ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 281, No. 1, August 15, pp. 144-1X,1990
Purification and Characterization of Three Ribonucleases from Human Kidney: Comparison with Urine Ribonucleases’ Keiko Mizuta,2 Shuichi Awazu, Toshihiro
Yasuda, and Koichiro
Kishi3
Department of Legal Medicine, Fukui Medical School, Matsuoku-cho, Fukui 910-l 1, Japan
Received February
51990,
and in revised form April 13,199O
Three ribonucleases (RNases) with different molecular masses were isolated from human kidney. The enzymes were purified to an electrophoretically homogeneous state, and their respective molecular masses were found to be 18,000 (tentatively named RNase HK-l), 20,000 (RNase HK-BA), and 22,000 (RNase HK-2B) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the amino acid compositions, aminoterminal sequences, and enzymological properties of the enzymes indicate that RNase HK-1 is related to “nonsecretory” RNase, and that RNases HK-2A and HK-2B are both related to “secretory” RNase. Furthermore, RNase HK-1 showed cross-reactivity with an antibody specific to nonsecretory RNase from human urine, whereas RNases HK-2A and HK-2B showed cross-reactivity with another antibody specific to human urine secretory RNase. However, the carbohydrate compositions of RNases HK-PA and HK-2B were markedly different from that of the secretory urine RNase. This finding seems to indicate that the kidney is 0 1990 Academic Press. Inc. not the origin of the urine enzyme.
Ribonucleases are widely distributed in various organs and body fluids, including serum, urine, saliva, and cerebrospinal fluid. Since Reddi and Holland first observed that RNase activity was elevated in sera of patients with pancreatic cancer (l), many workers have reported on the relationship between the activity of RNase in serum i This work was supported in part by grants from the Japan Brain Foundation and Tokyo Immunopharmacology Institute, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. * Present address: Department of Biochemistry and Biophysics, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima, 734 Japan. s To whom correspondence should be addressed. 144
or urine and pancreatic diseases: several workers have supported the hypothesis (2-4), whereas others have found little or no correlation (5-7). The molecular multiplicity of RNases has made it difficult to prove the correlation between RNase activity and disease states. Akagi et al. isolated five RNases with different molecular masses of 8500,13,000,20,000,32,000, and 45,000, as determined by gel filtration, from normal human serum (8). Blank and Dekker also showed the multiplicity of RNases in human serum and urine, reporting six serum RNases with molecular masses of 14,000,16,000,20,000, 25,000, 28,000, and 31,000, respectively, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9). Urine RNases consist of two major components, one with a molecular mass of 31,000 (SDS-PAGE)4 named secretory RNase, UL (lo), RNase C (ll), or Band A (12), and the other with a molecular mass of 16,000 (SDSPAGE) named nonsecretory RNase, Us (lo), RNase U (ll), or Band D (12). We have recently purified the nonsecretory RNase of urine and demonstrated genetic polymorphism (13). The nonsecretory RNase of urine has been sequenced and the presence of five short carbohydrate chains established (14). One of the secretory RNases, UL in human urine, has been isolated and its amino acid sequence analyzed (15). The results indicated that UL had almost the same polypeptide chain as human pancreatic RNase (16). However, since their glycosylation characteristics were quite different from each other, it was suggested that the urine enzyme, UL, did not originate in the pancreas (15, 17). The origin of serum RNases also remains unclear. Therefore, for a true understanding of RNases in normal and pathological states, it is important to isolate and characterize RNases from various organs, and to determine the origin of both ’ Abbreviations used: APUP-agarose, agarose-5’-(4-amino phenyl phosphoryl) uridine 2’(3’)-phosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 0003-9s61/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
HUMAN
KIDNEY
urine and serum RNases. Recently, Yasuda et al. purified two distinct types of RNase from human erythrocytes and demonstrated that the enzyme present in a higher content was enzymologically very similar to nonsecretory RNase in human urine (18). Also, Sorrentino et al. purified an RNase from human liver and showed that its enzymatic properties were very similar to those of nonsecretory urine RNase (19). Finally, protein-sequencing studies have yielded evidence that several functionally diverse proteins belong to a superfamily of ribonucleases. Angiogenin, a protein that stimulates the formation of blood vessels, is structurally related to pancreatic (secretory) RNase (20), retaining some nuclease activity (21). Furthermore, an eosinophil-derived neurotoxin with significant RNase activity may be identical with liver (nonsecretory) RNase (22). In this paper, we describe the isolation to homogeneity and characterization of three RNases from human kidney, since human kidney RNases have not yet been purified to homogeneity. We also compare the immunological and biochemical properties of these enzymes with RNases from human urine. MATERIALS
AND
METHODS
Materials All chemicals were of reagent grade. Cellulose phosphate Pll was purchased from Whatman (Maidstone, UK), poly(G)-agarose, poly(G), and molecular weight calibration marker kits were from Pharmacia (Uppsala, Sweden), agarose-B-(l-amino phenyl phosphoryl) uridine 2’(3’)-phosphate (APUP-agarose) was from ICN Immuno Biologicals (Lisle, NY), heparin-actigel from Sterogene Biochemicals (San Gabriel, CA) and Bio-Gel P-100 from Bio-Rad (Richmond, CA). Poly(A), poly(U), and poly(C) were obtained from Seikagaku-Kogyo (Tokyo, Japan) and yeast RNA was from Kojin (Tokyo, Japan).
Assay of RNase Activity The standard assay was performed according to the procedure reported by Uchida andEgami (23) with some modifications. RNA solution (1.2%, 50 ~1) was added to 150 ~1 of reaction mixture consisting of 50 mM Tris-HCl buffer, pH 7.5, 5 mM EDTA, and enzyme. The reaction was performed at 37°C and terminated by adding 50 ~1 of 25% perchloric acid containing 0.75% uranyl acetate. The reaction tubes were chilled in an ice-bath for 15 min, then centrifuged at 1600g for 5 min. A 100~~1 aliquot of the supernatant was diluted with 2.5 ml of distilled water and the absorbance of this solution at 260 nm was measured. One unit of RNase activity was defined as an increase by 1.0 of the absorbance at 260 nm per minute.
Purification of RNases Human kidneys were obtained at autopsy from a 58-year-old male (24 h after death due to burning), a 54-year-old male (14 h after death due to brain contusion), a 38-year-old male (8 h after death due to brain contusion) and a 32-year-old male (24 h after death due to loss of blood). All of these were proved to be histologically normal. The kidneys were stored at -80°C until use. All subsequent steps were carried out at 0-5°C. Step 1. The kidneys (295 g) from the 58-year-old male were partially thawed overnight in a refrigerator then cut into small pieces, the
145
RIBONUCLEASES
3 ! b -1.0
2E I a it 1 -0.5 is s 0
50 Fraction
100
0
‘0.0
number
FIG. 1. Cellulose phosphate column chromatography of RNase-active fractions. Fraction Nos. 76-88 (RNase HK-1 fraction), Nos. lOO110 (RNase HK-PB fraction), and Nos. 111-120 (RNase HK-PA fraction) were pooled separately and dialyzed for further purification. blood was rinsed out with cold saline, and the pieces were suspended in 590 ml of 0.25 M HzS04, containing 1 mM MgCls. The mixture was homogenized with an Ultra-turrax dispenser and centrifuged for 30 min at 12,OOOg.The supematant was collected and its pH was adjusted to 7 with NH,OH. Step 2. The crude extract from step 1 was fractionated with ammonium sulfate. The precipitate obtained between 0.3 and 0.9 saturation was collected and dissolved in ca. 150 ml of water and dialyzed against 5 mM sodium phosphate buffer, pH 6.0. Step 3. The sample from step 2 was centrifuged and the supernatant was applied at 0.2 ml/min to a cellulose phosphate column (2.6 X 37 cm) preequilibrated with 5 mM sodium phosphate buffer, pH 6.0. The column was washed with 100 ml of the same buffer, and then eluted with a linear concentration gradient of NaCl from 0 to 1 M in 3 liters of the same buffer. RNase activity was eluted at a broad range of NaCl concentration from 0.5 to 0.9 M as two peaks (Fig. 1). The second peak showed an asymmetrical pattern in terms of RNase activity. The first RNase-active peak, named RNase HK-1 fraction, and two portions of the second RNase-active peak, the former half named RNase HK-2B and the latter HK-PA, were collected separately and dialyzed against 5 mM Tris-HCl buffer, pH 7.0. Step 4. Each sample from step 3 was centrifuged. The supernatant was finally adjusted to a concentration of 50 mM Tris-HCl buffer, pH 7.0, containing 20 mM NaCl, and then applied at 0.3 ml/min to a heparin-actigel column (1.8 X 8 cm) preequilibrated with 50 mM Tris-HCl buffer, pH 7.0, containing 20 mM NaCI. The column was washed with 50 ml of the same buffer, and then eluted with a linear concentration gradient of NaCl from 20 to 500 mM (for RNase HK-1) or to 700 mM (for RNases HK-2A and HK-2B). The enzyme active fractions were pooled and individually dialyzed against distilled water.
Step 5. Each sample from step 4 was concentrated in uocuo at 30°C to a small volume and applied to a Bio-Gel P-106 column (1.6 X 90 cm) preequilibrated with 10 mM Tris-HCl buffer, pH 7.5, containing 0.3 M NaCl. The enzymes were eluted with the equilibration buffer at a flow rate of 3 ml/h and RNase-active fractions were pooled. The RNase HK-1 fraction was dialyzed against 10 mM sodium phosphate buffer, pH 7.5, and RNases HK-2A and HK-PB fractions were dialyzed against 20 mM sodium acetate buffer, pH 5.0. Step 6. The HK-1 fraction from step 5 was applied to a poly(G)agarose column (0.9 X 7 cm) preequilibrated with 10 mM sodium phosphate buffer, pH 7.5, and eluted with a linear gradient of O-l M NaCl in 100 ml of the same buffer. Peak fractions showing RNase activity were pooled and concentrated by ultrafiltration. The obtained RNase was designated RNase HK-1. If necessary, the enzyme was desalted on a Sephadex G-25 column buffered with 0.2 M acetic acid and lyophilized.
146
MIZUTA
ET AL.
TABLE I Purification Protein (md
Step 1. Extract 2. Ammonium sulfate fractionation 3. Cellulose phosphate 1st fraction (HK-1) 2nd fraction (HK-2B) 3rd fraction (HK-2A) 4. Heparin-actigel HK-1 HK-2A HK-2B 5. Bio-Gel P-100 HK-1 HK-LA HK-2B RNase HK-1 6. Poly(G)-agarose RNases HKBA and HK-2B 7. APUP-agarose HK-2A HK-2B 8. Poly(G)-agarose HK-2A HK-2B
of Human Kidney
RNases
Total activity” (unit)
Specific activity (unit/mg)
13,973
4440
0.318
3,126
3192
1.02
3.21
71.9
28.5 36.2 43.4
38.9
286 675
180
30.9 9.3 7.3
191
1729
9.05
56.0 42.6
645 588
11.5
Purification (-fold)
13.8
15.1
1371
90.8
1.92 5.70
412 326
214.6 57.2
1
Yield (W) 100
13.2 14.5
1.24
612
0.34 0.47
176 162
494 518 345
1553 1629 1085
13.8 4.0 3.6
0.90
506
562
1767
11.4
0.16 0.23
88 122
552 530
1736 1667
2.0 2.7
0.137 0.198
79 108
577 545
1814 1714
2.4
0 Activities of RNases were measured in 0.1 M Tris-HCl buffer, pH 7.0, for RNase HK-1, or in 0.1 M Tris-HCl HK-2A and HK-2B using RNA as substrate, as described under Materials and Methods. Step 7. RNases HK-2A and HK-2B fractions from step 5 were separately applied to an APUP-agarose column (0.9 X 6 cm) preequilibrated with 20 mM sodium acetate buffer, pH 5.0, and eluted with a linear concentration gradient of NaCl from 0 to 1.2 M in the same buffer. The RNases were each eluted with ca. 0.6 M NaCl and dialyzed against 10 mM sodium phosphate buffer, pH 7.5.
1.8
buffer, pH 8.0 for RNases
Step 8. Each of the samples from step 7 was applied to a poly(G)agarose column (0.9 X 7 cm) preequilibrated with 10 mM sodium phosphate buffer, pH 7.6. The enzymes were eluted with 1 M NaCl in the same buffer. The obtained RNases were designated RNase HK-2A and RNase HK-BB, respectively. These enzymes were desalted and lyophilized for proteochemical analysis
Protein Characterization
12
3
4
12
3
4
5
6
FIG. 2. SDS-PAGE of purified RNases HK-1, HK-2A, and HK-2B. (A) Protein staining with Coomassie blue R250. Lane 1, RNase HK1; lane 2, RNase EK-ZA, lane 3, RNase HK-2B; lane 4, molecular weight markers: 1, phosphorylase b (94 kDa); 2, bovine serum albumin (67 kDa); 3, ovalbumin (43 kDa); 4, carbonic anhydrase (30 kDa); 5, trypsin inhibitor (20 kDa); 6, a-lactalbumin (14.4 kDa). (B) Activity staining on SDS-polyacrylamide gel casting poly(C) (lanes l-3) or poly(U) (lanes 4-6). Lanes 1 and 4; RNase HK-1, lanes 2 and 5; RNase HK-BA, lanes 3 and 6; RNase HK-2B. The arrow indicates the origin of electrophoretogram.
SDS-PAGE was conducted in 12.5% polyacrylamide gel according to the method of Laemmli (24). Samples were dissolved in buffer containing 10 mM Tris-HCl, pH 6.8,10% glycerol, 2% SDS, 0.2% 2-mercaptoethanol, and a trace of bromphenol blue. Electrophoresis was done for 80 min at a constant current of 20 mA. In order to detect the RNase activity, poly(C) or poly(U) (ca. 0.2 mg/ml) was added to the separating gel solution prior to polymerization (9). After the electrophoretic run, the gel was incubated for 3 h in 50 mM Tris-HCl buffer, pH 7.5, at 37°C and then stained with 0.2% toluidine blue. Amino acid analysis was done using a Hitachi 835 amino acid analyzer equipped with a column (0.26 X 25 cm) of Hitachi X2619 resin (Tokyo, Japan) using ca. 30-pg samples that had been hydrolyzed at 110°C in 6 N HCl for 24 h. Automatic Edman degradation of native RNases (ca. 10 gg) was performed on an automated gas-phase protein sequencer (Applied Biosystems 477A, Foster City, MI); phenylthiohydantoin derivatives were identified with an HPLC apparatus attached to the sequencer. Amino sugars liberated from the enzymes by hydrolysis in 6 N HCl were quantitated with a Hitachi 835 amino acid analyzer. The composition of neutral sugar was determined by gas chromatography-electron ionization mass spectrometry. Analysis was performed on a JMSDX 300 instrument (JEOL, Tokyo, Japan). Analytical conditions and
147
HUMAN KIDNEY RIBONUCLEASES TABLE
II
Amino Acid Compositions of RNasesfrom Human Kidney Urine RNases
Kidney RNases
Amino acid HK-1
Nonsecretory”
Secretory *
HK-2A
HK-2B
11.3
11.3
15.7
11.7
5.0
5.5 11.7
5.6 11.4
11.7
10.8
10.3
2.3 5.3 5.3 6.6 2.8 4.0 3.8 2.6 3.8 4.0 3.5 5.6
4.1 3.5 8.3 5.8 4.2 1.8 1.8 3.4 3.2 6.5 3.9
4.4 3.2 7.6 6.6 4.4 2.9 1.5 3.0 3.5 6.2 3.7
ND’
9.1 ND
10.2 ND
8.6
5.1
4.2
9.0 4.5 10.5 1.5 4.5 6.8 6.0 3.0 5.3 3.8 3.0 3.8 3.0 3.8 6.0 0.8 9.0
6.3 12.4 10.2 3.9 3.1 7.8 6.3 3.9 2.3 1.6 3.9 3.1 6.3 3.9 7.8 0 5.5
mol% Asx Thr Ser Glx
16.2 8.9
GUY
Ala Val CYS
Met Ile Leu ‘G-r
Phe LYS His
Arg Trp Pro
’ Calculated from the sequence from Ref. (14). * Calculated from the sequence from Ref. (15). ’ ND, not determined.
preparation of the alditol trifluoroacetate derivatives of neutral sugars were described in a previous paper (26). Protein concentration was determined according to the method of Lowry et al. (26) using bovine serum albumin as a standard.
Production
of Antibodies
and Immunoblotting
Nonsecretory (RNase 1) and secretory (RNase 2) RNases were both purified from humanurine (13). Antibodiesspecificto the purified RNases were raised in rabbits as described in a previous paper (27). Immunoblotting after SDS-PAGE was performed according to the method described in our previous paper (28).
detection. Table I is a typical example from among the four preparations. Figure 2 shows the SDS-PAGE patterns of three purified RNases under reducing conditions. When these enzymes were subjected to SDS-PAGE in poly(U)-cast gel, all of them showed very similar activities, whereas in poly(C)-cast gel, RNase HK-1 showed only very weak activity in comparison with RNases HK-2A and HK-2B. Each enzyme band detected by enzymological staining corresponded to the band detected by protein staining, and vice versa. Molecuhr
Mass
The molecular masses of RNases HK-1, HK-BA, and HK-2B were estimated to be 18,000, 20,090 and 22,000, respectively, on SDS-PAGE under reducing conditions (Fig. 2). Chemical Composition
In Table II, the amino acid compositions of the purified kidney RNases are shown in comparison with those of previously reported urine RNaaes. The amino acid composition of RNase HK-1 was observed to be very similar to that of nonsecretory RNase from human urine. On the other hand, there was close similarity in the amino acid compositions of RNases HK-2A and HK2B and secretory RNase from human urine. However, the carbohydrate compositions of the kidney RNases were very different from the compositions of urine RNases, as shown in Table III. Fucose was the major neutral sugar of RNase HK-1. RNases HK-2A and HK2B both contained more mannose than fucose. RNase HK-2B contained a larger amount of carbohydrate than RNase HK-2A. The amino sugar contents of the three
TABLE III
Carbohydrate Compositions of RNasesfrom Human Kidney
RESULTS Enzyme Purification
The preparation of three RNases from 295 g of autopsied human kidney is summarized in Table I. We tried to isolate these RNases individually from kidneys obtained from four different individuals. The specific activity of the crude preparations (step 1) ranged from 0.32 to 0.5 units/mg protein, although the elution patterns of RNase activity on the cellulose phosphate column differed markedly among the four preparations, the RNase HK-1 accounting for 39,58, 73, and 84% of the total activity. However, three kinds of RNase were demonstrated to exist in each of the preparations by electrophoresis followed by enzymological and immunological
Kidney RNases Carbohydrate component
HK-1
HK-2A
Urine RNases
HK-PB
Nonsecretory”
Secretory*
Residues/molecule Glucosamine’ Galactosamine’ Fucose Mannose Galactose
3.0 NDd
1.5 ND
2.3
4.4
ND
ND
2.2 0.45
1.2 1.6
2.5 3.0
1.4 2.8
Trace”
Trace
Trace
’ Taken from Ref. (13). * Taken from Ref. (15). ’ The amino sugars were calculated as the N-acetyl form. d ND, not detected. eTrace, trace amount was detected.
16.7 3.3 8.9
10.9
148
MIZUTA Human
5
ET AL. 10
15
20
25
30
Kidney RNase HK-1
KPPQFT?AQ?FETQHI?HT
SQQ?TNANQVINNY
Nonsecretory urinary RNase
KPPQFT?AQWFETQHINMT
SQQCTNANQVINNY
Liver RNase
RPPQPT?AQWFETQHINM(T)(T)QQ?TNA
Human Kidney RNase HK-2A
KESRAK(K)FQRQHMDS?SSPS
Kidney RNase HK-2B
KESRAKKPQRQHMDSDSSP
Secretory urinary RNase
KESRAKKPQRQHMDSDSSPS
Pancreatic RNase
KESRAKKFQRQHMDSDSSPS
FIG. 3.
N-terminal amino acid sequences of human kidney RNases. For comparison, sequences of human urinary RNases (14, IS), human liver RNase (19) and human pancreatic RNase (16) are also shown. Question marks indicate the undetermined positions. Parentheses indicate tentatively identified residues.
kidney RNases were entirely in the form of glucosamine with no apparent galactosamine residues. The N-terminal amino acid sequence of RNase HK-1 was very similar to those of urine nonsecretory RNase (14) and liver RNase (19), while RNases HK-2A and HK-2B had an N-terminal amino acid sequence very similar to those of urine secretory RNase (15) and pancreatic RNase (16) (Fig. 3).
As shown in Table IV, poly(A) and poly(G) were not hydrolyzed by any of the kidney RNases described. RNase HK-1 exhibited greater preference for poly(U) than poly(C) as a substrate. On the other hand, RNases HK2A and HK-2B reacted much more actively with poly(C) than with poly(U). The three kidney RNases all appeared to be specific for cleavage at pyrimidine residues in RNA.
Enzymatic Properties
Immunological Properties
The effects of pH on the activities of the purified RNases were examined using yeast RNA as a substrate. The pH optimum for RNase HK-1 was 6.5-7.0, while those for RNases HK-2A and HK-2B were both 8.0 (Fig. 4). For measuring the substrate specificity of the kidney RNases, the yeast RNA substrate was replaced by synthetic homopolyribonucleotides in the standard assay.
Figure 5 shows the results of immunoblotting of kidney RNases with two kinds of anti-urinary RNase antibody with different specificities after SDS-PAGE. The agarose gel diffusion test showed that RNase HK-1 was reactive with anti-RNase 1 (nonsecretory RNase from human urine) antibody as well as with anti-RNase HK1 antibody, but not with anti-RNase 2 (secretory RNase from human urine). In contrast, RNases HK-2A and
4
6
8 PH
10
I
I
I
I
4
6
8
10
PH
4
6
8
10
PH
FIG. 4. Effects of pH on activities of RNases HK-1, HK-PA, and HK-2B. (A) RNase HK-1. (B) RNase HK-2A. (C) RNase HK-PB. The buffers used were 0.1 M sodium acetate buffer (0), 0.1 M sodium cacodylate buffer (O), 0.1 M Tris-HCl buffer (A) and 0.1 M sodium glycine buffer (A). The other experimental conditions were as described under Materials and Methods.
HUMAN TABLE Substrate
Specificity”
IV
of Human Activity*
Substrate RNA POlYC) POlYW) Poly(A) Poly (G)
RNase HK- 1 100 6 43 ND’ ND
KIDNEY
Kidney (%)
RNase HK-2A 100 4100 12 ND ND
RNases
RNase HK-2B 100 4400 15 ND ND
a The reaction mixture (0.2 ml) consisted of 0.2 mg of the substrate indicated, 0.1 M Tris-HCl buffer, pH 7.0, for RNase HK-1 or 0.1 M Tris-HCl buffer, pH 8.0, for RNases HK-PA and HK-PB. The other conditions were as described under Materials and Methods. * Activity is expressed relative to that for RNA. ’ ND, not detected.
HK-2B were both reactive with anti-RNase 2, but not with anti-RNase 1 or with anti-RNase HK-1 antibody (data not shown). Furthermore, the inhibitory effects of the antibodies on RNase catalytic activities are shown in Fig. 6. The anti-RNase 1 antibody completely blocked the enzyme activity of the purified RNase HK-1, whereas it was ineffective on the activities of RNases HK-2A and HK-2B. Conversely, the anti-RNase 2 antibody completely blocked the activities of RNases HK2A and HK-2B, but not the activity of RNase HK-1. As shown in Fig. 7, these results indicate that the RNase reactive with anti-RNase 1 antibody accounts for about 50% of the total RNase activity in the kidney extract, while the RNase reactive with the anti-RNase 2 antibody accounts for about 45%. At least 90% of the total RNase activity in the kidney was inhibited in the presence of both the anti-RNase 1 and anti-RNase 2 antibodies.
149
RIBONUCLEASES
urine contains both types. Purification and characterization has been described by several groups (10, 11,14, 15). We have separately purified these enzymes from human urine (13) and designated the nonsecretory type as RNase 1 and the secretory type as RNase 2. Considering the enzymatic properties of these kidney RNases and the classification criteria for human RNase, RNase HK1 is considered to be the nonsecretory type, and both RNases HK-2A and HK-2B the secretory type. Furthermore, their similarities of amino acid composition, Nterminal amino acid sequence, and reactivities against two kinds of RNase-specific antibody reinforced the validity of this classification. Sorrentino et al. (19) found that the amount of nonsecretory RNase in the kidney was about 60-70% of the total activity, which is somewhat higher than the result of Morita et al. (30), which was about 20-30%. In the present study, the content of the nonsecretory enzyme, RNase HK-1, was 58% at the step of cellulose phosphate column chromatography, which allows good separation between secretory and nonsecretory RNases (Table I). However, the amount of the secretory enzyme varied markedly among the preparations from four different individuals, and accordingly the content of the nonsecretory enzyme ranged from 39 to 84% in the four preparations. We were unable to identify any factors, e.g. age, sex, cause of death of the individuals, or time elapsed between death and autopsy which could be related to this great variation. Morita et al. speculated that a new RNase, belonging neither to the secretory nor nonsecretory type (30), could be present, although we found no evidence for the existence of any such new RNase in the kidney during the purification process. Figure 7 also shows that the total of the secretory and nonsecretory RNase activities reached more than 90% of the total RNase activity in the crude extract from human kidney. The polypeptide portions of the kidney RNases were very similar to that of either nonsecretory or secretory
DISCUSSION
In this study, three kidney RNases were isolated for the first time and comprehensively characterized, following the prediction of the presence of at least two kinds of RNase (19,27,30). These enzymes have molecular masses of 18,000, 20,000, and 22,000, respectively, as determined by SDS-PAGE, and are designated RNases HK-1, HK-BA, and HK-BB, respectively. RNase HK-1 is poly(U)-preferential, showing maximal activity at pH 6.5-7.0. Both RNases HK-2A and HK-2B are highly poly(C)-preferential, having an optimal pH around 8.0. Sierakowska and Shugar (29) classified mammalian RNases into two types, namely “secretory” and “nonsecretory.” The former, resembling pancreatic RNase, is characterized by a high pH optimum and preference for poly(C) as a substrate, whereas the latter has a lower pH optimum and preference for poly(U). Human
I.1
12
3
_I ‘I
12
3
FIG. 5. Immunoblotting patterns of kidney RNases with two kinds of anti-urine RNase antibody after SDS-polyacrylamide gel electrophoresis. (A) Anti-RNase 1 (nonsecretory type) antibody. (B) AntiRNase 2 (secretory type) antibody. Samples were RNase HK-1 (lane 11, RNase HK-2A (lane 2), and RNase HK-PB (lane 3). The arrow indicates the origin of electrophoretogram.
150
MIZUTA
Sloe >r ‘2 80 5 -!
ET AL.
A /---
?I60-
/ A
-0.5
0
0.5
Log (~1 antibody)
1.0
- 0.5
0 0.5 Log(,uul antibody)
1.0
FIG. 6. Inhibitory effects of anti-urine RNase antibodies on enzyme activities of purified human kidney RNases. Incubation mixtures (20 ~1) consisting of 0.26 pg of the purified enzyme; RNase HK-1 (A), RNase HK-PA (0), or RNase HK-PB (O), 20 mM Tris-HCl buffer, pH 7.0, 0.15 M NaCl, and serially diluted antibody were held at 20°C for 2 h. They were then centrifuged and their RNase activities in the supernatant (10 ~1) were determined in 0.1 M Tris-HCl buffer, pH 7.0, for RNase HK-1, or in 0.1 M Tris-HCl buffer, pH 8.0 for RNases HK-2A and HK2B. The other conditions were as described under Materials and Methods. (A) Anti-RNase 1 antibody. (B) Anti-RNase 2 antibody.
RNase, reflecting the analytical data for their amino acid compositions and N-terminal amino acid sequences. On the other hand, the carbohydrate compositions of the two types of kidney RNase were each quite different from those of previously reported and wellcharacterized urine RNases. RNase HK-1 was composed of about 2 fucose, 0.5 mannose, and 3 glucosamine residues per molecule (Table III), whereas in contrast, urine RNase 1 of the nonsecretory type contained about 1 fucase, 3 mannose, and 4 glucosamine residues per mole-
I 2100 Y )I
I
I
1
I
YAYA
A
:‘=r802 i GOz ‘6 4o s g 20E .s 0 I-( -1.0
A/ d
. s-0
P
A
l
OH
ACKNOWLEDGMENTS
/
L@
- 0.5
L 0
1 0.5
I
The authors express their gratitude to Miss Y. Ikehara and Mrs. F. Nakamura for their excellent technical and secretarial assistance. The authors also thank Miss K. Nakano and Miss Y. Tanaka for amino acid analysis and sequence determination.
1.0
Log (&I antibody) FIG. 7.
cule (13). Although RNases HK-2A and HK-2B contained 2.3 and 4.3% neutral sugars and 1.6 and 2.3% amino sugars, respectively, these sugar contents are much lower than those of secretory RNase from human urine (10, 11, 15). These differences in sugar composition indicated strongly that neither of these two urine RNases seems to originate from the kidney, but, further investigation is required in order to elucidate the origin of the enzymes. Furthermore, the difference in molecular weight between RNases HK-2A and HK-2B may be principally due to the carbohydrate moiety, rather than to the polypeptide moiety. These results show that direct and straightforward studies, including carbohydrate and proteochemical analyses for human organs and tissues, instead of other indirect methods involving enzyme assay or immunoassay, are indispensable for elucidating the origin of the various RNases present in body fluids, including serum and urine, because of the high multiplicity or heterogeneity of human RNases.
Inhibitory effects of anti-urine RNase antibodies on RNase activities of crude extract from human kidney. Crude extract from human kidney (Table I, 10 ~1) was incubated with anti-RNase 2 antibody (O), anti-RNase 2 antibody (0), or anti-RNase 1 antibody and antiRNase 2 antibody (A). The residual activity was determined in 0.1 M Tris-HCl buffer, pH 7.5. The other conditions were as described in the legend to Fig. 6.
REFERENCES 1. Reddi, K. K., and Holland,
J. F. (1976) Proc. Nutl. Acad. Sci. USA
73,2308-2310. 2. Renner, I. G., Mock, A., Reitherman, R., and Douglas, A. P. (1978) Gastroenterology 74,1142. 3. Warshaw, A. L., Lee, K.-H., Wood, W. C., and Cohen, A. M. (1980) Amer. J. Surg. 139,27-32.
HUMAN 4. Hishiki,
S., Sakaguchi,
KIDNEY
S., and Kanno, T. (1984) Clin. Chim. Acta
136,155-164. 5. Peterson, L. M. (1979) Proc. Natl. Acad. Sci. USA 76,2630-2634. 6. Weickmann, J. L., Olson, E. M., and Glitz, D. G. (1984) Cancer
Res. 44,1682-X87. 7. Kurihara, M., Ogawa, M., Ohta, T., Kurokawa, E., Kitahara, T., Murata, A., Matsuda, K., Kosaki, G., Watanabe, T., and Wada, H. (1984) Cancer Res. 44,2240-2243. 8. Akagi, K., Murai, K., Hirao, N., and Yamanaka, M. (1976) Bio-
chim. Biophys. Acta 442,368-378. 9. Blank, A., and Dekker, C. A. (1981) Biochemistry 20,2261-2267. 10. Iwama, M., Kunihiro, M., Ohgi, K., and Irie, M. (1981) J. Biochem. 89,1005-1016. 11. Cranston, J. W., Perini, F., Crisp, E. R., and Hixson, C. V. (1980)
Biochim. Biophys. Actu 616,239-258. 12. Sugiyama, R. H., Blank, A., and Dekker, C. A. (1981) Biochemistry 20,2268-2274. 13. Yasuda, T., Sato, W., and Kishi, K. (1988) Biochim. Biophys. Acta 966,185-194. 14. Beintema, J. J., Hofsteenge, J., Iwama, M., Morita, T., Ohgi, K., Irie, M., Sugiyama, R. H., Schieven, G. L., Dekker, C. A., and Glitz, D. G. (1988) Biochemistry 27,4530-4538. 15. Beintema, J. J., Blank, A., Schieven, G. L., Dekker, C. A., Sorrentino, S., and Libonati, M. (1988) Biochem. J. 265,501-505. 16. Beintema, J. J., Wietzes, P., Weickmann, J. L., and Glitz, D. G. (1984) Anal. B&hem. 136.48-64. 17. Hitoi, A., Yamashita, K., Niwata, Y., Irie, M., Kochibe, N., and Kobata, A. (1987) J. Biochem. 101,29-41.
151
RIBONUCLEASES 18. Yasuda, T., Mizuta,
K., and Kishi, K. (1990) Arch. Biochem. Bio-
phys. 279,130-137. 19. Sorrentino, S., Tucker, Chem. 263,16,125-16,131. 20. Strydom, D. J., Fett, J. thune, J. L., Rio&m, J. 24,5488-5494. 21. Shapiro, R., Riordan, J. 25,3527-3532.
G. K., and Glitz,
D. G. (1988) J. Biol.
W., Lobb, R. R., Alderman, E. M., BeF., and Vallee, B. L. (1986) Biochemistry F., and Vallee, B. L. (1986) Biochemistry
22. Gleich, G. J., Loegering, D. A., Bell, M. P., Checkel, J. L., Ackerman, S. J., and McKean, D. J. (1986) Proc. Nutl. Acad. Sci. USA 83,3146-3150. 23. Uchida, T., and Egami, F. (1966) in Procedures in Nucleic Acid Research (Cantoni, G. L., and Davis, D. R., Eds.), pp. 46-55, Harper and Row, New York/London. 24. Laemmli,
U. K. (1970) Nature 227,680-685.
25. Mizuta, K., Yasuda, T., and Kishi, K. (1989) Biochem. Genet. 27, 731-743. 26. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 27. Kishi, K., and Iseki, S. (1983) Proc. Japan Acad. 59B, 240-242. 28. Yasuda, T., Mizuta,
K., Ikehara,
Y., and Kishi,
K. (1989) Anal.
Biochem. 183,84-88. 29. Sierakowska, H., and Shugar, D. (1977) in Progress of Nucleic Acid Research and Molecular Biology (Cohen, W. E., Ed.), Vol. 20, pp. 59-130, Academic Press, Orlando, FL. 30. Morita,
T., Niwata,
Y., Ohgi, K., Ogawa, M., and Irie, M. (1986)
J. Biochem. 99,17-25.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 281, No. 1, August 15, pp. 144-1X,1990
Purification and Characterization of Three Ribonucleases from Human Kidney: Comparison with Urine Ribonucleases’ Keiko Mizuta,2 Shuichi Awazu, Toshihiro
Yasuda, and Koichiro
Kishi3
Department of Legal Medicine, Fukui Medical School, Matsuoku-cho, Fukui 910-l 1, Japan
Received February
51990,
and in revised form April 13,199O
Three ribonucleases (RNases) with different molecular masses were isolated from human kidney. The enzymes were purified to an electrophoretically homogeneous state, and their respective molecular masses were found to be 18,000 (tentatively named RNase HK-l), 20,000 (RNase HK-BA), and 22,000 (RNase HK-2B) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the amino acid compositions, aminoterminal sequences, and enzymological properties of the enzymes indicate that RNase HK-1 is related to “nonsecretory” RNase, and that RNases HK-2A and HK-2B are both related to “secretory” RNase. Furthermore, RNase HK-1 showed cross-reactivity with an antibody specific to nonsecretory RNase from human urine, whereas RNases HK-2A and HK-2B showed cross-reactivity with another antibody specific to human urine secretory RNase. However, the carbohydrate compositions of RNases HK-PA and HK-2B were markedly different from that of the secretory urine RNase. This finding seems to indicate that the kidney is 0 1990 Academic Press. Inc. not the origin of the urine enzyme.
Ribonucleases are widely distributed in various organs and body fluids, including serum, urine, saliva, and cerebrospinal fluid. Since Reddi and Holland first observed that RNase activity was elevated in sera of patients with pancreatic cancer (l), many workers have reported on the relationship between the activity of RNase in serum i This work was supported in part by grants from the Japan Brain Foundation and Tokyo Immunopharmacology Institute, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. * Present address: Department of Biochemistry and Biophysics, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima, 734 Japan. s To whom correspondence should be addressed. 144
or urine and pancreatic diseases: several workers have supported the hypothesis (2-4), whereas others have found little or no correlation (5-7). The molecular multiplicity of RNases has made it difficult to prove the correlation between RNase activity and disease states. Akagi et al. isolated five RNases with different molecular masses of 8500,13,000,20,000,32,000, and 45,000, as determined by gel filtration, from normal human serum (8). Blank and Dekker also showed the multiplicity of RNases in human serum and urine, reporting six serum RNases with molecular masses of 14,000,16,000,20,000, 25,000, 28,000, and 31,000, respectively, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9). Urine RNases consist of two major components, one with a molecular mass of 31,000 (SDS-PAGE)4 named secretory RNase, UL (lo), RNase C (ll), or Band A (12), and the other with a molecular mass of 16,000 (SDSPAGE) named nonsecretory RNase, Us (lo), RNase U (ll), or Band D (12). We have recently purified the nonsecretory RNase of urine and demonstrated genetic polymorphism (13). The nonsecretory RNase of urine has been sequenced and the presence of five short carbohydrate chains established (14). One of the secretory RNases, UL in human urine, has been isolated and its amino acid sequence analyzed (15). The results indicated that UL had almost the same polypeptide chain as human pancreatic RNase (16). However, since their glycosylation characteristics were quite different from each other, it was suggested that the urine enzyme, UL, did not originate in the pancreas (15, 17). The origin of serum RNases also remains unclear. Therefore, for a true understanding of RNases in normal and pathological states, it is important to isolate and characterize RNases from various organs, and to determine the origin of both ’ Abbreviations used: APUP-agarose, agarose-5’-(4-amino phenyl phosphoryl) uridine 2’(3’)-phosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 0003-9s61/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
HUMAN
KIDNEY
urine and serum RNases. Recently, Yasuda et al. purified two distinct types of RNase from human erythrocytes and demonstrated that the enzyme present in a higher content was enzymologically very similar to nonsecretory RNase in human urine (18). Also, Sorrentino et al. purified an RNase from human liver and showed that its enzymatic properties were very similar to those of nonsecretory urine RNase (19). Finally, protein-sequencing studies have yielded evidence that several functionally diverse proteins belong to a superfamily of ribonucleases. Angiogenin, a protein that stimulates the formation of blood vessels, is structurally related to pancreatic (secretory) RNase (20), retaining some nuclease activity (21). Furthermore, an eosinophil-derived neurotoxin with significant RNase activity may be identical with liver (nonsecretory) RNase (22). In this paper, we describe the isolation to homogeneity and characterization of three RNases from human kidney, since human kidney RNases have not yet been purified to homogeneity. We also compare the immunological and biochemical properties of these enzymes with RNases from human urine. MATERIALS
AND
METHODS
Materials All chemicals were of reagent grade. Cellulose phosphate Pll was purchased from Whatman (Maidstone, UK), poly(G)-agarose, poly(G), and molecular weight calibration marker kits were from Pharmacia (Uppsala, Sweden), agarose-B-(l-amino phenyl phosphoryl) uridine 2’(3’)-phosphate (APUP-agarose) was from ICN Immuno Biologicals (Lisle, NY), heparin-actigel from Sterogene Biochemicals (San Gabriel, CA) and Bio-Gel P-100 from Bio-Rad (Richmond, CA). Poly(A), poly(U), and poly(C) were obtained from Seikagaku-Kogyo (Tokyo, Japan) and yeast RNA was from Kojin (Tokyo, Japan).
Assay of RNase Activity The standard assay was performed according to the procedure reported by Uchida andEgami (23) with some modifications. RNA solution (1.2%, 50 ~1) was added to 150 ~1 of reaction mixture consisting of 50 mM Tris-HCl buffer, pH 7.5, 5 mM EDTA, and enzyme. The reaction was performed at 37°C and terminated by adding 50 ~1 of 25% perchloric acid containing 0.75% uranyl acetate. The reaction tubes were chilled in an ice-bath for 15 min, then centrifuged at 1600g for 5 min. A 100~~1 aliquot of the supernatant was diluted with 2.5 ml of distilled water and the absorbance of this solution at 260 nm was measured. One unit of RNase activity was defined as an increase by 1.0 of the absorbance at 260 nm per minute.
Purification of RNases Human kidneys were obtained at autopsy from a 58-year-old male (24 h after death due to burning), a 54-year-old male (14 h after death due to brain contusion), a 38-year-old male (8 h after death due to brain contusion) and a 32-year-old male (24 h after death due to loss of blood). All of these were proved to be histologically normal. The kidneys were stored at -80°C until use. All subsequent steps were carried out at 0-5°C. Step 1. The kidneys (295 g) from the 58-year-old male were partially thawed overnight in a refrigerator then cut into small pieces, the
145
RIBONUCLEASES
3 ! b -1.0
2E I a it 1 -0.5 is s 0
50 Fraction
100
0
‘0.0
number
FIG. 1. Cellulose phosphate column chromatography of RNase-active fractions. Fraction Nos. 76-88 (RNase HK-1 fraction), Nos. lOO110 (RNase HK-PB fraction), and Nos. 111-120 (RNase HK-PA fraction) were pooled separately and dialyzed for further purification. blood was rinsed out with cold saline, and the pieces were suspended in 590 ml of 0.25 M HzS04, containing 1 mM MgCls. The mixture was homogenized with an Ultra-turrax dispenser and centrifuged for 30 min at 12,OOOg.The supematant was collected and its pH was adjusted to 7 with NH,OH. Step 2. The crude extract from step 1 was fractionated with ammonium sulfate. The precipitate obtained between 0.3 and 0.9 saturation was collected and dissolved in ca. 150 ml of water and dialyzed against 5 mM sodium phosphate buffer, pH 6.0. Step 3. The sample from step 2 was centrifuged and the supernatant was applied at 0.2 ml/min to a cellulose phosphate column (2.6 X 37 cm) preequilibrated with 5 mM sodium phosphate buffer, pH 6.0. The column was washed with 100 ml of the same buffer, and then eluted with a linear concentration gradient of NaCl from 0 to 1 M in 3 liters of the same buffer. RNase activity was eluted at a broad range of NaCl concentration from 0.5 to 0.9 M as two peaks (Fig. 1). The second peak showed an asymmetrical pattern in terms of RNase activity. The first RNase-active peak, named RNase HK-1 fraction, and two portions of the second RNase-active peak, the former half named RNase HK-2B and the latter HK-PA, were collected separately and dialyzed against 5 mM Tris-HCl buffer, pH 7.0. Step 4. Each sample from step 3 was centrifuged. The supernatant was finally adjusted to a concentration of 50 mM Tris-HCl buffer, pH 7.0, containing 20 mM NaCl, and then applied at 0.3 ml/min to a heparin-actigel column (1.8 X 8 cm) preequilibrated with 50 mM Tris-HCl buffer, pH 7.0, containing 20 mM NaCI. The column was washed with 50 ml of the same buffer, and then eluted with a linear concentration gradient of NaCl from 20 to 500 mM (for RNase HK-1) or to 700 mM (for RNases HK-2A and HK-2B). The enzyme active fractions were pooled and individually dialyzed against distilled water.
Step 5. Each sample from step 4 was concentrated in uocuo at 30°C to a small volume and applied to a Bio-Gel P-106 column (1.6 X 90 cm) preequilibrated with 10 mM Tris-HCl buffer, pH 7.5, containing 0.3 M NaCl. The enzymes were eluted with the equilibration buffer at a flow rate of 3 ml/h and RNase-active fractions were pooled. The RNase HK-1 fraction was dialyzed against 10 mM sodium phosphate buffer, pH 7.5, and RNases HK-2A and HK-PB fractions were dialyzed against 20 mM sodium acetate buffer, pH 5.0. Step 6. The HK-1 fraction from step 5 was applied to a poly(G)agarose column (0.9 X 7 cm) preequilibrated with 10 mM sodium phosphate buffer, pH 7.5, and eluted with a linear gradient of O-l M NaCl in 100 ml of the same buffer. Peak fractions showing RNase activity were pooled and concentrated by ultrafiltration. The obtained RNase was designated RNase HK-1. If necessary, the enzyme was desalted on a Sephadex G-25 column buffered with 0.2 M acetic acid and lyophilized.
146
MIZUTA
ET AL.
TABLE I Purification Protein (md
Step 1. Extract 2. Ammonium sulfate fractionation 3. Cellulose phosphate 1st fraction (HK-1) 2nd fraction (HK-2B) 3rd fraction (HK-2A) 4. Heparin-actigel HK-1 HK-2A HK-2B 5. Bio-Gel P-100 HK-1 HK-LA HK-2B RNase HK-1 6. Poly(G)-agarose RNases HKBA and HK-2B 7. APUP-agarose HK-2A HK-2B 8. Poly(G)-agarose HK-2A HK-2B
of Human Kidney
RNases
Total activity” (unit)
Specific activity (unit/mg)
13,973
4440
0.318
3,126
3192
1.02
3.21
71.9
28.5 36.2 43.4
38.9
286 675
180
30.9 9.3 7.3
191
1729
9.05
56.0 42.6
645 588
11.5
Purification (-fold)
13.8
15.1
1371
90.8
1.92 5.70
412 326
214.6 57.2
1
Yield (W) 100
13.2 14.5
1.24
612
0.34 0.47
176 162
494 518 345
1553 1629 1085
13.8 4.0 3.6
0.90
506
562
1767
11.4
0.16 0.23
88 122
552 530
1736 1667
2.0 2.7
0.137 0.198
79 108
577 545
1814 1714
2.4
0 Activities of RNases were measured in 0.1 M Tris-HCl buffer, pH 7.0, for RNase HK-1, or in 0.1 M Tris-HCl HK-2A and HK-2B using RNA as substrate, as described under Materials and Methods. Step 7. RNases HK-2A and HK-2B fractions from step 5 were separately applied to an APUP-agarose column (0.9 X 6 cm) preequilibrated with 20 mM sodium acetate buffer, pH 5.0, and eluted with a linear concentration gradient of NaCl from 0 to 1.2 M in the same buffer. The RNases were each eluted with ca. 0.6 M NaCl and dialyzed against 10 mM sodium phosphate buffer, pH 7.5.
1.8
buffer, pH 8.0 for RNases
Step 8. Each of the samples from step 7 was applied to a poly(G)agarose column (0.9 X 7 cm) preequilibrated with 10 mM sodium phosphate buffer, pH 7.6. The enzymes were eluted with 1 M NaCl in the same buffer. The obtained RNases were designated RNase HK-2A and RNase HK-BB, respectively. These enzymes were desalted and lyophilized for proteochemical analysis
Protein Characterization
12
3
4
12
3
4
5
6
FIG. 2. SDS-PAGE of purified RNases HK-1, HK-2A, and HK-2B. (A) Protein staining with Coomassie blue R250. Lane 1, RNase HK1; lane 2, RNase EK-ZA, lane 3, RNase HK-2B; lane 4, molecular weight markers: 1, phosphorylase b (94 kDa); 2, bovine serum albumin (67 kDa); 3, ovalbumin (43 kDa); 4, carbonic anhydrase (30 kDa); 5, trypsin inhibitor (20 kDa); 6, a-lactalbumin (14.4 kDa). (B) Activity staining on SDS-polyacrylamide gel casting poly(C) (lanes l-3) or poly(U) (lanes 4-6). Lanes 1 and 4; RNase HK-1, lanes 2 and 5; RNase HK-BA, lanes 3 and 6; RNase HK-2B. The arrow indicates the origin of electrophoretogram.
SDS-PAGE was conducted in 12.5% polyacrylamide gel according to the method of Laemmli (24). Samples were dissolved in buffer containing 10 mM Tris-HCl, pH 6.8,10% glycerol, 2% SDS, 0.2% 2-mercaptoethanol, and a trace of bromphenol blue. Electrophoresis was done for 80 min at a constant current of 20 mA. In order to detect the RNase activity, poly(C) or poly(U) (ca. 0.2 mg/ml) was added to the separating gel solution prior to polymerization (9). After the electrophoretic run, the gel was incubated for 3 h in 50 mM Tris-HCl buffer, pH 7.5, at 37°C and then stained with 0.2% toluidine blue. Amino acid analysis was done using a Hitachi 835 amino acid analyzer equipped with a column (0.26 X 25 cm) of Hitachi X2619 resin (Tokyo, Japan) using ca. 30-pg samples that had been hydrolyzed at 110°C in 6 N HCl for 24 h. Automatic Edman degradation of native RNases (ca. 10 gg) was performed on an automated gas-phase protein sequencer (Applied Biosystems 477A, Foster City, MI); phenylthiohydantoin derivatives were identified with an HPLC apparatus attached to the sequencer. Amino sugars liberated from the enzymes by hydrolysis in 6 N HCl were quantitated with a Hitachi 835 amino acid analyzer. The composition of neutral sugar was determined by gas chromatography-electron ionization mass spectrometry. Analysis was performed on a JMSDX 300 instrument (JEOL, Tokyo, Japan). Analytical conditions and
147
HUMAN KIDNEY RIBONUCLEASES TABLE
II
Amino Acid Compositions of RNasesfrom Human Kidney Urine RNases
Kidney RNases
Amino acid HK-1
Nonsecretory”
Secretory *
HK-2A
HK-2B
11.3
11.3
15.7
11.7
5.0
5.5 11.7
5.6 11.4
11.7
10.8
10.3
2.3 5.3 5.3 6.6 2.8 4.0 3.8 2.6 3.8 4.0 3.5 5.6
4.1 3.5 8.3 5.8 4.2 1.8 1.8 3.4 3.2 6.5 3.9
4.4 3.2 7.6 6.6 4.4 2.9 1.5 3.0 3.5 6.2 3.7
ND’
9.1 ND
10.2 ND
8.6
5.1
4.2
9.0 4.5 10.5 1.5 4.5 6.8 6.0 3.0 5.3 3.8 3.0 3.8 3.0 3.8 6.0 0.8 9.0
6.3 12.4 10.2 3.9 3.1 7.8 6.3 3.9 2.3 1.6 3.9 3.1 6.3 3.9 7.8 0 5.5
mol% Asx Thr Ser Glx
16.2 8.9
GUY
Ala Val CYS
Met Ile Leu ‘G-r
Phe LYS His
Arg Trp Pro
’ Calculated from the sequence from Ref. (14). * Calculated from the sequence from Ref. (15). ’ ND, not determined.
preparation of the alditol trifluoroacetate derivatives of neutral sugars were described in a previous paper (26). Protein concentration was determined according to the method of Lowry et al. (26) using bovine serum albumin as a standard.
Production
of Antibodies
and Immunoblotting
Nonsecretory (RNase 1) and secretory (RNase 2) RNases were both purified from humanurine (13). Antibodiesspecificto the purified RNases were raised in rabbits as described in a previous paper (27). Immunoblotting after SDS-PAGE was performed according to the method described in our previous paper (28).
detection. Table I is a typical example from among the four preparations. Figure 2 shows the SDS-PAGE patterns of three purified RNases under reducing conditions. When these enzymes were subjected to SDS-PAGE in poly(U)-cast gel, all of them showed very similar activities, whereas in poly(C)-cast gel, RNase HK-1 showed only very weak activity in comparison with RNases HK-2A and HK-2B. Each enzyme band detected by enzymological staining corresponded to the band detected by protein staining, and vice versa. Molecuhr
Mass
The molecular masses of RNases HK-1, HK-BA, and HK-2B were estimated to be 18,000, 20,090 and 22,000, respectively, on SDS-PAGE under reducing conditions (Fig. 2). Chemical Composition
In Table II, the amino acid compositions of the purified kidney RNases are shown in comparison with those of previously reported urine RNaaes. The amino acid composition of RNase HK-1 was observed to be very similar to that of nonsecretory RNase from human urine. On the other hand, there was close similarity in the amino acid compositions of RNases HK-2A and HK2B and secretory RNase from human urine. However, the carbohydrate compositions of the kidney RNases were very different from the compositions of urine RNases, as shown in Table III. Fucose was the major neutral sugar of RNase HK-1. RNases HK-2A and HK2B both contained more mannose than fucose. RNase HK-2B contained a larger amount of carbohydrate than RNase HK-2A. The amino sugar contents of the three
TABLE III
Carbohydrate Compositions of RNasesfrom Human Kidney
RESULTS Enzyme Purification
The preparation of three RNases from 295 g of autopsied human kidney is summarized in Table I. We tried to isolate these RNases individually from kidneys obtained from four different individuals. The specific activity of the crude preparations (step 1) ranged from 0.32 to 0.5 units/mg protein, although the elution patterns of RNase activity on the cellulose phosphate column differed markedly among the four preparations, the RNase HK-1 accounting for 39,58, 73, and 84% of the total activity. However, three kinds of RNase were demonstrated to exist in each of the preparations by electrophoresis followed by enzymological and immunological
Kidney RNases Carbohydrate component
HK-1
HK-2A
Urine RNases
HK-PB
Nonsecretory”
Secretory*
Residues/molecule Glucosamine’ Galactosamine’ Fucose Mannose Galactose
3.0 NDd
1.5 ND
2.3
4.4
ND
ND
2.2 0.45
1.2 1.6
2.5 3.0
1.4 2.8
Trace”
Trace
Trace
’ Taken from Ref. (13). * Taken from Ref. (15). ’ The amino sugars were calculated as the N-acetyl form. d ND, not detected. eTrace, trace amount was detected.
16.7 3.3 8.9
10.9
148
MIZUTA Human
5
ET AL. 10
15
20
25
30
Kidney RNase HK-1
KPPQFT?AQ?FETQHI?HT
SQQ?TNANQVINNY
Nonsecretory urinary RNase
KPPQFT?AQWFETQHINMT
SQQCTNANQVINNY
Liver RNase
RPPQPT?AQWFETQHINM(T)(T)QQ?TNA
Human Kidney RNase HK-2A
KESRAK(K)FQRQHMDS?SSPS
Kidney RNase HK-2B
KESRAKKPQRQHMDSDSSP
Secretory urinary RNase
KESRAKKPQRQHMDSDSSPS
Pancreatic RNase
KESRAKKFQRQHMDSDSSPS
FIG. 3.
N-terminal amino acid sequences of human kidney RNases. For comparison, sequences of human urinary RNases (14, IS), human liver RNase (19) and human pancreatic RNase (16) are also shown. Question marks indicate the undetermined positions. Parentheses indicate tentatively identified residues.
kidney RNases were entirely in the form of glucosamine with no apparent galactosamine residues. The N-terminal amino acid sequence of RNase HK-1 was very similar to those of urine nonsecretory RNase (14) and liver RNase (19), while RNases HK-2A and HK-2B had an N-terminal amino acid sequence very similar to those of urine secretory RNase (15) and pancreatic RNase (16) (Fig. 3).
As shown in Table IV, poly(A) and poly(G) were not hydrolyzed by any of the kidney RNases described. RNase HK-1 exhibited greater preference for poly(U) than poly(C) as a substrate. On the other hand, RNases HK2A and HK-2B reacted much more actively with poly(C) than with poly(U). The three kidney RNases all appeared to be specific for cleavage at pyrimidine residues in RNA.
Enzymatic Properties
Immunological Properties
The effects of pH on the activities of the purified RNases were examined using yeast RNA as a substrate. The pH optimum for RNase HK-1 was 6.5-7.0, while those for RNases HK-2A and HK-2B were both 8.0 (Fig. 4). For measuring the substrate specificity of the kidney RNases, the yeast RNA substrate was replaced by synthetic homopolyribonucleotides in the standard assay.
Figure 5 shows the results of immunoblotting of kidney RNases with two kinds of anti-urinary RNase antibody with different specificities after SDS-PAGE. The agarose gel diffusion test showed that RNase HK-1 was reactive with anti-RNase 1 (nonsecretory RNase from human urine) antibody as well as with anti-RNase HK1 antibody, but not with anti-RNase 2 (secretory RNase from human urine). In contrast, RNases HK-2A and
4
6
8 PH
10
I
I
I
I
4
6
8
10
PH
4
6
8
10
PH
FIG. 4. Effects of pH on activities of RNases HK-1, HK-PA, and HK-2B. (A) RNase HK-1. (B) RNase HK-2A. (C) RNase HK-PB. The buffers used were 0.1 M sodium acetate buffer (0), 0.1 M sodium cacodylate buffer (O), 0.1 M Tris-HCl buffer (A) and 0.1 M sodium glycine buffer (A). The other experimental conditions were as described under Materials and Methods.
HUMAN TABLE Substrate
Specificity”
IV
of Human Activity*
Substrate RNA POlYC) POlYW) Poly(A) Poly (G)
RNase HK- 1 100 6 43 ND’ ND
KIDNEY
Kidney (%)
RNase HK-2A 100 4100 12 ND ND
RNases
RNase HK-2B 100 4400 15 ND ND
a The reaction mixture (0.2 ml) consisted of 0.2 mg of the substrate indicated, 0.1 M Tris-HCl buffer, pH 7.0, for RNase HK-1 or 0.1 M Tris-HCl buffer, pH 8.0, for RNases HK-PA and HK-PB. The other conditions were as described under Materials and Methods. * Activity is expressed relative to that for RNA. ’ ND, not detected.
HK-2B were both reactive with anti-RNase 2, but not with anti-RNase 1 or with anti-RNase HK-1 antibody (data not shown). Furthermore, the inhibitory effects of the antibodies on RNase catalytic activities are shown in Fig. 6. The anti-RNase 1 antibody completely blocked the enzyme activity of the purified RNase HK-1, whereas it was ineffective on the activities of RNases HK-2A and HK-2B. Conversely, the anti-RNase 2 antibody completely blocked the activities of RNases HK2A and HK-2B, but not the activity of RNase HK-1. As shown in Fig. 7, these results indicate that the RNase reactive with anti-RNase 1 antibody accounts for about 50% of the total RNase activity in the kidney extract, while the RNase reactive with the anti-RNase 2 antibody accounts for about 45%. At least 90% of the total RNase activity in the kidney was inhibited in the presence of both the anti-RNase 1 and anti-RNase 2 antibodies.
149
RIBONUCLEASES
urine contains both types. Purification and characterization has been described by several groups (10, 11,14, 15). We have separately purified these enzymes from human urine (13) and designated the nonsecretory type as RNase 1 and the secretory type as RNase 2. Considering the enzymatic properties of these kidney RNases and the classification criteria for human RNase, RNase HK1 is considered to be the nonsecretory type, and both RNases HK-2A and HK-2B the secretory type. Furthermore, their similarities of amino acid composition, Nterminal amino acid sequence, and reactivities against two kinds of RNase-specific antibody reinforced the validity of this classification. Sorrentino et al. (19) found that the amount of nonsecretory RNase in the kidney was about 60-70% of the total activity, which is somewhat higher than the result of Morita et al. (30), which was about 20-30%. In the present study, the content of the nonsecretory enzyme, RNase HK-1, was 58% at the step of cellulose phosphate column chromatography, which allows good separation between secretory and nonsecretory RNases (Table I). However, the amount of the secretory enzyme varied markedly among the preparations from four different individuals, and accordingly the content of the nonsecretory enzyme ranged from 39 to 84% in the four preparations. We were unable to identify any factors, e.g. age, sex, cause of death of the individuals, or time elapsed between death and autopsy which could be related to this great variation. Morita et al. speculated that a new RNase, belonging neither to the secretory nor nonsecretory type (30), could be present, although we found no evidence for the existence of any such new RNase in the kidney during the purification process. Figure 7 also shows that the total of the secretory and nonsecretory RNase activities reached more than 90% of the total RNase activity in the crude extract from human kidney. The polypeptide portions of the kidney RNases were very similar to that of either nonsecretory or secretory
DISCUSSION
In this study, three kidney RNases were isolated for the first time and comprehensively characterized, following the prediction of the presence of at least two kinds of RNase (19,27,30). These enzymes have molecular masses of 18,000, 20,000, and 22,000, respectively, as determined by SDS-PAGE, and are designated RNases HK-1, HK-BA, and HK-BB, respectively. RNase HK-1 is poly(U)-preferential, showing maximal activity at pH 6.5-7.0. Both RNases HK-2A and HK-2B are highly poly(C)-preferential, having an optimal pH around 8.0. Sierakowska and Shugar (29) classified mammalian RNases into two types, namely “secretory” and “nonsecretory.” The former, resembling pancreatic RNase, is characterized by a high pH optimum and preference for poly(C) as a substrate, whereas the latter has a lower pH optimum and preference for poly(U). Human
I.1
12
3
_I ‘I
12
3
FIG. 5. Immunoblotting patterns of kidney RNases with two kinds of anti-urine RNase antibody after SDS-polyacrylamide gel electrophoresis. (A) Anti-RNase 1 (nonsecretory type) antibody. (B) AntiRNase 2 (secretory type) antibody. Samples were RNase HK-1 (lane 11, RNase HK-2A (lane 2), and RNase HK-PB (lane 3). The arrow indicates the origin of electrophoretogram.
150
MIZUTA
Sloe >r ‘2 80 5 -!
ET AL.
A /---
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/ A
-0.5
0
0.5
Log (~1 antibody)
1.0
- 0.5
0 0.5 Log(,uul antibody)
1.0
FIG. 6. Inhibitory effects of anti-urine RNase antibodies on enzyme activities of purified human kidney RNases. Incubation mixtures (20 ~1) consisting of 0.26 pg of the purified enzyme; RNase HK-1 (A), RNase HK-PA (0), or RNase HK-PB (O), 20 mM Tris-HCl buffer, pH 7.0, 0.15 M NaCl, and serially diluted antibody were held at 20°C for 2 h. They were then centrifuged and their RNase activities in the supernatant (10 ~1) were determined in 0.1 M Tris-HCl buffer, pH 7.0, for RNase HK-1, or in 0.1 M Tris-HCl buffer, pH 8.0 for RNases HK-2A and HK2B. The other conditions were as described under Materials and Methods. (A) Anti-RNase 1 antibody. (B) Anti-RNase 2 antibody.
RNase, reflecting the analytical data for their amino acid compositions and N-terminal amino acid sequences. On the other hand, the carbohydrate compositions of the two types of kidney RNase were each quite different from those of previously reported and wellcharacterized urine RNases. RNase HK-1 was composed of about 2 fucose, 0.5 mannose, and 3 glucosamine residues per molecule (Table III), whereas in contrast, urine RNase 1 of the nonsecretory type contained about 1 fucase, 3 mannose, and 4 glucosamine residues per mole-
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l
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ACKNOWLEDGMENTS
/
L@
- 0.5
L 0
1 0.5
I
The authors express their gratitude to Miss Y. Ikehara and Mrs. F. Nakamura for their excellent technical and secretarial assistance. The authors also thank Miss K. Nakano and Miss Y. Tanaka for amino acid analysis and sequence determination.
1.0
Log (&I antibody) FIG. 7.
cule (13). Although RNases HK-2A and HK-2B contained 2.3 and 4.3% neutral sugars and 1.6 and 2.3% amino sugars, respectively, these sugar contents are much lower than those of secretory RNase from human urine (10, 11, 15). These differences in sugar composition indicated strongly that neither of these two urine RNases seems to originate from the kidney, but, further investigation is required in order to elucidate the origin of the enzymes. Furthermore, the difference in molecular weight between RNases HK-2A and HK-2B may be principally due to the carbohydrate moiety, rather than to the polypeptide moiety. These results show that direct and straightforward studies, including carbohydrate and proteochemical analyses for human organs and tissues, instead of other indirect methods involving enzyme assay or immunoassay, are indispensable for elucidating the origin of the various RNases present in body fluids, including serum and urine, because of the high multiplicity or heterogeneity of human RNases.
Inhibitory effects of anti-urine RNase antibodies on RNase activities of crude extract from human kidney. Crude extract from human kidney (Table I, 10 ~1) was incubated with anti-RNase 2 antibody (O), anti-RNase 2 antibody (0), or anti-RNase 1 antibody and antiRNase 2 antibody (A). The residual activity was determined in 0.1 M Tris-HCl buffer, pH 7.5. The other conditions were as described in the legend to Fig. 6.
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