]. Biochem., 80, 19-26 (1976). Dedicated to Prof. N. Shimazono on his 70th birthday.
Purification and Properties of an Alkaline Ribonuclease from the Hepatic Cytosol Fraction of Bullfrog, Rana catesbeiana
Department of Biochemistry, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113 Received for publication, December 22, 1975
In the hepatic cytosol fraction of bullfrog, Rana catesbeiana, an alkaline RNase [EC 3.1.4. 22] exists in two forms. One is the free form of RNase, which elutes from a carboxymethyl-cellulose column at a concentration of 0.2 M NaCl. The other is a masked or latent form (RNase-RNase inhibitor complex) which is not adsorbed on the carboxymethyl-cellulose column and which can be converted to the free form of RNase by the addition of />-chloromercuribenzoate. Electrophoretically pure RNase was obtained by the following procedure. The unadsorbed fraction of hepatic cytosol on a column of carboxymethyl-cellulose was treated with />-chloromercuribenzoate and then applied to a second carboxymethyl-cellulose column. The molecular weight of RNase was determined to be approximately 12,000 by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. From the results of gel filtration, the molecular weight of the RNaseRNase inhibitor complex was 130,000. The RNase hydrolyzed poly C, poly U, and poly I, but not poly A or poly G. When poly C was used as a substrate, 2', 3'-cyclic CMP as an intermediate and 3'-CMP as a final product were identified. The results of amino acid analysis indicated the presence of an unusual component. The general properties of the RNase and the RNase-RNase inhibitor complex are also reported.
Since Roth ( / ) and de Lamirande et al. ( 2 ) demonstrated the presence of two types of RNase in mammalian liver, i.e. alkaline RNase and acid RNase, evidence for the occurrence of several RNases differing in pH optimum, , r,
.,
^
1
.
. ,D.
intracellular localization, divalent cation requirement and so on in mammalian liver has been summarized (3). An alkaline RNase from bovine liver has been characterized and reported to have catalytic properties similar to those of pancreatic RNase (4). It has been stated by Roth (5) that the RNase inhibitor
• , • ,n,
Present address: Department of Physiological Chemistry and Nutrition, Faculty of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113. Abbreviations used : SDS, sodium dodecyl sulfate ; PCMB, />-chloromercuribenzoate. Vol. 80, No. 1, 1976
"""•>-»* "J « v / 8 I i v e r supernatant is not present in free form but in a complex form bound with RNase. In this paper, we will describe (I) the separation of an alkaline RNase and the
in fr0
19
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Hiroshi NAGANO,1 Hiroyuki KIUCHI, Yasuko ABE, and Ryoiti SHUKUYA
H. NAGANO, H. KIUCHI, Y. ABE, and R. SHUKUYA
20
RNase-RNase inhibitor complex from bullfrog liver cytosol, (II) the properties of the complex, (III) the purification and characterization of the alkaline RNase. MATERIALS AND METHODS
Protein was determined by the method of Lowry et al. ( 7 ) with bovine serum albumin as a standard. Sodium chloride concentration in column effluents was determined using a conductivity meter. Assay of RNase Activity—The reaction mixture for activity measurements of RNase contained 0.1 mmole of Tris-HCl buffer, pH 7.5, 2 mg of yeast RNA and an appropriate amount of RNase solution in a final volume of 0.8 ml. After incubation at 37° for 10 min, 0.2 ml of ice-cold 25% perchloric acid-0.25% uranium acetate mixture was added. The precipitate of RNA and protein was discarded by centrifugation at 3,000 rpm for 10 min after standing in ice for 30 min. The supernatant solution was diluted 10 times with distilled water and its absorbance at 260 nm was determined. As a control, enzyme solution was added after the addition of the perchloric aciduranium acetate mixture. A unit of RNase activity is denned as one absorbance unit released under the above conditions. The latent RNase activity was determined using the above reaction mixture containing 1 mM PCMB. Purification of an Alkaline RNase—Freshly obtained bullfrog liver (70 g) was homogenized in 4 volumes of 0.25 M sucrose using a Teflon homogenizer. A precipitate was removed by centrifugation at 105,000X0 for 60
Polyacrylamide Gel Electrophoresis — The polyacrylamide gel electrophoresis procedure of Reisfeld et al. for basic proteins (8) was used. Condensing and sample gels were not used. The sample protein (0.07 mg) in 0.1 ml of 12% glycerol was applied directly to a separating gel. Electrophoresis was performed at 4 mA per tube (5 m m x 5 cm) until the tracking dye (pyronine Y) moved to the bottom of the gel tube. Protein bands were stained with Amido Black. The staining of RNase activity on polyacrylamide gels was performed according to the method of Wolf (9). Electrophoresis in the presence of SDS to estimate polypeptide chain molecular weights was conducted by the method of Shapiro et al. (10). Molecular Weight Determination by Gel Filtration — According to the method of Andrews (11), gel filtration using Sephadex G-75 was employed to estimate the molecular weight of alkaline RNase. To a column (3x 86 cm) of Sephadex G-75 equilibrated with 50 mM phosphate buffer, pH 7.2, a sample solution (1.6 ml) containing 0.5 ml of RNase solution (40 units), 0.1 ml of human hemoglobin /. Biochtm.
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Adult bullfrogs and tadpoles were purchased from Nozawaya and Shiihashi, Tokyo, respectively. Ampholine carrier ampholyte (pH 3— 10, 40% w/v), cytochrome c, and a-chymotrypsin [EC 3.4.21.1] were from LKB, Sankyo and Sigma Co., respectively. Rabbit muscle pyruvate kinase [EC 2.7.1.40], yeast alcohol dehydrogenase [EC 1.1.1.1], poly A, poly G, poly I, poly U, poly C, 5'-CMP, 3'-CMP, 2'(3')CMP, 2', 3'-cyclic CMP, and cytidine were from Boehringer Mannheim. Yeast RNA, from Wako Pure Chemicals, was purified by the method of Kirby (6).
min. The supernatant solution was dialyzed twice against 5,000 ml of 10 mM phosphate buffer, pH 6.5, and applied to a column (6.5 X 29 cm) of CM-cellulose which had been equilibrated with 10 mM phosphate buffer, pH 6.5. Elution was carried out with the same buffer. Fractions of 16 ml were collected. The unadsorbed fraction containing the latent RNase was pooled (710 ml) and treated with PCMB (1 mM final concentration). The treated fraction was again applied to a column (4.5x34 cm) of CM-cellulose, washed with 500 ml of 10 mM phosphate buffer, pH 6.5, and eluted with a linear gradient prepared from 1,000 ml of 10 mM phosphate buffer, pH 6.5, in the mixing chamber and 1,000 ml of the same buffer containing 0.5 M NaCl in the reservoir. Fractions of 22 ml were collected. The fraction containing RNase activity was pooled, dialyzed against 5,000 ml of 10 mM phosphate buffer, pH 6.5, and applied to a third column (3.5x20 cm) of CM-cellulose previously equilibrated with the same buffer. The conditions of elution were the same as for the second CM-cellulose column chromatography.
BULLFROG LIVER RNase
Paper Chromatography for Identification of the Catalytic Products—Toyo chromatography paper No. 51 was used with the following solvents. Solvents I (12), saturated ammonium sulfate-0.5M sodium acetate-2-propanol (40 : 9 : 1, v/v/v); solvent II (13), solid ammonium sulfate-0.1 M potassium phosphate buffer, pH 7.0 (2 : 5, w/v); solvent III (13), 95% ethanol-1 M ammonium acetate buffer, pH 7.5 (5 : 2, v/v). Chromatographs were run for 11—27 hr using ascending systems. Amino Acid Analysis—About 0.88 mg of the purified alkaline RNase was mixed with 2 ml of 6 N HC1 in a hydrolysis tube. Hydrolysis was performed in vacuo at 110° for 24, 48, or 72 hr. Amino acid analysis was carried out by the method of Moore and Stein (14) using a Hitachi KLA-5 amino acid analyzer. Performic acid oxidation was conducted according to the method of Hirs (75). Electrofocusing—%'or determination of the isoelectric point of the alkaline RNase, electrofocusing was performed using a carrier amVol. 80, No. 1, 1976
pholyte with a pH range of 3—10 and an electrofocusing column of 110 ml capacity. The column solution was prepared as described in the LKB instruction manual. The protein was electrofocused for 48 hr at 1,000 V. Fractions of 2 ml were collected. A Beck man expandomatic SS-2 pH meter was used for pH determination. RESULTS Alkaline RNase and Its Complex with RNase Inhibitor—Liver supernatant after centrifugation at 105,000X0 for 60 min was applied to a column of CM-cellulose equilibrated with 10 mM phosphate buffer, pH 6.5. As shown in Fig. 1A, RNase activity which developed with PCMB (Peak I) was not adsorbed. RNase eluted at 0.2 M NaCl (Peak II) exhibited the same activity in the presence and in the absence of PCMB. When Peak I or Peak II was rechromatographed under the same conditions, the elution pattern did not change. The amounts of RNase activity found in Peak I (latent form) and Peak II (free form) were calculated to be 0.6 ±0.2 unit/mg wet weight and 3.5±0.8 units/mg wet weight, respectively, using the larval form of bullfrog liver. Peak I of Fig. 1A (unadsorbed fraction) was treated with 1 mM PCMB and applied to a second CM-cellulose column (Fig. IB). RNase activity was eluted at 0.2 M NaCl; it exhibited the same activity in the presence and absence of PCMB. The following points are suggested by the above results. (1) RNase found in Peak I of Fig. 1A may be a complex of RNase and RNase inhibitor, because RNase inhibitor is known to be inactivated by PCMB. (2) RNase activity eluted with 0.2 M NaCl (Fig. 1A) may be due to free RNase. (3) The free form of RNase and the RNase released from the bound form appear to be identical, because, as shown in Fig. 1, these activities are eluted at the same concentration of NaCl. The molecular weight, pH optimum, heat stability, base specificity, and optimal temperature of both RNases were identical. (4) As RNase inhibitor released from the complex is inactivated by PCMB, its elution position could not be determined in Fig. IB.
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solution (molecular weight, 64,000) obtained after hemolysis with 3 volumes of water, 2 mg of bovine pancreatic a-chymotrypsin (molecular weight, 21,600) and 2 mg of horse heart cytochrome c (molecular weight, 12,300) was applied. Fractions of 2.9 ml were collected. The elution patterns of hemoglobin and cytochrome c were determined from the absorbances at 500 nm and 410 nm, respectively. The activity of a-chymotrypsin was determined using N-acetyltyrosine ethyl ester as a substrate. For estimation of the molecular weight of the RNase-RNase inhibitor complex, a column (2.8x24.4 cm) of Sephadex G-200 equilibrated with 50 mM phosphate buffer, pH 7.2, was used. To the complex solution (1.1 ml) prepared as shown in Fig. 1A (48 units of RNase activity in the presence of PCMB), 0.2 mg of rabbit muscle pyruvate kinase (molecular weight, 220,000), 0.3 mg of yeast alcohol dehydrogenase (molecular weight, 150,000) and 0.1 ml of human hemolyzate prepared as described above, were added. Fractions of 2 ml were collected. The position of the complex was determined by assaying RNase activity in the presence of 1 mM PCMB.
21
H. NAGANO, H. KIUCHI, Y. ABE, and R. SHUKUYA
22
20 40 60 FRACTION NUMBER
Fig. 1. CM-cellulose column chromatographic profiles of the free and latent forms of RNase. (A) Separation of the free and latent RNase on a CMcellulose column from bullfrog liver supernatant. The supernatant of 20% homogenate from 0.34 g of fresh liver was applied to a column (2x5 cm) of CM-cellulose equilibrated with 10 mM phosphate buffer, pH 6.5. Elution was carried out with a linear gradient prepared from 100 ml of 10 mM phosphate buffer, pH 6.5 in the mixing chamber and 100 ml of the same buffer containing 0.5 M NaCl in the reservoir. Fractions of 3.1 ml were collected. O, RNase activity in the presence of 1 mM PCMB; • , RNase activity in the absence of PCMB ; , NaCl concentration. (B) Rechromatography of the latent RNase treated with 1 mM PCMB. PCMB was added to the Peak I of Fig. 1A (16.6 ml) to a final concentration of 1 mM and the mixture was applied to a column of CM-cellulose. The chromatographic conditions and symbols are as described in Fig. 1A.
Molecular Size of the Complex—The molecular size of the RNase-RNase inhibitor complex (latent RNase) was determined approximately. Peak I fraction of Fig. 1A was applied to a column of Sephadex G-200. RNase activity in the presence of PCMB was eluted just behind of yeast alcohol dehydrogenase, suggesting an approximate molecular weight of 130,000 daltons. Stability of the Complex—Figure 2 shows the stability of the complex at various pH's.
20 40 60 80 TEMPERATURE (•)
100
Fig. 3. Heat stability of the RNase-RNase inhibitor complex. A solution (0.5 ml) containing 0.2 ml of 0.5 M Tris-HCl buffer, pH 7.5, and 10 ft] of Peak I of Fig. 1A (about 1.1 units of activity in the presence of PCMB) was treated for 10 min at the indicated temperature. RNA solution ( • ) or RNA solution and PCMB (O) were added to the heattreated solution to a final volume of 0.8 ml.
/. Biochem.
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0
Fig. 2. pH-stability curve of the RNase-RNase inhibitor complex. A solution containing 0.2 ml of 50 mM acetate buffer (pH 4.5-6.5) or 50 mM TrisHC1 buffer (pH 6.5-9.5) and 5 fi\ of the unadsorbed fraction from CM-cellulose column chromatography (about 0.7 unit in the presence of PCMB) was incubated at 37° for 10 min. After incubation, 0.2 ml of 0.5 M Tris-HCl buffer, pH 7.5, was added and RNase activity in the presence (O) or absence ( • ) of 1 mM PCMB was assayed as described in the text.
BULLFROG LIVER RNase TABLE I. Summary of the purification procedure for alkaline RNase.
Fraction
Volume ^ p r o t e i n
Supernatant
290
First CM-cellulose Second CM-cellulose Third CM-cellulose
710 620
3,400 2,630 11.6
273
2.3
Reactivity 43,500" 39,050 34,100 20,500
Recovery
Specificity
(100)" 89.7 78.9 47.4
12.6 14.8 2,940 9,100
Purification
m1.2 233 722
About half of the complex released RNase on treatment at pH 5.0 for 10 min at 37°. The complex is stable at pH 6.5—8.0 under the conditions used. Heat treatment at 40° for 10 min at pH 6.5 inactivated 50% of the inhibitor and treatment at 60° for 10 min decomposed the complex completely (Fig. 3). Purification of Alkaline RNase—Purification of the RNase from bullfrog liver was carried out by the following procedure. (1) The unadsorbed fraction of hepatic cytosol was obtained using a CM-cellulose column. This fraction contains the latent form of RNase and obviously does not contain material which can be adsorbed on a CM-cellulose column. (2) The unadsorbed fraction was treated with 1 mM PCMB. The released RNase can be adsorbed on a CM-cellulose column as shown in Fig. IB. However, nearly all other components will not be retained. Thus, pure RNase can be obtained easily. The purification procedure is summarized in Table I. As the RNase fraction obtained from the second CM-cellulose column chromatography showed multiple protein bands on a polyacrylamide gel, a third CM-cellulose column chromatography was carried out under the same conditions as for the second column. Finally, 2.3 mg of protein was obtained from 70 g of liver. This RNase was subjected to polyacrylamide gel electrophoresis according to the method of Reisfeld etal. (8). As shown in Fig. 4A, electrophoresis showed one major band and a faint band which migrated slightly faster than the major band. Both protein bands showed RNase activity (Fig. 4B). The major band was estimated to represent more than 98% of the total as determined densitoVol. 80, No. 1, 1976
Fig. 4. Polyacrylamide gel electrophoreses of the alkaline RNase in /)-alanine/acetic acid buffer (pH 4.5). (A) Protein bands were stained with Amido Black. (B) Staining of RNase activity. The method of Wolf (9) was employed for activity staining.
metrically. Therefore, the final preparation appears to be practically homogeneous. Molecular Weight of the Alkaline RNase— Two different methods were employed for determination of the molecular weight. A plot of log molecular weight versus elution volume was nearly linear from the results of gel filtration using Sephadex G-75. The RNase activity was eluted at the same position as cytochrome c, indicating that the molecular weight of the RNase is about 12,000. A molecular weight of approximately 12,000 was also estimated from a plot of log molecular weight against relative mobility on polyacrylamide gel electrophoresis in the presence of SDS. RNase again migrated to the same position as cytochrome c. These results indicate that the RNase is composed of a single polypeptide chain. Isoelectric Point— Isoelectric point of RNase was determined by electrofocusing. As shown
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Free form of RNase activity is excluded.
H. NAGANO, H. KIUCHI, Y. ABE, and R. SHUKUYA
24
TABLE III. Substrate specificity of the alkaline RNase. RNase activity was assayed under the standard conditions using yeast RNA or homopolyribonucleotides as substrates. The amount of enzyme used was 0.18 unit (0.02 fig of protein). Substrate RNA
60
20 30 40 50 FRACTION NUMBER
Fig. 5. Electrofocusing of the alkaline RNase. A crude sample containing about 6,000 activity units (5 mg of protein) was subjected to electrofocusing. TABLE II. Amino acid analysis.1 Amino acid
Bullfrog liver
Bovine pancreasb
Tryptophan Lysine Histidine Arginine Aspartic acid Threonine d Serine d Glutamic acid Proline Glycine Alanine Half-cystine" Valine Methionine Isol;ucine Leucine Tyrosine Phenylalanine "X"
NDC
0
7
10
3
4
4
4
10
15
7
10
7
15
11
12
6
4
14
3
13
12
2
8
8
9
1
4
5
3
2
2
6
6
3
3
3
a
The nearest integer, assuming a molecular weight of approximately 12,000. ° Ref. 20. c ND, not determined. d Extrapolated to zero time of hydrolysis. • Determined after performic acid oxidation.
in Fig. 5, a p / of 9.4 was determined. Amino Acid Analysis — The results of amino acid analysis are summarized in Table
1
C U A G I
1.25 2.5
0 0 0.09
II. The nearest integer of each amino acid is given, assuming a molecular weight of 12,000. •A basic substance "X" whose elution position was just before that of lysine, corresponding to ornithine, was found. Its content in RNase is 3 moles/mole if it is ornithine. For reference, the amino acid composition of pancreatic RNase is listed in the same table. Bullfrog liver RNase shows lower contents of balfcystine, serine, tyrosine and methionine and higher contents of leucine and glycine than pancreatic RNase. Base Specificity—Synthetic homopolymers were used as substrates to determine the substrate specificity (Table III). Bullfrog RNase acts preferentially on pyrimidine homopolymers, and it is interesting to note that poly I is hydrolyzed to some extent. Pancreatic RNase also hydrolyzes poly A and poly I, but the activity is extremely low (16). Using the assay conditions described in "METHODS," DNA was not depolymerized. Product and Intermediate Analysis—The product of the RNase reaction was identified as 3'-CMP with poly C as a substrate. A schematic presentation of the results of paper chromatography is given in Fig. 6. Three spots appeared, and two of them were found to be 3'-CMP (spot 1) and 2', 3'-cyclic CMP (spot 2). Although spot 3 could not be identified, it is presumed to be oligo C. No spots corresponding to cytidine, 2'-CMP or 5'-CMP were detected. Non-enzymatic hydrolysis of poly C did not occur under the same condiJ. Biochem.
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10
Poly Poly Poly Poly Poly
Relative activity
BULLFROG LIVER RNase
2-0XWP
A
25
CO
3'-CMP 5'-CMP
CD
o
^'-oCMP CytBne
)
P*iC sarnpte
2 O
O j
B
3'-CMP
-
5:-CMP
-
O
CD
2\3'-cCMP Cytxfce
•
pdyC
(
arrpte
-
2-(3-)-CMP3'-CMP
-
5'-CMP
-
CD
-
pdyC
(
sanrle
•
02 0.4 NaCl ( M )
3
2
1
o
o
o
CO CD CD
C
O
^'-cCMPCytuJne
o
3 C5
1 O
o
2
O
0 10 20 30cm Fig. 6. Analysis of the products of hydrolysis of poly C by paper chromatography. A solution (0.1 ml) containing 0.25 mg of poly C, 45.6 units of the purified RNase, and 12.5 /imoles of Tris-HCl buffer, pH 7.5, was incubated at 37° for 10 min, then a 20 fi\ aliquot was spotted on the paper. Various standards (200 pig each) were used. (A) Solvent I; (B) Solvent II; (C) Solvent III.
tions. Upon brief hydrolysis (1 min), spot 2 was detected but not spot 1, indicating that 2', 3'-cyclic CMP is an intermediate. pH Optimum of the RNase Reaction— Under the assay conditions described in "METHODS," the pH optimum was found to be pH 7.0-7.5 (Fig. 7A). It was characteristic of the enzyme that the activity at pH 7.5 decreased and a new activity peak appeared at pH 4.5-5.0 in the presence of NaCl. The activity peak in the acidic region became prominent in the presence of 1 mM ZnCli and 0.35 M NaCl (see below, Fig. 7A). Other General Properties of the RNase— The optimum temperature of the RNase was found to be 65—70° under the standard assay conditions. RNase was stable to heat treatVol. 80, No. 1, 1976
Fig. 7. pH optimum (A) and optimal NaCl concentration (B) for the RNase reaction. (A) 50 mM acetate buffer (pH 4.0-6.5) and 50 mM Tris-HCl buffer (pH 6.5-9.5) were used. RNase activity was determined in the presence of 0.1 M NaCl (O), 0.35 M NaCl (A), or 0.35M NaCl + 1 mM ZnCl, ( • ) at the indicated pH. Solid lines indicate Tris-HCl buffer and dotted lines indicate acetate buffer. (B) RNase activity was measured in 50 mM Tris-HCl buffer, pH 7.5 (O), or 50 mM acetate buffer, pH 4.5 ( • ) , at the indicated concentration of NaCl.
ment at temperatures lower than 70° for 10 min. The critical point for heat inactivation was 70°. Divalent cations were not required for activity. EDTA had no effects on the RNase activity. Of divalent cations tested, Zn1+ was most inhibitory. The presence of 1 mM ZnCU inhibited 80% of the RNase activity at pH 7.5 but had no effect on the activity at pH 4.5. As shown in Fig. 7B, the optimal concentration of NaCl was 0.15 M under the assay conditions at pH 7.5. However, the RNase activity at pH 4.5 exhibited a maximum at 0.4 M NaCl. DISCUSSION Two kinds of alkaline RNase have been reported in rat liver. RNase II is characterized by a pH optimum of 8.0, heat stability and localization in the supernatant and mitochondrial fraction (/, 2, 22). The other, RNase III, is characterized by a pH optimum of 9.5, heat lability and localization in the particulate
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2-p'KMP
3 O
26
H. NAGANO, H. KIUCHI, Y. ABE, and R. SHUKUYA
Although bullfrog liver RNase resembles pancreatic RNase in mode of action, molecular weight, p / and so on (20), bullfrog liver RNase hydrolyzes poly I, and a difference was also observed in the sensitivity to RNase inhibitor from bullfrog muscle. This inhibitor inhibits bullfrog RNase but not pancreatic RNase (unpublished data). The molecular weight of the RNase inhibitor could not be determined because bullfrog liver contains no free RNase inhibitor. Therefore we could not estimate the stoichiometry of RNase and RNase inhibitor. Assuming that the molecular weights of bullfrog RNase inhibitor and rat liver RNase inhibitor (molecular weight, 50,000, see Ref. 21) are similar, 6—7 molecules of RNase may bind per molecule of RNase inhibitor. An alternative possibility is that a molecule of RNase binds with a molecule of RNase inhibitor to form a complex with a molecular weight of 62,000 and dimer of this complex is then formed. According to our unpublished results, RNase inhibitor in bullfrog muscle (molecular weight, 140,000) binds with bullfrog liver RNase (molecular weight, 12,000) to form a complex with a molecular size of approximately 210,000 daltons, indicating that roughly 6 molecules of RNase are inactivated by one molecule of
RNase inhibitor from muscle. The results of amino acid analysis indicate the presence of an unusual basic component corresponding to ornithine. It is not clear whether this component is a contaminant or a normal constituent of liver RNase. Four analyses using the same preparation gave similar results. Further studies are now in progress. This work was supported in part by a grant for scientific research from the Ministry of Education, Science and Culture, of Japan. REFERENCES 1. Roth, J.S. (1954) /. Biol. Chem. 208, 181-194 2. de Lamirande, G., Allard, C , da Costa, H.C., & Cantero, A. (1954) Science 119, 351-353 3. Barnard, E.A. (1969) Annual Review of Biochemistry 38, 677-732 4. Maver, M.E. & Greco, A.E. (1982) / . Biol. Chem. 237, 736-741 5. Roth, J.S. (1962) Biochim. Biophys. Ada 61, 903-915 6. Kirby, K.S. (1956) Biochem. J. 64, 405-408 7. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 8. Reisfeld, R.A., Lewis, U.J., & Williams, D.E. (1962) Nature 195, 281-283 9. Wolf, G. (1968) Experientia 24, 890-891 10. Shapiro, A.L., Vinuela, E., & Maizel, J.V., Jr. (1967) Biochem. Biophys. Res. Commun. 28,815-820 11. Andrews, P. (1985) Biochem. J. 96, 595-606 12. Markham, R. & Smith, J.D. (1952) Biochem. J. 52, 558-565 13. Goldspink, D.F. & Pennington, R.J. (1970) Biochem. J. 118, 9-13 14. Moore, S. & Stein, W.H. (1963) Methods Enzymol. 6, 819-831 15. Hirs, C.H.W. (1987) Methods Enzymol. 11, 59-62 16. Beers, R.F., Jr. (1950) /. Biol. Chem. 235, 23932398 17. Zytko, J., de Lamirande, G., Allard, C , & Cantero, A. (1958) Biochim. Biophys. Ada 27, 495-503 18. Reid, E. & Nodes, J.T. (1959) Ann. N.Y. Acad. Sd. 81, 618-633 19. Kaplan, H.S. & Heppel, L.A. (1956) / . Biol. Chem. 222, 907-922 23. Richards, F.M. & Wyckoff, H.W. (1971) The Enzyme (Boyer, P.D., ed.) 3rd edition, Vol. 4, pp. 647-806, Academic Press, New York 21. Gribnau, A.A.M., Schoenmakers, J.G.G., & Bloemendal, H. (1959) Arch. Biochem. Biophys. 130, 48-52 22. Rahman, Y.E. (1987) Biochim. Biophys. Ada 146, 477-483 23. Rahman, Y.E. (1966) Biochim. Biophys. Ada 119, 470-479 / . Biochem.
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fraction (23). The RNase presented here is similar to RNase II, as regards pH optimum, heat stability and intracellular localization. Liver alkaline RNase has been reported to hydrolyze polypyrimidine nucleotides and to produce 2', 3'-cyclic pyrimidine nucleotide (17, 18) or 3'-pyrimidine nucleotide (4, 19) as a final product. Bullfrog liver RNase splits poly C to form 3'-CMP with the intermediate formation of 2', 3'-cyclic CMP. An unidentified spot on paper chromatograms corresponding to oligocytidilate (Fig. 6) has been also detected using bovine liver RNase with poly C as a substrate (4). Poly I is a good substrate for bullfrog liver RNase, while poly A, poly G, and DNA are not. Even if excess RNase is used, as described in the legend to Fig. 6, poly A and poly G are not hydrolyzed. Three spots were also observed on paper chromatograms with poly I as a substrate. Two appeared to be 3'-IMP and 2', 3'-cyclic IMP.
Purification and Properties of an Alkaline Ribonuclease from the Hepatic Cytosol Fraction of Bullfrog, Rana catesbeiana
Department of Biochemistry, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113 Received for publication, December 22, 1975
In the hepatic cytosol fraction of bullfrog, Rana catesbeiana, an alkaline RNase [EC 3.1.4. 22] exists in two forms. One is the free form of RNase, which elutes from a carboxymethyl-cellulose column at a concentration of 0.2 M NaCl. The other is a masked or latent form (RNase-RNase inhibitor complex) which is not adsorbed on the carboxymethyl-cellulose column and which can be converted to the free form of RNase by the addition of />-chloromercuribenzoate. Electrophoretically pure RNase was obtained by the following procedure. The unadsorbed fraction of hepatic cytosol on a column of carboxymethyl-cellulose was treated with />-chloromercuribenzoate and then applied to a second carboxymethyl-cellulose column. The molecular weight of RNase was determined to be approximately 12,000 by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. From the results of gel filtration, the molecular weight of the RNaseRNase inhibitor complex was 130,000. The RNase hydrolyzed poly C, poly U, and poly I, but not poly A or poly G. When poly C was used as a substrate, 2', 3'-cyclic CMP as an intermediate and 3'-CMP as a final product were identified. The results of amino acid analysis indicated the presence of an unusual component. The general properties of the RNase and the RNase-RNase inhibitor complex are also reported.
Since Roth ( / ) and de Lamirande et al. ( 2 ) demonstrated the presence of two types of RNase in mammalian liver, i.e. alkaline RNase and acid RNase, evidence for the occurrence of several RNases differing in pH optimum, , r,
.,
^
1
.
. ,D.
intracellular localization, divalent cation requirement and so on in mammalian liver has been summarized (3). An alkaline RNase from bovine liver has been characterized and reported to have catalytic properties similar to those of pancreatic RNase (4). It has been stated by Roth (5) that the RNase inhibitor
• , • ,n,
Present address: Department of Physiological Chemistry and Nutrition, Faculty of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113. Abbreviations used : SDS, sodium dodecyl sulfate ; PCMB, />-chloromercuribenzoate. Vol. 80, No. 1, 1976
"""•>-»* "J « v / 8 I i v e r supernatant is not present in free form but in a complex form bound with RNase. In this paper, we will describe (I) the separation of an alkaline RNase and the
in fr0
19
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Hiroshi NAGANO,1 Hiroyuki KIUCHI, Yasuko ABE, and Ryoiti SHUKUYA
H. NAGANO, H. KIUCHI, Y. ABE, and R. SHUKUYA
20
RNase-RNase inhibitor complex from bullfrog liver cytosol, (II) the properties of the complex, (III) the purification and characterization of the alkaline RNase. MATERIALS AND METHODS
Protein was determined by the method of Lowry et al. ( 7 ) with bovine serum albumin as a standard. Sodium chloride concentration in column effluents was determined using a conductivity meter. Assay of RNase Activity—The reaction mixture for activity measurements of RNase contained 0.1 mmole of Tris-HCl buffer, pH 7.5, 2 mg of yeast RNA and an appropriate amount of RNase solution in a final volume of 0.8 ml. After incubation at 37° for 10 min, 0.2 ml of ice-cold 25% perchloric acid-0.25% uranium acetate mixture was added. The precipitate of RNA and protein was discarded by centrifugation at 3,000 rpm for 10 min after standing in ice for 30 min. The supernatant solution was diluted 10 times with distilled water and its absorbance at 260 nm was determined. As a control, enzyme solution was added after the addition of the perchloric aciduranium acetate mixture. A unit of RNase activity is denned as one absorbance unit released under the above conditions. The latent RNase activity was determined using the above reaction mixture containing 1 mM PCMB. Purification of an Alkaline RNase—Freshly obtained bullfrog liver (70 g) was homogenized in 4 volumes of 0.25 M sucrose using a Teflon homogenizer. A precipitate was removed by centrifugation at 105,000X0 for 60
Polyacrylamide Gel Electrophoresis — The polyacrylamide gel electrophoresis procedure of Reisfeld et al. for basic proteins (8) was used. Condensing and sample gels were not used. The sample protein (0.07 mg) in 0.1 ml of 12% glycerol was applied directly to a separating gel. Electrophoresis was performed at 4 mA per tube (5 m m x 5 cm) until the tracking dye (pyronine Y) moved to the bottom of the gel tube. Protein bands were stained with Amido Black. The staining of RNase activity on polyacrylamide gels was performed according to the method of Wolf (9). Electrophoresis in the presence of SDS to estimate polypeptide chain molecular weights was conducted by the method of Shapiro et al. (10). Molecular Weight Determination by Gel Filtration — According to the method of Andrews (11), gel filtration using Sephadex G-75 was employed to estimate the molecular weight of alkaline RNase. To a column (3x 86 cm) of Sephadex G-75 equilibrated with 50 mM phosphate buffer, pH 7.2, a sample solution (1.6 ml) containing 0.5 ml of RNase solution (40 units), 0.1 ml of human hemoglobin /. Biochtm.
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Adult bullfrogs and tadpoles were purchased from Nozawaya and Shiihashi, Tokyo, respectively. Ampholine carrier ampholyte (pH 3— 10, 40% w/v), cytochrome c, and a-chymotrypsin [EC 3.4.21.1] were from LKB, Sankyo and Sigma Co., respectively. Rabbit muscle pyruvate kinase [EC 2.7.1.40], yeast alcohol dehydrogenase [EC 1.1.1.1], poly A, poly G, poly I, poly U, poly C, 5'-CMP, 3'-CMP, 2'(3')CMP, 2', 3'-cyclic CMP, and cytidine were from Boehringer Mannheim. Yeast RNA, from Wako Pure Chemicals, was purified by the method of Kirby (6).
min. The supernatant solution was dialyzed twice against 5,000 ml of 10 mM phosphate buffer, pH 6.5, and applied to a column (6.5 X 29 cm) of CM-cellulose which had been equilibrated with 10 mM phosphate buffer, pH 6.5. Elution was carried out with the same buffer. Fractions of 16 ml were collected. The unadsorbed fraction containing the latent RNase was pooled (710 ml) and treated with PCMB (1 mM final concentration). The treated fraction was again applied to a column (4.5x34 cm) of CM-cellulose, washed with 500 ml of 10 mM phosphate buffer, pH 6.5, and eluted with a linear gradient prepared from 1,000 ml of 10 mM phosphate buffer, pH 6.5, in the mixing chamber and 1,000 ml of the same buffer containing 0.5 M NaCl in the reservoir. Fractions of 22 ml were collected. The fraction containing RNase activity was pooled, dialyzed against 5,000 ml of 10 mM phosphate buffer, pH 6.5, and applied to a third column (3.5x20 cm) of CM-cellulose previously equilibrated with the same buffer. The conditions of elution were the same as for the second CM-cellulose column chromatography.
BULLFROG LIVER RNase
Paper Chromatography for Identification of the Catalytic Products—Toyo chromatography paper No. 51 was used with the following solvents. Solvents I (12), saturated ammonium sulfate-0.5M sodium acetate-2-propanol (40 : 9 : 1, v/v/v); solvent II (13), solid ammonium sulfate-0.1 M potassium phosphate buffer, pH 7.0 (2 : 5, w/v); solvent III (13), 95% ethanol-1 M ammonium acetate buffer, pH 7.5 (5 : 2, v/v). Chromatographs were run for 11—27 hr using ascending systems. Amino Acid Analysis—About 0.88 mg of the purified alkaline RNase was mixed with 2 ml of 6 N HC1 in a hydrolysis tube. Hydrolysis was performed in vacuo at 110° for 24, 48, or 72 hr. Amino acid analysis was carried out by the method of Moore and Stein (14) using a Hitachi KLA-5 amino acid analyzer. Performic acid oxidation was conducted according to the method of Hirs (75). Electrofocusing—%'or determination of the isoelectric point of the alkaline RNase, electrofocusing was performed using a carrier amVol. 80, No. 1, 1976
pholyte with a pH range of 3—10 and an electrofocusing column of 110 ml capacity. The column solution was prepared as described in the LKB instruction manual. The protein was electrofocused for 48 hr at 1,000 V. Fractions of 2 ml were collected. A Beck man expandomatic SS-2 pH meter was used for pH determination. RESULTS Alkaline RNase and Its Complex with RNase Inhibitor—Liver supernatant after centrifugation at 105,000X0 for 60 min was applied to a column of CM-cellulose equilibrated with 10 mM phosphate buffer, pH 6.5. As shown in Fig. 1A, RNase activity which developed with PCMB (Peak I) was not adsorbed. RNase eluted at 0.2 M NaCl (Peak II) exhibited the same activity in the presence and in the absence of PCMB. When Peak I or Peak II was rechromatographed under the same conditions, the elution pattern did not change. The amounts of RNase activity found in Peak I (latent form) and Peak II (free form) were calculated to be 0.6 ±0.2 unit/mg wet weight and 3.5±0.8 units/mg wet weight, respectively, using the larval form of bullfrog liver. Peak I of Fig. 1A (unadsorbed fraction) was treated with 1 mM PCMB and applied to a second CM-cellulose column (Fig. IB). RNase activity was eluted at 0.2 M NaCl; it exhibited the same activity in the presence and absence of PCMB. The following points are suggested by the above results. (1) RNase found in Peak I of Fig. 1A may be a complex of RNase and RNase inhibitor, because RNase inhibitor is known to be inactivated by PCMB. (2) RNase activity eluted with 0.2 M NaCl (Fig. 1A) may be due to free RNase. (3) The free form of RNase and the RNase released from the bound form appear to be identical, because, as shown in Fig. 1, these activities are eluted at the same concentration of NaCl. The molecular weight, pH optimum, heat stability, base specificity, and optimal temperature of both RNases were identical. (4) As RNase inhibitor released from the complex is inactivated by PCMB, its elution position could not be determined in Fig. IB.
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solution (molecular weight, 64,000) obtained after hemolysis with 3 volumes of water, 2 mg of bovine pancreatic a-chymotrypsin (molecular weight, 21,600) and 2 mg of horse heart cytochrome c (molecular weight, 12,300) was applied. Fractions of 2.9 ml were collected. The elution patterns of hemoglobin and cytochrome c were determined from the absorbances at 500 nm and 410 nm, respectively. The activity of a-chymotrypsin was determined using N-acetyltyrosine ethyl ester as a substrate. For estimation of the molecular weight of the RNase-RNase inhibitor complex, a column (2.8x24.4 cm) of Sephadex G-200 equilibrated with 50 mM phosphate buffer, pH 7.2, was used. To the complex solution (1.1 ml) prepared as shown in Fig. 1A (48 units of RNase activity in the presence of PCMB), 0.2 mg of rabbit muscle pyruvate kinase (molecular weight, 220,000), 0.3 mg of yeast alcohol dehydrogenase (molecular weight, 150,000) and 0.1 ml of human hemolyzate prepared as described above, were added. Fractions of 2 ml were collected. The position of the complex was determined by assaying RNase activity in the presence of 1 mM PCMB.
21
H. NAGANO, H. KIUCHI, Y. ABE, and R. SHUKUYA
22
20 40 60 FRACTION NUMBER
Fig. 1. CM-cellulose column chromatographic profiles of the free and latent forms of RNase. (A) Separation of the free and latent RNase on a CMcellulose column from bullfrog liver supernatant. The supernatant of 20% homogenate from 0.34 g of fresh liver was applied to a column (2x5 cm) of CM-cellulose equilibrated with 10 mM phosphate buffer, pH 6.5. Elution was carried out with a linear gradient prepared from 100 ml of 10 mM phosphate buffer, pH 6.5 in the mixing chamber and 100 ml of the same buffer containing 0.5 M NaCl in the reservoir. Fractions of 3.1 ml were collected. O, RNase activity in the presence of 1 mM PCMB; • , RNase activity in the absence of PCMB ; , NaCl concentration. (B) Rechromatography of the latent RNase treated with 1 mM PCMB. PCMB was added to the Peak I of Fig. 1A (16.6 ml) to a final concentration of 1 mM and the mixture was applied to a column of CM-cellulose. The chromatographic conditions and symbols are as described in Fig. 1A.
Molecular Size of the Complex—The molecular size of the RNase-RNase inhibitor complex (latent RNase) was determined approximately. Peak I fraction of Fig. 1A was applied to a column of Sephadex G-200. RNase activity in the presence of PCMB was eluted just behind of yeast alcohol dehydrogenase, suggesting an approximate molecular weight of 130,000 daltons. Stability of the Complex—Figure 2 shows the stability of the complex at various pH's.
20 40 60 80 TEMPERATURE (•)
100
Fig. 3. Heat stability of the RNase-RNase inhibitor complex. A solution (0.5 ml) containing 0.2 ml of 0.5 M Tris-HCl buffer, pH 7.5, and 10 ft] of Peak I of Fig. 1A (about 1.1 units of activity in the presence of PCMB) was treated for 10 min at the indicated temperature. RNA solution ( • ) or RNA solution and PCMB (O) were added to the heattreated solution to a final volume of 0.8 ml.
/. Biochem.
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0
Fig. 2. pH-stability curve of the RNase-RNase inhibitor complex. A solution containing 0.2 ml of 50 mM acetate buffer (pH 4.5-6.5) or 50 mM TrisHC1 buffer (pH 6.5-9.5) and 5 fi\ of the unadsorbed fraction from CM-cellulose column chromatography (about 0.7 unit in the presence of PCMB) was incubated at 37° for 10 min. After incubation, 0.2 ml of 0.5 M Tris-HCl buffer, pH 7.5, was added and RNase activity in the presence (O) or absence ( • ) of 1 mM PCMB was assayed as described in the text.
BULLFROG LIVER RNase TABLE I. Summary of the purification procedure for alkaline RNase.
Fraction
Volume ^ p r o t e i n
Supernatant
290
First CM-cellulose Second CM-cellulose Third CM-cellulose
710 620
3,400 2,630 11.6
273
2.3
Reactivity 43,500" 39,050 34,100 20,500
Recovery
Specificity
(100)" 89.7 78.9 47.4
12.6 14.8 2,940 9,100
Purification
m1.2 233 722
About half of the complex released RNase on treatment at pH 5.0 for 10 min at 37°. The complex is stable at pH 6.5—8.0 under the conditions used. Heat treatment at 40° for 10 min at pH 6.5 inactivated 50% of the inhibitor and treatment at 60° for 10 min decomposed the complex completely (Fig. 3). Purification of Alkaline RNase—Purification of the RNase from bullfrog liver was carried out by the following procedure. (1) The unadsorbed fraction of hepatic cytosol was obtained using a CM-cellulose column. This fraction contains the latent form of RNase and obviously does not contain material which can be adsorbed on a CM-cellulose column. (2) The unadsorbed fraction was treated with 1 mM PCMB. The released RNase can be adsorbed on a CM-cellulose column as shown in Fig. IB. However, nearly all other components will not be retained. Thus, pure RNase can be obtained easily. The purification procedure is summarized in Table I. As the RNase fraction obtained from the second CM-cellulose column chromatography showed multiple protein bands on a polyacrylamide gel, a third CM-cellulose column chromatography was carried out under the same conditions as for the second column. Finally, 2.3 mg of protein was obtained from 70 g of liver. This RNase was subjected to polyacrylamide gel electrophoresis according to the method of Reisfeld etal. (8). As shown in Fig. 4A, electrophoresis showed one major band and a faint band which migrated slightly faster than the major band. Both protein bands showed RNase activity (Fig. 4B). The major band was estimated to represent more than 98% of the total as determined densitoVol. 80, No. 1, 1976
Fig. 4. Polyacrylamide gel electrophoreses of the alkaline RNase in /)-alanine/acetic acid buffer (pH 4.5). (A) Protein bands were stained with Amido Black. (B) Staining of RNase activity. The method of Wolf (9) was employed for activity staining.
metrically. Therefore, the final preparation appears to be practically homogeneous. Molecular Weight of the Alkaline RNase— Two different methods were employed for determination of the molecular weight. A plot of log molecular weight versus elution volume was nearly linear from the results of gel filtration using Sephadex G-75. The RNase activity was eluted at the same position as cytochrome c, indicating that the molecular weight of the RNase is about 12,000. A molecular weight of approximately 12,000 was also estimated from a plot of log molecular weight against relative mobility on polyacrylamide gel electrophoresis in the presence of SDS. RNase again migrated to the same position as cytochrome c. These results indicate that the RNase is composed of a single polypeptide chain. Isoelectric Point— Isoelectric point of RNase was determined by electrofocusing. As shown
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Free form of RNase activity is excluded.
H. NAGANO, H. KIUCHI, Y. ABE, and R. SHUKUYA
24
TABLE III. Substrate specificity of the alkaline RNase. RNase activity was assayed under the standard conditions using yeast RNA or homopolyribonucleotides as substrates. The amount of enzyme used was 0.18 unit (0.02 fig of protein). Substrate RNA
60
20 30 40 50 FRACTION NUMBER
Fig. 5. Electrofocusing of the alkaline RNase. A crude sample containing about 6,000 activity units (5 mg of protein) was subjected to electrofocusing. TABLE II. Amino acid analysis.1 Amino acid
Bullfrog liver
Bovine pancreasb
Tryptophan Lysine Histidine Arginine Aspartic acid Threonine d Serine d Glutamic acid Proline Glycine Alanine Half-cystine" Valine Methionine Isol;ucine Leucine Tyrosine Phenylalanine "X"
NDC
0
7
10
3
4
4
4
10
15
7
10
7
15
11
12
6
4
14
3
13
12
2
8
8
9
1
4
5
3
2
2
6
6
3
3
3
a
The nearest integer, assuming a molecular weight of approximately 12,000. ° Ref. 20. c ND, not determined. d Extrapolated to zero time of hydrolysis. • Determined after performic acid oxidation.
in Fig. 5, a p / of 9.4 was determined. Amino Acid Analysis — The results of amino acid analysis are summarized in Table
1
C U A G I
1.25 2.5
0 0 0.09
II. The nearest integer of each amino acid is given, assuming a molecular weight of 12,000. •A basic substance "X" whose elution position was just before that of lysine, corresponding to ornithine, was found. Its content in RNase is 3 moles/mole if it is ornithine. For reference, the amino acid composition of pancreatic RNase is listed in the same table. Bullfrog liver RNase shows lower contents of balfcystine, serine, tyrosine and methionine and higher contents of leucine and glycine than pancreatic RNase. Base Specificity—Synthetic homopolymers were used as substrates to determine the substrate specificity (Table III). Bullfrog RNase acts preferentially on pyrimidine homopolymers, and it is interesting to note that poly I is hydrolyzed to some extent. Pancreatic RNase also hydrolyzes poly A and poly I, but the activity is extremely low (16). Using the assay conditions described in "METHODS," DNA was not depolymerized. Product and Intermediate Analysis—The product of the RNase reaction was identified as 3'-CMP with poly C as a substrate. A schematic presentation of the results of paper chromatography is given in Fig. 6. Three spots appeared, and two of them were found to be 3'-CMP (spot 1) and 2', 3'-cyclic CMP (spot 2). Although spot 3 could not be identified, it is presumed to be oligo C. No spots corresponding to cytidine, 2'-CMP or 5'-CMP were detected. Non-enzymatic hydrolysis of poly C did not occur under the same condiJ. Biochem.
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10
Poly Poly Poly Poly Poly
Relative activity
BULLFROG LIVER RNase
2-0XWP
A
25
CO
3'-CMP 5'-CMP
CD
o
^'-oCMP CytBne
)
P*iC sarnpte
2 O
O j
B
3'-CMP
-
5:-CMP
-
O
CD
2\3'-cCMP Cytxfce
•
pdyC
(
arrpte
-
2-(3-)-CMP3'-CMP
-
5'-CMP
-
CD
-
pdyC
(
sanrle
•
02 0.4 NaCl ( M )
3
2
1
o
o
o
CO CD CD
C
O
^'-cCMPCytuJne
o
3 C5
1 O
o
2
O
0 10 20 30cm Fig. 6. Analysis of the products of hydrolysis of poly C by paper chromatography. A solution (0.1 ml) containing 0.25 mg of poly C, 45.6 units of the purified RNase, and 12.5 /imoles of Tris-HCl buffer, pH 7.5, was incubated at 37° for 10 min, then a 20 fi\ aliquot was spotted on the paper. Various standards (200 pig each) were used. (A) Solvent I; (B) Solvent II; (C) Solvent III.
tions. Upon brief hydrolysis (1 min), spot 2 was detected but not spot 1, indicating that 2', 3'-cyclic CMP is an intermediate. pH Optimum of the RNase Reaction— Under the assay conditions described in "METHODS," the pH optimum was found to be pH 7.0-7.5 (Fig. 7A). It was characteristic of the enzyme that the activity at pH 7.5 decreased and a new activity peak appeared at pH 4.5-5.0 in the presence of NaCl. The activity peak in the acidic region became prominent in the presence of 1 mM ZnCli and 0.35 M NaCl (see below, Fig. 7A). Other General Properties of the RNase— The optimum temperature of the RNase was found to be 65—70° under the standard assay conditions. RNase was stable to heat treatVol. 80, No. 1, 1976
Fig. 7. pH optimum (A) and optimal NaCl concentration (B) for the RNase reaction. (A) 50 mM acetate buffer (pH 4.0-6.5) and 50 mM Tris-HCl buffer (pH 6.5-9.5) were used. RNase activity was determined in the presence of 0.1 M NaCl (O), 0.35 M NaCl (A), or 0.35M NaCl + 1 mM ZnCl, ( • ) at the indicated pH. Solid lines indicate Tris-HCl buffer and dotted lines indicate acetate buffer. (B) RNase activity was measured in 50 mM Tris-HCl buffer, pH 7.5 (O), or 50 mM acetate buffer, pH 4.5 ( • ) , at the indicated concentration of NaCl.
ment at temperatures lower than 70° for 10 min. The critical point for heat inactivation was 70°. Divalent cations were not required for activity. EDTA had no effects on the RNase activity. Of divalent cations tested, Zn1+ was most inhibitory. The presence of 1 mM ZnCU inhibited 80% of the RNase activity at pH 7.5 but had no effect on the activity at pH 4.5. As shown in Fig. 7B, the optimal concentration of NaCl was 0.15 M under the assay conditions at pH 7.5. However, the RNase activity at pH 4.5 exhibited a maximum at 0.4 M NaCl. DISCUSSION Two kinds of alkaline RNase have been reported in rat liver. RNase II is characterized by a pH optimum of 8.0, heat stability and localization in the supernatant and mitochondrial fraction (/, 2, 22). The other, RNase III, is characterized by a pH optimum of 9.5, heat lability and localization in the particulate
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2-p'KMP
3 O
26
H. NAGANO, H. KIUCHI, Y. ABE, and R. SHUKUYA
Although bullfrog liver RNase resembles pancreatic RNase in mode of action, molecular weight, p / and so on (20), bullfrog liver RNase hydrolyzes poly I, and a difference was also observed in the sensitivity to RNase inhibitor from bullfrog muscle. This inhibitor inhibits bullfrog RNase but not pancreatic RNase (unpublished data). The molecular weight of the RNase inhibitor could not be determined because bullfrog liver contains no free RNase inhibitor. Therefore we could not estimate the stoichiometry of RNase and RNase inhibitor. Assuming that the molecular weights of bullfrog RNase inhibitor and rat liver RNase inhibitor (molecular weight, 50,000, see Ref. 21) are similar, 6—7 molecules of RNase may bind per molecule of RNase inhibitor. An alternative possibility is that a molecule of RNase binds with a molecule of RNase inhibitor to form a complex with a molecular weight of 62,000 and dimer of this complex is then formed. According to our unpublished results, RNase inhibitor in bullfrog muscle (molecular weight, 140,000) binds with bullfrog liver RNase (molecular weight, 12,000) to form a complex with a molecular size of approximately 210,000 daltons, indicating that roughly 6 molecules of RNase are inactivated by one molecule of
RNase inhibitor from muscle. The results of amino acid analysis indicate the presence of an unusual basic component corresponding to ornithine. It is not clear whether this component is a contaminant or a normal constituent of liver RNase. Four analyses using the same preparation gave similar results. Further studies are now in progress. This work was supported in part by a grant for scientific research from the Ministry of Education, Science and Culture, of Japan. REFERENCES 1. Roth, J.S. (1954) /. Biol. Chem. 208, 181-194 2. de Lamirande, G., Allard, C , da Costa, H.C., & Cantero, A. (1954) Science 119, 351-353 3. Barnard, E.A. (1969) Annual Review of Biochemistry 38, 677-732 4. Maver, M.E. & Greco, A.E. (1982) / . Biol. Chem. 237, 736-741 5. Roth, J.S. (1962) Biochim. Biophys. Ada 61, 903-915 6. Kirby, K.S. (1956) Biochem. J. 64, 405-408 7. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 8. Reisfeld, R.A., Lewis, U.J., & Williams, D.E. (1962) Nature 195, 281-283 9. Wolf, G. (1968) Experientia 24, 890-891 10. Shapiro, A.L., Vinuela, E., & Maizel, J.V., Jr. (1967) Biochem. Biophys. Res. Commun. 28,815-820 11. Andrews, P. (1985) Biochem. J. 96, 595-606 12. Markham, R. & Smith, J.D. (1952) Biochem. J. 52, 558-565 13. Goldspink, D.F. & Pennington, R.J. (1970) Biochem. J. 118, 9-13 14. Moore, S. & Stein, W.H. (1963) Methods Enzymol. 6, 819-831 15. Hirs, C.H.W. (1987) Methods Enzymol. 11, 59-62 16. Beers, R.F., Jr. (1950) /. Biol. Chem. 235, 23932398 17. Zytko, J., de Lamirande, G., Allard, C , & Cantero, A. (1958) Biochim. Biophys. Ada 27, 495-503 18. Reid, E. & Nodes, J.T. (1959) Ann. N.Y. Acad. Sd. 81, 618-633 19. Kaplan, H.S. & Heppel, L.A. (1956) / . Biol. Chem. 222, 907-922 23. Richards, F.M. & Wyckoff, H.W. (1971) The Enzyme (Boyer, P.D., ed.) 3rd edition, Vol. 4, pp. 647-806, Academic Press, New York 21. Gribnau, A.A.M., Schoenmakers, J.G.G., & Bloemendal, H. (1959) Arch. Biochem. Biophys. 130, 48-52 22. Rahman, Y.E. (1987) Biochim. Biophys. Ada 146, 477-483 23. Rahman, Y.E. (1966) Biochim. Biophys. Ada 119, 470-479 / . Biochem.
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fraction (23). The RNase presented here is similar to RNase II, as regards pH optimum, heat stability and intracellular localization. Liver alkaline RNase has been reported to hydrolyze polypyrimidine nucleotides and to produce 2', 3'-cyclic pyrimidine nucleotide (17, 18) or 3'-pyrimidine nucleotide (4, 19) as a final product. Bullfrog liver RNase splits poly C to form 3'-CMP with the intermediate formation of 2', 3'-cyclic CMP. An unidentified spot on paper chromatograms corresponding to oligocytidilate (Fig. 6) has been also detected using bovine liver RNase with poly C as a substrate (4). Poly I is a good substrate for bullfrog liver RNase, while poly A, poly G, and DNA are not. Even if excess RNase is used, as described in the legend to Fig. 6, poly A and poly G are not hydrolyzed. Three spots were also observed on paper chromatograms with poly I as a substrate. Two appeared to be 3'-IMP and 2', 3'-cyclic IMP.
