 1 g of original wet tissue weight) and it was shaken vigorously, the allowed to stand for 18-24 hr at 4°. In the case of the acetone activation experiment, deionized water was replaced by various concentrations of acetone solution; the procedure was as above. The homogenate was centrifuged at 7,000 x g for 30 min, and the supernatant was dialyzed for 2 days against deionized water. The EXPERIMENTS AND RESULTS precipitate formed during dialysis was removed by Assay of Kallikrein Activities—The vasodilator centrifugation. The vasodilator activity of the activity was determined as described previously supernatant was assayed and the activities in the {14,15) by measuring the blood flow increase at the whole, the medulla and the cortex of the kidneys of femoral artery of a dog. The activity is expressed various animals are shown in Table I. In general, in terms of kallikrein units: KU. The esterolytic the kidney cortex contained more kallikrein than the activity was assayed by colorimetric measurement medulla, and dog kidney was especially active. with chromotropic acid using TAME (Protein The perfused kidney did not show a marked differResearch Foundation, Osaka) as a substrate (75). ence in activity compared with the unperfused one, The activity is expressed in terms of esterolytic units so the following experiments were performed using (EU) equal to /*moles of TAME hydrolyzed per unperfused kidneys. On activation with acetone min at 30° in 0.05 M Tris-HCl buffer, pH 8.0. The (10, 20, 30, and 60%, V/V), the maximum activity kinin-forming activity was estimated with bovine was found at 20%, amounting to approximately 3 kininogen as a substrate, and the contractile re- times that of the water extract, so the acetone consponse of an isolated guinea pig ileum was recorded. centration for activation was fixed at 20%. As a standard, synthetic bradykinin (BRS 640, Purification of DRK—All steps were carried Sandoz Pharmaceuticals, Switzerland) was em- out at 0-4°. ployed ; the activity is expressed in terms of bradyStep 1. Activation by acetone: From about kinin equivalent, as described previously (75). 30 kidneys, 280 g of kidney cortex was obtained, Determination of Protein Concentration—Pro- and the homogenate was activated with acetone as tein concentration was determined spectrophoto- described above. The activity was 0.03 KU and metrically by measuring the absorbance at 280 nm, 0.006 EU per / W using a Hitachi spectrophotometer, model 124 with a cell of 1 cm light path, assuming tentatively that TABLE I. Kallikrein contents in the kidneys of various animals. The vasodilator activity was determined withthe value of £ t ^* = 10.0. out any activation treatment of the tissue homogenates. Chemicals—All chemicals used in this investigation were of analytical or reagent grade. Activity (KU per gram wet weight) Preliminary Experiments for the Purification of Species — Cortex Medulla Whole DRK—Healthy adult dogs of either sex, weighing 8-15 kg, were killed by bleeding and the kidneys — Dog 1.4-2.8 0.06-0.16 were removed immediately. Subsequently, the — Cow 0.25-0.32 1.1-1.7 kidneys were washed thoroughly or one kidney of — Rabbit 1.4 0.9 each pair was psrfused with ice-cold saline for 3-4 Rat 0.4 0.5-0.8 0.3 hr to remove as much blood as possible. The Hog 0.2-0.4 0.3 — kidneys of cows, horses, and hogs were obtained from a slaughterhouse, and those of rabbits, guinea Guinea pig — — 0.3 pigs, and rats (Donryu strain) were also employed. Horse — 0.1 0.08 The cortex and medulla were separated with scisHuman 0.1 0.12 0.07 sors. The tissues were weighed, crushed with sea sand, frozen overnight, and thawed. This freeze- —, not examined. J. Biochem.
1279
DOG RENAL KALLIKREIN
Step 2. DEAE-cellulose adsorption: Four liters of deionized water was added to the homogenate. After adjusting the pH to 7.0-7.5, 20 g of DEAE-cellulosc (0.89 meq/g, Brown Co., U.S.A.) was added to the solution as described previously (16) and packed in a column (3x35 cm) after adsorption for 2 hr. The initial eluate with 0.05 M Tris-HCl buffer had no vasodilator activity. On the other hand, the eluate with 0.5 M buffer possessed the greatest vasodilator activity (Table IT). However, this eluate gave only 10% recovery of the esterolytic activity. This suggested that there was considerable contamination by TAME esterases other than kaUikrein in the kidney tissue. Step 3. Fractionation with acetone: The active fraction eluted with 0.5 M buffer was dialyzed for 1 day against tap water and for 1 more day against deionized water. The contents of the dialysis sac were diluted with deionized water to make a final protein concentration of approximately 10/lj80 per ml, and the diluted solution was adjusted to pH 7.0. Acetone was added to make a final concentration of 50% (V/V) and the precipitate formed was centrifuged off at 15,000 xg for 30 min. Cold acetone was again added to the supernatant to 80% (V/V) and the precipitate was collected by centrifugation. About 9-fold purification was achieved by this procedure with 84% recovery of the vasodilator activity.
Step 4. Sephadex G-75 gel filtration: The acetone powder of the above precipitate was dissolved and filtrated through a Sephadex G-75 column (Pharmacia AB, Sweden), as shown in Fig. 1. Though the esterolytic activity was distributed in several fractions, the vasodilator activity formed a single peak (fractions 27-36) with 58 % recovery. Step 5. DEAE-Sephadex A-50 chromatography: The pooled fraction of DRK was dialyzed overnight against deionized water and concentrated to approximately 5 ml as described previously (16). This concentrated solution was applied to a DEAE-Sephadex A-50 column (3.5 ±0.5 meq/g, Pharmacia AB, Sweden). As shown in Fig. 2, most of the activity was eluted and these fractions (tubes 52-59) were combined. Step 6. Sephadex G-100 gel filtration: The active fraction was dialyzed and concentrated as mentioned in step 5, and final purification was performed on a Sephadex G-100 column, as shown in Fig. 3. The overall results of these purification steps are summarized in Table II. The purified DRK preparation showed an activity of 65.5 KU per Ats0. This value is about 2,200 times higher than that of this initial homogenate, but the purification factor for the esterolytic activity was only 190. Most of the non-kallikrein TAME esterase(s) was 0.1.
1.5
0.1
/V/
JL \ r \ ''
1.0
20 •
15
i °'2
10
§ 0,1
5
0 0
20
10
60
80
3
/
',
0,05
0.05
i
0,5
0,3
S
I
0.1
_
20
/
.'
10
60
80
100
100 FRACTION MffBER (3.7 ML/TUBE)
FRACTION HS1BER (I.ZHL/TUBE)
Fig. 1. Gel filtration of the acetone (50-80%) precipitate on Sephadex G-75. About 500 mg of the dried powder was dissolved in 12 ml of 0.02 M acetic acidammonium acetate buffer (pH 7.4), and applied to a Sephadex G-75 column (2.5x90 cm). Elution was carried out with the same buffer at a flow rate of 10 ml per hr, and fractions of 4.2 ml were collected. • , vasodilator activity; O, esterolytic activity; , absorbancy at 280 nm. Vol. 80, No. 6, 1976
Fig. 2. DEAE-Sephadex A-50 column chromatography following gel filtration on Sephadex G-75. The concentrated active fraction (total Aua = 306) was applied to a DEAE-Sephadex column (1.5x30 cm) previously equilibrated with 0.02 M Tris-HCl buffer (pH. 7.7) containing 0.05 M NaCl. The column was eluated with a linear gradient of 0.05 to 0.4 M NaCl in the above buffer (400 ml), and fractions of 3.7 ml were collected. • , vasodilator activity; o , esterolytic activity; , absorbancy at 280 nm; , concentration of NaCl.
C. MORIWAKI, K. MIYAZAKI, Y. MATSUDA, H. MORIYA, Y. FUJIMOTO, and H. UEKI
1280
0.15 .
.0.15
A n
0.10 •
0,05 -
•0,05
X 0
10
.0.10
15 •
5
20
30
•
10
FRACTION NUWER (3,9 ML/TUBE) Fig. 3. Sephadex G-100 gel filtration following D E A E - S e p h a d e x A-50 column c h r o m a t o g r a p h y . A Sephadex G-100 column ( 1 . 5 x 9 0 c m ) was equilibrated with 0.05 M Tris-HCl buffer ( p H 7.4) containing 0.05 M NaCl. T h e flow rate was 10 ml per hr a n d fractions of 3.9 ml were
collected. • , vasodilator activity; O , esterolytic activity; sorbancy at 280 nm. TABLE II.
, ab-
Summary of the purification of D R K . Recovery
Steps
Homogenate and acetone-activated
Protein
100
(%)
Activity Vasodilator
Activity KU
EU
100
100
0.03
KU/.4m
Esterolytic
(P.F.)«
(1)
EU/,418O
0.006
(P.F.)i
(1)
DEAE-celluIose 0.05 M 0.5 M
5.7
0
12
0.0
13.5
86
10
0.19
0.013 (6)
0.004
(0
Acetone fractionation 0-50%
4.6
2
3
0.02
50-80%
1.5
84
7
1.7
(60)
0.030
(5)
0.1
58
2.3
17.7
(600)
0.139
(20)
Sephadex G-75
0.005
DEAE-Sephadex A-50
0.02
34
1.6
56.6
(1900)
0.477
(80)
Sephadex G-100
0.01
18
1.4
65.5
(2200)
1.124
(190)
* P.F. : purification factor.
removed by the DEAE-cellulose absorption procedure. The kinin-releasing activity of the purified DRK preparation was determined as 2.5 ft% bradykinin equivalents per min psr At30. Its caseinolytic activity (17) was negligible. Criteria for the Homogeneity of Purified DRK—
Disc electrophoresis of DRK was carried out by the
method of Orstein (18) and Davis (19) in 7.5% (W/V) polyacrylamide gel with 0.05 M Tris-glycinate buffer at pH 8.6 and the gel was stained with Coomassie Brilliant Blue. The preparation gave a single band, with a very faint sub-band. Sedimentation equilibrium analysis (Fig. 5-B) gave a linear function between the logarithm of the fringe disJ. Biochem.
DOG RENAL KALLIKREIN
1281
o
0.6 -
I
1 oo°°° o o
0,2
pi t
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/
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°
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20
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I
5 3
A 2
u1 \ 30
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1-
N 10
50
FRACTION NUMBER ( 2 HL/TUBE )
Fig. 4. Isoelectric fractionation of DRK. Isoelectric focusing was performed with carrier ampholyte (pH 3-5) on LKB 8100 equipment. Electrophoresis was carried out at 500 volts and 6-8° for 40 hr. O, pH; , absorbancy at 280 nm; • , vasodilator activity. placement of DRK and the distance from the center of the rotor (R), suggesting the homogeneity of this preparation. However, isoelectric fractionation gave three active peaks with pi's of 3.9, 4.1, and 4.3. This result indicates that DRK may consist of multiple form. Amino Acid Analysis—The purified DRK preparation (158 fig) was hydrolyzed at 110° for 32 hr in an evacuated, sealed tube with redistilled 6 N HC1, and subsequently analyzed in a Hitachi KLA3B amino acid analyser, according to the modified method of Spackman et al. (20). The recovery of amino acids was 66 %. The results of the analysis are shown in Table III. Estimation of the Molecular Weight—The molecular weight of DRK and dog urinary kallikrein were estimated to be 3.8 xlO4 and 2.6 xlO4, respectively, by Sephadex G-100 gel filtration (Fig. 5-A). Furthermore, an estimate of the molecular weight by ultracentrifugation (sedimentation equilibrium method) was made using a Spinco model E ultracentrifuge as described previously (27). The molecular weight calculated from Fig. 5-B was 3.8x10*. Some Properties of DRK in Comparison with Those of Kallikreins from Other Sources—Kallikreins: Dog urinary kallikrein was prepared by the method of Matsuda et al. (unpublished). Its vasodilator and esterolytic activities per AIM were Vol. 80, No. 6, 1976
TABLE III. Amino acid composition of the purifisd DRK. Aminr> npiri
Lys His Arg Asp Thrb Serb GIu Pro Gly Ala Cys Val Met lie Leu Tyr Phe Trp c
Mole (%)
Residues1 per molecule
Nearest integer
4.17 2.57 2.89 10.37 4.97 6.42 11.23 5.78 9.52 6.31 5.67 6.63 1.50 4.28 6.42 3.42 3.00 4.92
13.75 8.46 9.51 33.80 16.22 21.16 37.03 19.05 33.01 21.15 18.70 21.86 5.00 14.62 21.16 11.28 9.87 16.22
14 8- 9 9-10 34 16 21 37 19 33 21 19 22 5 15 21 11 10 16
» Calculated on the basis of a molecular weight of 3.8x10*. b The hydrolytic losses of threonine and serine were corrected using factors of 5.0% and 14.8%, respectively. « Tryptophan content was determined spectrophotometrically according to Goodwin and Morton (22).
C. MOR1WAKI, K. MIYAZAKI, Y. MATSUDA, H. MORIYA, Y. FUJ1MOTO, and H. UEKI
1282
QA)
2.0
\
V)B)
urinary k 1.5
-
k. C)\
D)
i
n
1
2 3 4 5 10 Molecular We1ght( xlO-4) Fig. 5A: Estimation of the approximate molecular weight of DRK by Sephadex G-100 gel filtration. One mg each of cytochrome c, SBTI, ovalbumin, bovine serum albumin and blue dextran and 0.5 mg each of DRK and partially purified dog urinary kallikrein was dissolved in 1 ml of Tris-HCl buffer (0.1 M, pH 7.7), and applied to a Sephadex G-100 column (1.5x90 cm). The flow rate was maintained at 11 ml per hr. Eluates of each sample were checked for absorbancy at 280 nm for SBTI, ovalbumin and bovine serum albumin, at 410 nm for cytochrome c and at 620 nm for blue dextran. The maximum activity was recorded for the kallikreins. A, Cytochrome c; B, SBTI; C, Ovalbumin; D, Bovine serum albumin.
168 KU and 4.15 EU, respectively. Dog pancreatic killikrein was partially purified by the method of Yamashita et al. (23). Human urinary and hog pancreatic kallikreins were partially purified by the modified methods of Matsuda et al. (24) and Moriya et al. (25); the vasodilator activities of these preparations were 67.7 and 23.25 KU per ^ u o , respectively. Heat stability: D R K and dog urinary kallikrein were heated at 50, 60, and 75° for 10, 30, and 60 min in 0.05 M Tris-HCl buffer at pH 8.0, and the TAME esterolytic activity was determined. The initial activities of DRK and dog urinary kallikrein were 4 and 6.3 x 10"* EU, respectively. DRK was stable to heat treatment, i.e. more than 80 % of the activity was retained after heating at 50° for 60 min, 60° for 30 min or 75° for 10 min and 60% of the activity was retained even after treatment at 75° for 60 min. Dog urinary kallikrein showed similar heat stability, i.e., about 50% of the activity was retained after heating at 75° for 60 min. Human urinary kallikrein showed almost the same heat stability as D R K and dog urinary kallikrein (24).
2.0 •
3
10
l.o
50
51
52
Fig. 5B: Sedimentation equilibrium centrifugation of DRK. The initial protein concentration was 2 mg per 100 ml of 0.1 M phosphate buffer (pH 7.0). The plot was read from an interference photograph of the equilibliu-n distribution at 20,410 rpm and 11° for 192 min.
Fig. 6. The pH profiles of the activities of dog renal, urinary and pancreatic kallikreins. TAME-esterolytic activity was determined at various pH's in 0.04 M Britton-Robinson wide-range buffer. The dog renal, urinary and pancreatic kallikreins had esterolytic activities of 4, 6.3 and 7 x 10"' EU (estimated in 0.05 M TrisHCl buffer, pH 8.0, at 30°), respectively. Each enzyme was incubated with TAME at the indicated pH for 30 min at 30°. Maximum activity was taken as 100 in the figure. O, dog urinary kallikrein; • , dog renal kallikrein; A , dog pancreatic kallikrein.
J. Biochem.
DOG RENAL KALLIKREIN
1283
TABLE IV. Inhibitory effects of various proteinase inhibitors on dog kallikreins. The enzyme solutions were preincubated with Trasylol, trypsin inhibitors of soybean (SBTI) and ovomucoid (OTI), and potato kallikrein inhibitors (PKJ-56 and PKI-64) as described in the text, in a total volume of 0.4 ml. The incubation mixtures (0.1 ml) were assayed on the arterial blood flow in dogs. Inhibition % Inhibitors
Trasylol
SBTI OTI PKJ-56 PKI-64
10 40 200 400 2000 500 1000 500 1000 2 10
KIU
/ig /ig /ig
2 pg 10
Renal (0.8KU)
Urinary (0.8KU)
Pancreatic (2.0KU)
N.D. N.D. N.D. N.D. 22 N.D. N.D. N.D. 20 N.D. N.D. N.D. N.D.
N.D. N.D. N.D. 12 36 N.D. 12 N.D. 15 N.D. N.D. N.D. N.D.
N.D. N.D. 18 56 60 N.D. 20 N.D. N.D. 30 95 10 85
N.D. : not detectable.
Effect of pH on activity: The pH profiles of the activity of a purified preparation of DRK, partially purified dog urinary, and pancreatic kallikreins were compared. The results are shown in Fig. 6. Both DRK and urinary kallikrein showed maximum activity at pH 8.6, but the optimum pH of pancreatic kallikrein was 8.0. Effect of various inhibitors:
The effect of
Trasylol (Bayer AG, West Germany) on the vasodilator activity of DRK and other kallikrein was determined after preincubation at 37° for 20 min in 0.05 M Tris-HCl buffer, pH 8.0. As shown in Table IV, dog urinary and renal kallikreins were hardly inhibited by Trasylol, but pancreatic kallikrein was sensitive to Trasylol. Hog pancreatic kallikrein was so sensitive to the inhibitor that 0.5 KIU of Trasylol caused 50% inhibition, and the activity was inhibited completely by 10 KIU. Human urinary kallikrein was similar to dog urinary kallikrein in this respect. Other proteinase inhibitors, such as SBTI, OTI (Sigma Chemical Co., U.S.A.) and potato kallikrein inhibitors (26) were also examined. As shown in Table IV, DRK and Vol. 80, No. 6, 1976
urinary kallikrein were not inhibited by these inhibitors. On the other hand, dog pancreatic kallikrein was markedly inhibited by potato kallikrein inhibitors. DISCUSSION In the present investigation, renal kallikrein was found mainly in the kidney cortex; this is in complete agreement with other reports (10,11, 27). However, the kallikrein content (per gram wet weight) was relatively low in comparison with those of other tissues and the value of god kidney was calculated to be about one-twentieth that of the pancreas (8). One possible explanation for this is that the kallikrein might be located in subcellular particles, existing in an inactive form (8). We made many attempts to destroy the subcellular particles, e.g. by dilution, freezing and thawing, acetone treatment, acidic pH, enzymic treatment and drastic homogenation in waring blenders (10, 11, 27, 28). In this investigation, repeated freezing and thawing and acetone treatment were used at
1284
C. MORIWAKI, K. MIYAZAKI, Y. MATSUDA, H. MORIYA, Y. FUJIMOTO, and H. UEKI
neutral pH for the activation of DRK for convenience in the purification procedure. As there was no difference in the kallikrein contents of kidneys with and without perfusion kidneys which were simple rinsed were employed for the purification of DRK. Possible contamination with plasma kallikrein was excluded by adsorption on DEAE-cellulose, which is known to adsorb the glandular kallikreins but not plasma kallikrein (29, 30). Although the purified DRK seemed almost pure on disc electrophoresis, electrofccusing of the preparation using carrier ampholyte gave three different active fractions with pi's of 3.9, 4.1, and 4.3. The occurrence of kallikreins multiple forms has been reported by many investigations, e.g. in hog pancreas (31), rat pancreas (32), bovine serum (55), and hog submaxillary gland (34). Werle et al. obtained five electrophoretically distinguishable kallikreins from hog pancreas (35) and eight from the submaxillary gland (34), and they considered that the difference was due to different contents of sialic acid (35). More detailed experiments are required on the multiple forms of DRK. On analysis of the amino acid composition, a low recovery of amino acids was found (66%). Although this suggests the possible presence of components other than amino acids, further experiments are required to confirm this. The apparent molecular weight of DRK was calculated to be 3.8x10* both by gel filtration on Sephadex G-100 and by ultracentrifugal analysis. The molecular weight of rat renal kallikrein was estimated to be about 3.8 x 104 by Nustad (28) and 4x10* by Porcelli et al. (36). The latter authors reported that the molecular weight of rat urinary kallikrein was 3.2xlO4 (36), but they suggested that the urinary kallikrein might be a partial degradation product of renal kallikrein. In our investigation, DRK was found to be a larger molecular than dog urinary kallikrein, and this suggests the above possibility. Nustad reported that the rat kidney homogenate had two pH optima at 6.5 and 8.5, using BAEE as a substrate (27). In our experiment, only one pH optimum, 8.6, was found, and this was in agreement with the results of Webster and Pierce (37). If an acidic and unstable BAEE esterase was present, as reported Nustad for dog kidney, it might have been removed during the purification procedures.
In addition to optimum pH, many similarities were found between dog renal and urinary kallikreins in the present investigation, e.g., heat stability and behaviour towards various inhibitors. Trasylol inhibited dog pancreatic kallikrein, but not urinary and renal kallikreins. SBTI had not inhibitory action on DRK. The results of SBTI inhibition are in agreement with the report of Nustad (28), but not with that of Werle and Vogel (13). Other proteinase inhibitors extracted from ovomucoid and potato inhibited neither DRK nor dog urinary kallikrein. However, dog pancreatic kallikrein was quite sensitive to the potato kallikrein inhibitors. DRK appeared to be different from the pancreatic and plasma kallikreins based on this inhibition experiment, together with the results on molecular weight, isoelectric point and heat stability. The overall results suggest that the origin of urinary kallikrein is the kidney. The inactive renal kallikrein might be activated at the functional stage of the kidney, e.g. as a result of ion balance, hypertension and water excretion; the urinary kallikrein seems to be a degradation product of activated renal kallikrein. A recent investigation by Kaizu and Margolius (38) utilizing a heterogeneous population of rat renal cortical cells in suspension seems to support this view. Our investigations on the purification of dog urinary kallikrein are continuing. The authors would like to acknowledge the helpful suggestions and observations of Prof. Dr. M. Schachter, Department of Physiology, University of Alberta. We are indebted to Dr. N. Murata, Faculty of Science, University of Tokyo, for carrying out ultracentrifugation and amino acid analysis. REFERENCES 1. Webster, M. & Gilmore, J.P. (1964) .-inter. J. Physiol. 206, 714-719 2. Barraclough, M.A. & Mills, I.H. (1965) Clin. Sci. 28, 67-74 3. Willis, L.R., Ludeus, J.H., Hook, J.B., & Williamson, H.E. (1969) Amer. J. Physiol. 217, 1-5 4. Stein, J.H., Congbalay, R.C., Karsh, D.L., Osgood, R.W., & Ferris, T.F. (1972) J. Clin. Invest. 51, 1709-1721 5. Gill, J.R., Ludeus, J.H., Hook, J.B., & Williamson, H.E. (1969) Amer. J. Physiol. 209, 844-849 / . Biochem.
DOG RENAL KALLIKREIN 6. Geller, R.C., Margolius, H.S., Pisano, J.J., & Keiser, H.R. (1972) Ore. Res. 31, 857-861 7. Marin-Grez, M. & Carretero, O.A. (1973) in Kininogenases (Haberland, G.L. & Rohen, J.W., eds.) pp. 113-121, F.K. Schattauer Stuttgart, New York 8. Webster, M.E. (1970) in Handbook of Experimental Pharmacology Vol. XXV, Bradykinin, Kallidin and Kallikrein (Eichler, O., Farah, H., Herken, R , & Werch, A.D., eds.) pp. 131-155, Springer Verlag, New York 9. Kraut, R , Frey, E.K., & Werle, E. (1930) Z. Physiol. Chem. 189, 97-L06 10. De Calvalho, I.F. & Diniz, C.R. (1964) Ann. New York Acad. Sci. 116, 912-917 11. De Calvalho, I.F. & Diniz, C.R. (1966) Biochim. Biophys. Ada 128, 136-148 12. Nustad, K. (1970) Br. J. Pharmac. 39, 73-86 13. Werle, E. & Vogel, R. (1961) Archs int. Pharmacodyn. Ther. 131, 257-261 14. Moriya, H., Yamazaki, K., Fukushima, H., & Moriwaki, C. (1965) /. Biochem. 58, 208-213 15. Moriwaki, C , Hojima, Y., & Moriya, H. (1974) Chemical & Pharmaceutical Bulletin 22(5), 975-983 16. Fujimoto, Y., Moriya, R , & Moriwaki, C. (1973) J. Biochem. 74, 239-246 37. Kunitz, M. (1947) /. Gen. Physiol. 30, 291-310 18. Orstein, L. (1964) .inn. New York Acad. Sci. 121, 321-349 19. Davis, B.J. (1964) Ann. New York Acad. Sci. 121, 404-427 20. Spackman, D.H., Stein, W.H., & Moore, S. (1958) Anal. Chem. 30, 1190-1206 21. Fujimoto, Y., Moriwaki, C , & Moriya, H. (1973) /. Biochem. 74, 247-252
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