- Email: [email protected]
Degradation of RNA in rat reticulocytes
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Degradation II. Relationship
Between
of RNA
in Rat Reticulocytes
RNase and Its Corresponding
SATARO Faculty
146, 71-77 (1971)
of Pharmaceutical Received
GOTO
Ah3
Sciences,
December
DEN’ICHI
TJniversily
Inhibitor MIZUNO
of ?‘okyo, Honyo,
15, 1970; accepted
in Rat Reticulocyte
March
Tokyo,
Japan
16, 1971
A latent RNase was partially purified by ammonium sulfate fractionation and Sephadex G-75 gel filtration from rat reticulocyte lysate. By mixing free RNase and its inhibitor in vitro, the latent RNase was shown to be a complex of each component. The latent RNase was activated by SH reagents, but no dissociation of the complex was observed. It was also activated by G M urea and 0.1 M H&.&, and in these cases the enzyme was released as judged by gel filtration. From the results of this and precious paper, a possible mechanism of the regulation of RNase activity in rat reticulocyte is discussed.
cell may be simpler than in other cells or tissues. In this report, we describe a procedure for the purification of the latent RNase and factors which activate it and demonstrate that the latent RNase is a complex of RNase and its inhibitor.
It has been known that there are RSase inhibitors in various tissues of the rat, such as liver (1, 2), brain (3), kidney (I), adrenal (4, 5), adipose tissue (6), and parotid gland (7). The inhibitor in liver was purified and characterized extensively, although its role in the metabolism of RKA has been obscure. It was reported that the inhibitor \vas inactivated easily on heat treatment or dilution and was specifically inhibitory against alkaline RNase of animal tissues (1, 2, 8). The RXase inhibitor of rat liver has been used frequently for the preparation of polysomes from animal tissues to protect them from enzymatic degradation (9-11). It has been, therefore, suggested that the degradation of messenger RNA may be ascribed to the action of alkaline RNase (9, 12, 13). Although it was reported that the inhibitor level is changed under various conditions (4, 14, 1.5), it is not known how it may regulate ItNase activity in vivo. As described in the preceding paper, an RNase inhibitor is also present in rat reticulocytes. On heating the crude extract at 55” it masked the enzyme activity completely being itself activated. It seems useful to know the relationship between the RXase and its inhibitor in reticulocyte because cell structure and RNA degrading system of this
MATERIALS Preparation
AND
METHOT
of Rat Reticulocyte Lysate sup
Rat reticulocytes were induced and the cell Iysate sup was prepared as described before (16).
Assay of RNase and l,atent RNase RNase activity was measured as described before (16). Latent RNase was usually activated by 0.025 rnM p-chloromercuribenzoate (PCMB), which inactivates the RNase inhibitor. This concentration of PCMB was shown to activate t,he partially purified latent RNase completely and to have no effect on the RNase activity itself. The degree of activation is expressed as percentage of the maximum activity which is determined in the presence of optimum amount of PCMB.
Assay oj RNase Inhibitor The activit,y of RNase inhibitor was measured as an inhibitory activity t,o the RNase which was prepared by t.reatment of the crude extra.ct at 67”. This method is different from the case of rat 71
72
GOT0
AND
0 0 4 a 12 16 20 MOUNT OF 55'C LWilON (~11
FIG. 1. Inhibition of rat reticulocyte RNase by its inhibitor. The RNase was obtained by heating crude extract at 67” but not purified further. The inhibitor fraction was obtained by 55” treatment of crude extract. One hundred microliters of 67” fraction and indicated amount of 55” fraction were mixed and RNase activity was measured under standard assay condition.
liver RNase inhibitor in earlier studies, in which bovine pancreatic RNase was used instead of its corresponding RNase present in the same tissue. The assay system consists of 160 ~1 of the expH tract treated at 67”, 300 ~1 of 0.1 M Tris-Cl, 7.5, 10 ~1 of 0.1 M EDTA, an appropriate amount of inhibitor fraction and 32P- or ‘%-labeled E. coli rRNA. The method of assay is the same as described before (16). The inhibitory activity was expressed as the percentage of the activity of the added RNase. The relation between the amount of inhibitor fraction and the activity of the RNase was given in Fig. 1. The inhibition was linear to about 80%. To inhibit the enzyme activity completely, further twofold or more of the inhibitor fraction was needed. A similar tendency has been reported on the inhibition of bovine pancreatic RNase by rat liver supernatant (14).
MIZUNO
servation. All procedures were performed at O-5” unless otherwise stated. Fifty milliliters of frozen lysate sup was thawed at room temperature (E-20’) and the red aggregates were removed by centrifugation at 2,000~ for 10 min. To the resultant supernatant, 0.1 M sodium phosphate buffer, pH 5.6, was added to a final concentration of 0.02 nr. The mixture was heated at 55” for 5 min. After standing in an ice bath for 20 min, the aggregated materials were removed by centrifugation. Solid ammonium sulfate was then added to the supernatant with stirring to 55% saturation. After stirring for 20 min, the precipitate was collected by centrifugation at 10,OOOy for 20 min and dissolved in l-2 ml of 0.05 nr sodium phosphate buffer, pH 6.5. The solution was dialyzed against 500 ml of the same buffer for 4 hr, and the precipitate was removed by centrifugation. The supernatant was applied to a column of Sephadex G-75 and eluted by the same buffer. A typical elution pattern is shown in Fig. 2. In contact to the case of the 67” treatment [see the preceding paper (IS)] no activity was observed at the position of the faster peak [peak I in the Fig. 2 of the preceding paper (IS)] without PCMB in the assay
Other Methods Gel filtration on Sephadex G-75 was performed as described before (16). Protein was determined by the method of Lowry et al. (17), using bovine serum albumin as a reference standard. RESULTS
Partial Purzfication of the Latent RNase As reported in the previous paper (16), RNase activity of the crude extract of rat reticulocytes disappeared completely on heating at 55” for 5 min. Partial purification of the latent RNase was based on this ob-
FIG. 2. Gel filtration of the latent RNase on Sephadex G-75 (1.9 X 73 cm). One-half milliliter of dialyzed sample obtained after ammonium sulfate fractionation was applied and eluted by 0.05 M sodium phosphate buffer, pH 6.5. One hundred microliters of each fraction (5 ml) was used for the assay. Latent enzyme was activated by 2.5 X 1OW M PCMB. (O---O) activity in cpm. (A---A) A280. The position of free RNase was shown by an arrow.
RAT
RETICULOCYTE
RNase
mixture and no activity at all was found at the position of free RNase with or without PC?,IB. It is, therefore, suggested that all the RNase in the crude extract is complexed with its inhibitor after heating at 55”. Even when ammonium sulfate was added to 68 % saturation instead of 55 %, there was no activity at the position of free RNase. Therefore, it cannot be considered that free IiKase in the 55” treated crude extract did not precipitate because of the low concentration of ammonium sulfate. The specific activity and yield of the latent enzyme at each stage are shown in Table I. The RNase activity was measured in the presence of the optimal concentration of PCMB. The reason why the activity was increased greatly after heating at 55” ‘is not known. It was shown that the activated RKase had optimum pH of 6.5-7.5. E$ect of the Crude Extract Heated at 55” on the RNase Activity of the Extract Stored at 0” and That Heated at 67” The effect of the crude extract heated at 55” on the RNase activity was examined. As shown in Table II, the sample completely inhibited the enzyme activity of the extract heated at 67”, while the activity of the extract stored at 0” was not affected at all. This fact could be explained by considering that the activity of the “0”” sample is due to the RNase already complexed with its inhibitor and this activity was not affected by the inhibitor derived from the sample treated at 55” (see also Discussion). Formation of the Complex with Free RNase and Inhibitor The RNase inhibitor was purified partially from the crude extract of rat reticulocyte by a procedure described below. All the procedures were done at O-5” unless otherwise noticed. Fifty milliliters of frozen lysate sup was thawed at room temperature and the supernatant u-as obtained after centrifugation. The sup was mixed with 0.1 nr EDTA (final concn, 1 mnr), 0.05 M sodium phosphate buffer, pH 6.5 (final concn, 0.01 RI), and 1.5 M NaCl (final concn, 0.15 11). The mixture was then applied to DEAE-cellu-
AND
ITS
INHIBITOR
73
Total activity (cm)
Crude extract 55” treatment Ammonium snlfate Sephadex G-75
0.8 x 10” 3.7 x 105 1.8 x 105
Specific activity (CPdW protein) 5.7 x 102 6.6 X 103 5.2 x lo4
1.75 x 105
TABLE EFFECT
Total
protein (w)
0.G
2.8 x 105
II
OF THE CKUDE EXTR.\CT HE.ITED OK RNME ACTIVITY
AT
55’ fraction (PI)
0’ fraction Ml)
RNase activity (CPW
100 50 0
100 100 100
548 559 570
55”
67” fraction (Pl) 100 50 25 0
100 loo 100 100
11 0 G 784
lose column 0.9 X 2 cm, which was prepared by the method of Peterson and Sober (18) and buffered with the buffer described above. After washing with 10 ml of the same buffer, the inhibitor was eluted by the buffer containing 1.0 XI NaC1 and 1 mM EDTA. After concentrating the eluate to 0.4 ml in a collodion bag, 0.6 ml of H,O was added; it was applied to the column of Sephadex G-75, 1.0 X 80 cm and eluted by the buffer containing 0.05 31 sodium phosphate, pH 6.5 and 1 rnhf EDTA. The distribution of the inhibitor was shown in Fig. 3. Although the latent RNase could not be removed completely from the inhibitor fraction by these procedures, the maximum activity of the latent RNase present in the inhibitor was 0.2% of the “67”” RNase activity used in the assay (see Materials and Methods). Arrows indicated the positions of the latent RNase and the free RKase lvhich were checked with the same column. -Judging from the position of
74
GOT0
AND
0
MIZUNO
TUBE NO.
TUBE NO.
Fr~.r3.r,Gel filtration of RNase inhibitor on Sephadex G-75. One-half milliliter of the sample was applied to the column of Sephadex G-75, 1.0 X 80 cm and eluted by 0.05 M sodium phosphate buffer, pH 6.5 containing 1 mM of EDTA. Three microliters of each fraction was used for the assay. The activity of inhibitor is shown in percentage inhibition of RNase of 67” treated crude extract (100 ~1). The positions of latent RNase (A) and free RNase (B) were shown by arrows.
the inhibitor activity on the column, the approximate molecular weight of the inhibitor was 52,000-60,000, as determined by the method of Andrews (19). This partially purified inhibitor thus obtained and free RNase were mixed at 0” in vitro and analyzed by gel filtration to determine whether a complex of the two formed. Twenty-two milliliters of free RNase, 2.5 ml of inhibitor as recovered from the peak of Fig. 3, and 0.,5 ml of 0.1 JI EDTA were mixed in a glass flask and then the mixture was concentrated in a collodion bag completely. The activity of latent RNase contaminating in the inhibitor fraction was not more than 2% of the activity of the added free RNase. The condensate was dissolved in a solution containing 0.05 &L sodium phosphate, pH 6.5 and 1 rnM EDTA, and applied to the Sephadex G-75 column. As shown in Fig. 4, the activity was recovered only at the position of the latent RNase. Although the latency before gel filtration was complete, it was decreased to 73 % after the filtration. From these findings it was concluded that the latent RNase recovered in the peak of Fig. 2 is a complex of the RNase and its inhibitor. The approximate molecular weight of the complex is 56,000 from the eluting position on the gel filtration [see the previous paper (lci)].
FIG. 4. Formation of the complex from free RNase and its inhibitor. The free RNase and its inhibitor which were both purified by gel filtration were mixed in vitro and applied to the column of Sephadex G-75, 1.0 X 80 cm and eluted by 0.05 M sodium phosphate buffer, pH 6.5 containing 1 mM of EDTA. The enzyme assay was done either with (0-e) or without (0-O) PCMB. The position of free HNase was shown by an arrow.
Activating Factors of the Partially Puri$ed Latent RNase 1. SH reagents. The partially purified latent RNase was activated by 1 X 10M58 X 1OWM PCMB. No activation was shown by less than 2.5 X lo-” M, and the concentration of lo-* AI or more was inhibitory to the RNase itself. It was shown that 2.5 X lo+ M PCMB did not influence the activity of free RNase. As shown in Table III, 1 m&l of o-iodosobenzoate and N-ethylmaleimide activated the latent RNase completely. Other reagents examined showed little or no activation (Table III). 2. Metal ions. Metal ions such as Fe2+, Mg2+, Ca2+, Mn2+, and Co2+ did not activate latent enzyme at 1 miv1. Latent enzyme was activated completely by Cu2+ (3 X lOV1 X lop5 1~1)and Zn2+ (5 X 10-j-l X lob4 M). This was reversed by EDTA. 3. HzS04. The partially purified latent RNase was treated with various concentrations of sulfric acid, dialyzed against 0.05 M sodium phosphate buffer, pH 6.5 for 12 hr, and assayed with or without PCMB. Although it was not activated at 0.001 N, 40 and 100% activation occurred at 0.01 and 0.1 N, respectively. 4. Heat treatment. As reported previously (16), the RNase in crude extracts which is apparently inactivated by the 55” treatment is reactivated on further heating at higher
RAT RETICULOCYTE TABLE .~~TIV.~TI~N
OF
P.\RTI.~LLY
RNase
-
RNase AND ITS INHIBITOR
III PURIFIED
LATENT
BY SH REAGENTS
-4ddition
NOW p-Chloromercurihetl~oate Iodoacetate o-Iodosobenzoate N-Methylmaleimide Oxidized glutathione
Hz02 KPe (CN) 6
Concn (nm)
0.025 0.1 1.0 0.1 1.0 0.1 1.0
5.0 0.5, 5.0 1.0
% Activation
o-o.5 100 0.6 0.6
12 96
8 107
0 0 0
This has been interpreted to mean that the latent RNase might be activated through the denaturation of the inhibitor. To test this interpretation, the partially purified latent RNase was heated in 0.05 1\1sodium phosphate buffer, pH 6.5 at various temperatures for 5 min. After being chilled in an ice bath, the activity was measured with or without PCMB. The percentage activation and the total activity are shown in Fig. 5. Activation of the latent enzyme was most remarkable at 65-75”. This was similar to the observation on the crude extract (16). On the other hand, the total
temperature.
activity is greatly diminished at 70-75”. This suggested that the inactivation of the enzyme itself occurred at the same temperature as in the case of the free RNase. Comparing both curves in Fig. 5 it should be noticed that the temperature at which the maximum activation of latent RNase occurs is similar to the temperature at which the inactivation of RSase occurred. But it seemed that the inhibitor was more heatlabile than the enzyme itself because at 70”
40 % of the activation
was observed without This was confirmed by the prolonged heating of the
an obvious
75
loss of the activity.
latent enzyme at 67”. After 30 min of heating, about 60% was activated whereas the loss of the total activity was less than 20 %.
5. Urea. The latent
enzyme was mixed
with various concentrations of urea and dialyzed against the buffer. The samples were assayed with or without PCR4B (Fig. 6). Activation was observed at 3 JI urea or
FIG. 5. Effect of heat on latent RNase. The partially purified latent RNase was heated in 0.05 M sodium phosphate buffer, pH 6.5 at indicated temperature for 5 min. RNase activity was measured in the presence or absence of PCMB
and percentage activation was calculated (O-O). The total activity was measured in the presence of PCMB and percentage activity of nonheated sample was shown (A-A).
FIG. 6. Activation of latent RNase by urea. The partially purified latent RNase was treated with various concentrations of urea and dialyzed. The activity was measured in the presence or absence of PCMB and percentage activation was calculated.
more. The activation
was maximum
at 6 nI,
whereas no loss of activity was observed at this concentration. The latent enzyme was not activated at all by treatment with 0.5 &L NaCl or by three cycles of freezing and thawing. Dissociation of the Complex of RNase and Its Inhibitor In the experiments described above, it was not shown whether the complex was dissociated or not by activating factors. This point
was studied
by gel filtration.
76
GOT0
AND
MIZUNO
16
FIG. 7. Effect of PCMB on latent RNase. Analysis by gel filtration. The partially purified latent RNase was treated with PCMB and applied to the column of Sephadex G-75, 1.0 X 73 cm and eluted by 0.05 M sodium phosphate buffer, pH 6.5. RNase activity was measured in the presence (a---@) or absence (O--O) of PCMB. The position of free RNase was shown by an arrow.
After the treatment of the latent RNase with PCMB, all the activity was recovered in the position of the complex (Fig. 7). Thus the complex had not been dissociated. Although the latent enzyme was activated completely before the filtration, latency was partly restored after the filtration. This shows that the activation by PCMB is reversible. Similar results were obtained after treatment with CuCl*, o-iodosobenzoate, and N-ethylmaleimide. It was therefore concluded that activation by SH reagents did not result in the dissociation of the complex. In the case of the treatment with 6 M urea, all of the recovered activity was obtained at the position of free RNase (Fig. 8). This indicates dissociation of RNase from the complex. The inhibitor was not recovered after the urea treatment inside or outside of the bag. It was denatured probably irreversibly. In this experiment it was also observed that the dissociated enzyme was easily inactivated during the gel filtration. This may be due to the dilution of the enzyme. The inactivation could be avoided by the addition of bovine serum albumin as the case described below.
20
24 TUBENO.
28
32
36
FIG. 8. Effect of 6 M urea on latent RNase. Analysis by gel filtration. The partially purified latent RNase was treated with 6 M urea, dialyzed, and applied to the column of Sephadex G-75, 1.9 X 73 cm and eluted by 0.05 M sodium phosphate buffer, pH 6.5. RNase activity wm measured in the presence (O--O) or absence (O---O) of PCMB. The position of latent RNase was shown by an arrow.
After 0.1 N H&304 treatment, the complex was also dissociated. In this case however, eluting buffer should contain bovine serum albumin (final concentration, 150 pg/ml), otherwise the enzyme was inactivated during the gel filtration. DISCUSSION
As described in the Introduction, RNase inhibitors have been reported to be present in various tissues. However, the properties of these inhibitors have been examined bs using bovine pancreatic RNase. In this report we could show a relation between the RNase and its corresponding inhibitor derived from one and the same cell, rat reticulocyte. We showed that there are two types of activation of the latent RISase, activation with and without dissociation of complex. The inhibitor which is activated by the treatment at 55” inhibited the RNase activity in the sample treated at 67”, but the inhibition was not complete even if an excess amount of the inhibitor fraction was added. This may be due to the fact that the enzyme sample treated at 67” contains both free RNase and RNase which remains active even though complexed with its inhibitor. Indeed, about 20 % of the uninhibited RNase activity in the sample treated at 67” was shown to be present in the position of the complex after the gel filtration (see Fig. 2 in
RAT
RETICULOCYTE
RNase
the preceding paper). Therefore, incomplete inhibition could be explained by considering that the inhibitor in the complex is not exchanged easily with free inhibitor. A similar phenomenon was observed in the effect of the crude extract heated at 55” on RNase activity (Table II). Among factors which activate the latent RKase, SH reagents did not induce the dissociation of the enzyme. Therefore, SH groups in the inhibitor do not seem to play a principal role on the formation of the complex, though they seem important to the inhibitory action. On the other hand, after treatment with 6 51 urea, activation and dissociation of the enzyme occurred. Since the inhibitor is denatured irreversibly by this treatment, dissociation is not unexpected. Rat liver RKase inhibitor was reported to interact with bovine pancreatic RNase which had been inactivated by a modification at the active site (20). This suggests that the binding site of the inhibitor on the RNase is different from the active site of the enzyme. This is also the case in our experiment where a complete activation of the latent RNase could be observed without dissociation of the complex. The inhibitor may have attached to a sit’e on the enzyme other than active site and some subsequent structural changes which cause the inactivation of the enzyme may occur at the active site. Activation of the latent RNase with the release of the enzyme is observed only under rather severe conditions such as the treatment with 6 ALurea or 0.1 M H,SO+ Therefore, it can be considered that the release of the enzyme must be difficult in vim. On the other hand, the activation without release of the enzyme is observed by the treatment with SH reagents of rather low concentration. Metal ions such as Cu2+ and Zn2+ which react with SH groups in protein may play some roles on the activation without inducing the dissociation of the complex as a
AND
ITS
INHIBITOR
result of a possible condensation parts of the cell (21, 22).
77 at some
REFERENCES 1. ROTH, J. S., Biochink. Biophys. Acta 21, 34 (1956). 2. SHORTM.IN, K., Biochim. Biophys. Acta 61, 37 (1961). 3. ~~~~~~~~~~~~~~ Y., M.*sE, K., x-m SUZUNI, Y. Ezperientia 23, 526 (1967). 4. IMRIE, R. C., AND HUTCHISON, W. C., Biochim. Biophys. Acta 108, 106 (1965). 5. GIRIJ.~, N. S., AND SHEEXI~~IS~IN, A., Biochenk. J. 98, 562 (1966). 6. EICHEL, H. J., AND FIGUEROA, E. hl., BiOchi?fk. Biophys. Acta 61, 216 (1961). 7. RO~INO~ITCH, M. It., AND SCREE~NY, L. M., J. Biol. Chem. 243, 3441 (1968). 8. SHORTMSN, K., Biochim. Biophys. Acla 66, 88 (1962). G., .IND BLOE,W. S., REZELM.IN, 9. Bow, MENIX~L, H., Biochem. S. 96, 15~ (1965). P. V., HAMMOND, W. S., AND 10. NORTTHUP, VI.~, M. F. L., Proc. Xat. Acad. Sci. U.S.A 67, 273 (1967). G. R., AND SCH.ICHTER, II., Can. b. 11. L.I~FORD, Biochem. 46, 144 (1907). G., *\ND v.~x POTTER, R., Proc. 12. BLOBEL, Abut. Acad. Sci. U.S.A. 56, 1283 (1966). 13. HYMEW, W. C., .\ND KUFF, E. I~., Biochenk. Biophys. Res. Commun. 16, 506 (1964). A., .IND BUTCH, H., Riochem. 14. CH.IKR.IV~ITY, Pharmacol. 16, 1711 (1967). XI. M., .~ND BERTRAY, 15. HILZ, H., OLDERKOP, B., Hoppe-Seyler’s 2. Physiol. Chem. 349, 1475 (1968). II., ilr&. Biochem. 16. GOTO, S., .XND MIZUNO, Biophys. 146, 64 (1971). N. J., FAI~R, 17. LO~VRY, 0. H., ROSEBHOUGH, R. J., J. Biol. Chem. A., AND K.%ND.ILL, 183, 265 (1951). 18. PETERSON, E. A., AND SOILER, H. A., Methods Enzymol. 6, (1962). It., Biochem. J. 91, 222 (1964). 19. ANDREWS, I)., Biochem. J. 20. ROTH, J. S., AND HURLEY, 101, 112 (1966). 21. H.~RRISON, D. G., Ails LONG, C., J. Physiol. London 199, 367 (1968). G. A., AND REIS~, M. M., Biochim. 22. MORRILL, Biophys. Acta 179, 43 (1969).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Degradation II. Relationship
Between
of RNA
in Rat Reticulocytes
RNase and Its Corresponding
SATARO Faculty
146, 71-77 (1971)
of Pharmaceutical Received
GOTO
Ah3
Sciences,
December
DEN’ICHI
TJniversily
Inhibitor MIZUNO
of ?‘okyo, Honyo,
15, 1970; accepted
in Rat Reticulocyte
March
Tokyo,
Japan
16, 1971
A latent RNase was partially purified by ammonium sulfate fractionation and Sephadex G-75 gel filtration from rat reticulocyte lysate. By mixing free RNase and its inhibitor in vitro, the latent RNase was shown to be a complex of each component. The latent RNase was activated by SH reagents, but no dissociation of the complex was observed. It was also activated by G M urea and 0.1 M H&.&, and in these cases the enzyme was released as judged by gel filtration. From the results of this and precious paper, a possible mechanism of the regulation of RNase activity in rat reticulocyte is discussed.
cell may be simpler than in other cells or tissues. In this report, we describe a procedure for the purification of the latent RNase and factors which activate it and demonstrate that the latent RNase is a complex of RNase and its inhibitor.
It has been known that there are RSase inhibitors in various tissues of the rat, such as liver (1, 2), brain (3), kidney (I), adrenal (4, 5), adipose tissue (6), and parotid gland (7). The inhibitor in liver was purified and characterized extensively, although its role in the metabolism of RKA has been obscure. It was reported that the inhibitor \vas inactivated easily on heat treatment or dilution and was specifically inhibitory against alkaline RNase of animal tissues (1, 2, 8). The RXase inhibitor of rat liver has been used frequently for the preparation of polysomes from animal tissues to protect them from enzymatic degradation (9-11). It has been, therefore, suggested that the degradation of messenger RNA may be ascribed to the action of alkaline RNase (9, 12, 13). Although it was reported that the inhibitor level is changed under various conditions (4, 14, 1.5), it is not known how it may regulate ItNase activity in vivo. As described in the preceding paper, an RNase inhibitor is also present in rat reticulocytes. On heating the crude extract at 55” it masked the enzyme activity completely being itself activated. It seems useful to know the relationship between the RXase and its inhibitor in reticulocyte because cell structure and RNA degrading system of this
MATERIALS Preparation
AND
METHOT
of Rat Reticulocyte Lysate sup
Rat reticulocytes were induced and the cell Iysate sup was prepared as described before (16).
Assay of RNase and l,atent RNase RNase activity was measured as described before (16). Latent RNase was usually activated by 0.025 rnM p-chloromercuribenzoate (PCMB), which inactivates the RNase inhibitor. This concentration of PCMB was shown to activate t,he partially purified latent RNase completely and to have no effect on the RNase activity itself. The degree of activation is expressed as percentage of the maximum activity which is determined in the presence of optimum amount of PCMB.
Assay oj RNase Inhibitor The activit,y of RNase inhibitor was measured as an inhibitory activity t,o the RNase which was prepared by t.reatment of the crude extra.ct at 67”. This method is different from the case of rat 71
72
GOT0
AND
0 0 4 a 12 16 20 MOUNT OF 55'C LWilON (~11
FIG. 1. Inhibition of rat reticulocyte RNase by its inhibitor. The RNase was obtained by heating crude extract at 67” but not purified further. The inhibitor fraction was obtained by 55” treatment of crude extract. One hundred microliters of 67” fraction and indicated amount of 55” fraction were mixed and RNase activity was measured under standard assay condition.
liver RNase inhibitor in earlier studies, in which bovine pancreatic RNase was used instead of its corresponding RNase present in the same tissue. The assay system consists of 160 ~1 of the expH tract treated at 67”, 300 ~1 of 0.1 M Tris-Cl, 7.5, 10 ~1 of 0.1 M EDTA, an appropriate amount of inhibitor fraction and 32P- or ‘%-labeled E. coli rRNA. The method of assay is the same as described before (16). The inhibitory activity was expressed as the percentage of the activity of the added RNase. The relation between the amount of inhibitor fraction and the activity of the RNase was given in Fig. 1. The inhibition was linear to about 80%. To inhibit the enzyme activity completely, further twofold or more of the inhibitor fraction was needed. A similar tendency has been reported on the inhibition of bovine pancreatic RNase by rat liver supernatant (14).
MIZUNO
servation. All procedures were performed at O-5” unless otherwise stated. Fifty milliliters of frozen lysate sup was thawed at room temperature (E-20’) and the red aggregates were removed by centrifugation at 2,000~ for 10 min. To the resultant supernatant, 0.1 M sodium phosphate buffer, pH 5.6, was added to a final concentration of 0.02 nr. The mixture was heated at 55” for 5 min. After standing in an ice bath for 20 min, the aggregated materials were removed by centrifugation. Solid ammonium sulfate was then added to the supernatant with stirring to 55% saturation. After stirring for 20 min, the precipitate was collected by centrifugation at 10,OOOy for 20 min and dissolved in l-2 ml of 0.05 nr sodium phosphate buffer, pH 6.5. The solution was dialyzed against 500 ml of the same buffer for 4 hr, and the precipitate was removed by centrifugation. The supernatant was applied to a column of Sephadex G-75 and eluted by the same buffer. A typical elution pattern is shown in Fig. 2. In contact to the case of the 67” treatment [see the preceding paper (IS)] no activity was observed at the position of the faster peak [peak I in the Fig. 2 of the preceding paper (IS)] without PCMB in the assay
Other Methods Gel filtration on Sephadex G-75 was performed as described before (16). Protein was determined by the method of Lowry et al. (17), using bovine serum albumin as a reference standard. RESULTS
Partial Purzfication of the Latent RNase As reported in the previous paper (16), RNase activity of the crude extract of rat reticulocytes disappeared completely on heating at 55” for 5 min. Partial purification of the latent RNase was based on this ob-
FIG. 2. Gel filtration of the latent RNase on Sephadex G-75 (1.9 X 73 cm). One-half milliliter of dialyzed sample obtained after ammonium sulfate fractionation was applied and eluted by 0.05 M sodium phosphate buffer, pH 6.5. One hundred microliters of each fraction (5 ml) was used for the assay. Latent enzyme was activated by 2.5 X 1OW M PCMB. (O---O) activity in cpm. (A---A) A280. The position of free RNase was shown by an arrow.
RAT
RETICULOCYTE
RNase
mixture and no activity at all was found at the position of free RNase with or without PC?,IB. It is, therefore, suggested that all the RNase in the crude extract is complexed with its inhibitor after heating at 55”. Even when ammonium sulfate was added to 68 % saturation instead of 55 %, there was no activity at the position of free RNase. Therefore, it cannot be considered that free IiKase in the 55” treated crude extract did not precipitate because of the low concentration of ammonium sulfate. The specific activity and yield of the latent enzyme at each stage are shown in Table I. The RNase activity was measured in the presence of the optimal concentration of PCMB. The reason why the activity was increased greatly after heating at 55” ‘is not known. It was shown that the activated RKase had optimum pH of 6.5-7.5. E$ect of the Crude Extract Heated at 55” on the RNase Activity of the Extract Stored at 0” and That Heated at 67” The effect of the crude extract heated at 55” on the RNase activity was examined. As shown in Table II, the sample completely inhibited the enzyme activity of the extract heated at 67”, while the activity of the extract stored at 0” was not affected at all. This fact could be explained by considering that the activity of the “0”” sample is due to the RNase already complexed with its inhibitor and this activity was not affected by the inhibitor derived from the sample treated at 55” (see also Discussion). Formation of the Complex with Free RNase and Inhibitor The RNase inhibitor was purified partially from the crude extract of rat reticulocyte by a procedure described below. All the procedures were done at O-5” unless otherwise noticed. Fifty milliliters of frozen lysate sup was thawed at room temperature and the supernatant u-as obtained after centrifugation. The sup was mixed with 0.1 nr EDTA (final concn, 1 mnr), 0.05 M sodium phosphate buffer, pH 6.5 (final concn, 0.01 RI), and 1.5 M NaCl (final concn, 0.15 11). The mixture was then applied to DEAE-cellu-
AND
ITS
INHIBITOR
73
Total activity (cm)
Crude extract 55” treatment Ammonium snlfate Sephadex G-75
0.8 x 10” 3.7 x 105 1.8 x 105
Specific activity (CPdW protein) 5.7 x 102 6.6 X 103 5.2 x lo4
1.75 x 105
TABLE EFFECT
Total
protein (w)
0.G
2.8 x 105
II
OF THE CKUDE EXTR.\CT HE.ITED OK RNME ACTIVITY
AT
55’ fraction (PI)
0’ fraction Ml)
RNase activity (CPW
100 50 0
100 100 100
548 559 570
55”
67” fraction (Pl) 100 50 25 0
100 loo 100 100
11 0 G 784
lose column 0.9 X 2 cm, which was prepared by the method of Peterson and Sober (18) and buffered with the buffer described above. After washing with 10 ml of the same buffer, the inhibitor was eluted by the buffer containing 1.0 XI NaC1 and 1 mM EDTA. After concentrating the eluate to 0.4 ml in a collodion bag, 0.6 ml of H,O was added; it was applied to the column of Sephadex G-75, 1.0 X 80 cm and eluted by the buffer containing 0.05 31 sodium phosphate, pH 6.5 and 1 rnhf EDTA. The distribution of the inhibitor was shown in Fig. 3. Although the latent RNase could not be removed completely from the inhibitor fraction by these procedures, the maximum activity of the latent RNase present in the inhibitor was 0.2% of the “67”” RNase activity used in the assay (see Materials and Methods). Arrows indicated the positions of the latent RNase and the free RKase lvhich were checked with the same column. -Judging from the position of
74
GOT0
AND
0
MIZUNO
TUBE NO.
TUBE NO.
Fr~.r3.r,Gel filtration of RNase inhibitor on Sephadex G-75. One-half milliliter of the sample was applied to the column of Sephadex G-75, 1.0 X 80 cm and eluted by 0.05 M sodium phosphate buffer, pH 6.5 containing 1 mM of EDTA. Three microliters of each fraction was used for the assay. The activity of inhibitor is shown in percentage inhibition of RNase of 67” treated crude extract (100 ~1). The positions of latent RNase (A) and free RNase (B) were shown by arrows.
the inhibitor activity on the column, the approximate molecular weight of the inhibitor was 52,000-60,000, as determined by the method of Andrews (19). This partially purified inhibitor thus obtained and free RNase were mixed at 0” in vitro and analyzed by gel filtration to determine whether a complex of the two formed. Twenty-two milliliters of free RNase, 2.5 ml of inhibitor as recovered from the peak of Fig. 3, and 0.,5 ml of 0.1 JI EDTA were mixed in a glass flask and then the mixture was concentrated in a collodion bag completely. The activity of latent RNase contaminating in the inhibitor fraction was not more than 2% of the activity of the added free RNase. The condensate was dissolved in a solution containing 0.05 &L sodium phosphate, pH 6.5 and 1 rnM EDTA, and applied to the Sephadex G-75 column. As shown in Fig. 4, the activity was recovered only at the position of the latent RNase. Although the latency before gel filtration was complete, it was decreased to 73 % after the filtration. From these findings it was concluded that the latent RNase recovered in the peak of Fig. 2 is a complex of the RNase and its inhibitor. The approximate molecular weight of the complex is 56,000 from the eluting position on the gel filtration [see the previous paper (lci)].
FIG. 4. Formation of the complex from free RNase and its inhibitor. The free RNase and its inhibitor which were both purified by gel filtration were mixed in vitro and applied to the column of Sephadex G-75, 1.0 X 80 cm and eluted by 0.05 M sodium phosphate buffer, pH 6.5 containing 1 mM of EDTA. The enzyme assay was done either with (0-e) or without (0-O) PCMB. The position of free HNase was shown by an arrow.
Activating Factors of the Partially Puri$ed Latent RNase 1. SH reagents. The partially purified latent RNase was activated by 1 X 10M58 X 1OWM PCMB. No activation was shown by less than 2.5 X lo-” M, and the concentration of lo-* AI or more was inhibitory to the RNase itself. It was shown that 2.5 X lo+ M PCMB did not influence the activity of free RNase. As shown in Table III, 1 m&l of o-iodosobenzoate and N-ethylmaleimide activated the latent RNase completely. Other reagents examined showed little or no activation (Table III). 2. Metal ions. Metal ions such as Fe2+, Mg2+, Ca2+, Mn2+, and Co2+ did not activate latent enzyme at 1 miv1. Latent enzyme was activated completely by Cu2+ (3 X lOV1 X lop5 1~1)and Zn2+ (5 X 10-j-l X lob4 M). This was reversed by EDTA. 3. HzS04. The partially purified latent RNase was treated with various concentrations of sulfric acid, dialyzed against 0.05 M sodium phosphate buffer, pH 6.5 for 12 hr, and assayed with or without PCMB. Although it was not activated at 0.001 N, 40 and 100% activation occurred at 0.01 and 0.1 N, respectively. 4. Heat treatment. As reported previously (16), the RNase in crude extracts which is apparently inactivated by the 55” treatment is reactivated on further heating at higher
RAT RETICULOCYTE TABLE .~~TIV.~TI~N
OF
P.\RTI.~LLY
RNase
-
RNase AND ITS INHIBITOR
III PURIFIED
LATENT
BY SH REAGENTS
-4ddition
NOW p-Chloromercurihetl~oate Iodoacetate o-Iodosobenzoate N-Methylmaleimide Oxidized glutathione
Hz02 KPe (CN) 6
Concn (nm)
0.025 0.1 1.0 0.1 1.0 0.1 1.0
5.0 0.5, 5.0 1.0
% Activation
o-o.5 100 0.6 0.6
12 96
8 107
0 0 0
This has been interpreted to mean that the latent RNase might be activated through the denaturation of the inhibitor. To test this interpretation, the partially purified latent RNase was heated in 0.05 1\1sodium phosphate buffer, pH 6.5 at various temperatures for 5 min. After being chilled in an ice bath, the activity was measured with or without PCMB. The percentage activation and the total activity are shown in Fig. 5. Activation of the latent enzyme was most remarkable at 65-75”. This was similar to the observation on the crude extract (16). On the other hand, the total
temperature.
activity is greatly diminished at 70-75”. This suggested that the inactivation of the enzyme itself occurred at the same temperature as in the case of the free RNase. Comparing both curves in Fig. 5 it should be noticed that the temperature at which the maximum activation of latent RNase occurs is similar to the temperature at which the inactivation of RSase occurred. But it seemed that the inhibitor was more heatlabile than the enzyme itself because at 70”
40 % of the activation
was observed without This was confirmed by the prolonged heating of the
an obvious
75
loss of the activity.
latent enzyme at 67”. After 30 min of heating, about 60% was activated whereas the loss of the total activity was less than 20 %.
5. Urea. The latent
enzyme was mixed
with various concentrations of urea and dialyzed against the buffer. The samples were assayed with or without PCR4B (Fig. 6). Activation was observed at 3 JI urea or
FIG. 5. Effect of heat on latent RNase. The partially purified latent RNase was heated in 0.05 M sodium phosphate buffer, pH 6.5 at indicated temperature for 5 min. RNase activity was measured in the presence or absence of PCMB
and percentage activation was calculated (O-O). The total activity was measured in the presence of PCMB and percentage activity of nonheated sample was shown (A-A).
FIG. 6. Activation of latent RNase by urea. The partially purified latent RNase was treated with various concentrations of urea and dialyzed. The activity was measured in the presence or absence of PCMB and percentage activation was calculated.
more. The activation
was maximum
at 6 nI,
whereas no loss of activity was observed at this concentration. The latent enzyme was not activated at all by treatment with 0.5 &L NaCl or by three cycles of freezing and thawing. Dissociation of the Complex of RNase and Its Inhibitor In the experiments described above, it was not shown whether the complex was dissociated or not by activating factors. This point
was studied
by gel filtration.
76
GOT0
AND
MIZUNO
16
FIG. 7. Effect of PCMB on latent RNase. Analysis by gel filtration. The partially purified latent RNase was treated with PCMB and applied to the column of Sephadex G-75, 1.0 X 73 cm and eluted by 0.05 M sodium phosphate buffer, pH 6.5. RNase activity was measured in the presence (a---@) or absence (O--O) of PCMB. The position of free RNase was shown by an arrow.
After the treatment of the latent RNase with PCMB, all the activity was recovered in the position of the complex (Fig. 7). Thus the complex had not been dissociated. Although the latent enzyme was activated completely before the filtration, latency was partly restored after the filtration. This shows that the activation by PCMB is reversible. Similar results were obtained after treatment with CuCl*, o-iodosobenzoate, and N-ethylmaleimide. It was therefore concluded that activation by SH reagents did not result in the dissociation of the complex. In the case of the treatment with 6 M urea, all of the recovered activity was obtained at the position of free RNase (Fig. 8). This indicates dissociation of RNase from the complex. The inhibitor was not recovered after the urea treatment inside or outside of the bag. It was denatured probably irreversibly. In this experiment it was also observed that the dissociated enzyme was easily inactivated during the gel filtration. This may be due to the dilution of the enzyme. The inactivation could be avoided by the addition of bovine serum albumin as the case described below.
20
24 TUBENO.
28
32
36
FIG. 8. Effect of 6 M urea on latent RNase. Analysis by gel filtration. The partially purified latent RNase was treated with 6 M urea, dialyzed, and applied to the column of Sephadex G-75, 1.9 X 73 cm and eluted by 0.05 M sodium phosphate buffer, pH 6.5. RNase activity wm measured in the presence (O--O) or absence (O---O) of PCMB. The position of latent RNase was shown by an arrow.
After 0.1 N H&304 treatment, the complex was also dissociated. In this case however, eluting buffer should contain bovine serum albumin (final concentration, 150 pg/ml), otherwise the enzyme was inactivated during the gel filtration. DISCUSSION
As described in the Introduction, RNase inhibitors have been reported to be present in various tissues. However, the properties of these inhibitors have been examined bs using bovine pancreatic RNase. In this report we could show a relation between the RNase and its corresponding inhibitor derived from one and the same cell, rat reticulocyte. We showed that there are two types of activation of the latent RISase, activation with and without dissociation of complex. The inhibitor which is activated by the treatment at 55” inhibited the RNase activity in the sample treated at 67”, but the inhibition was not complete even if an excess amount of the inhibitor fraction was added. This may be due to the fact that the enzyme sample treated at 67” contains both free RNase and RNase which remains active even though complexed with its inhibitor. Indeed, about 20 % of the uninhibited RNase activity in the sample treated at 67” was shown to be present in the position of the complex after the gel filtration (see Fig. 2 in
RAT
RETICULOCYTE
RNase
the preceding paper). Therefore, incomplete inhibition could be explained by considering that the inhibitor in the complex is not exchanged easily with free inhibitor. A similar phenomenon was observed in the effect of the crude extract heated at 55” on RNase activity (Table II). Among factors which activate the latent RKase, SH reagents did not induce the dissociation of the enzyme. Therefore, SH groups in the inhibitor do not seem to play a principal role on the formation of the complex, though they seem important to the inhibitory action. On the other hand, after treatment with 6 51 urea, activation and dissociation of the enzyme occurred. Since the inhibitor is denatured irreversibly by this treatment, dissociation is not unexpected. Rat liver RKase inhibitor was reported to interact with bovine pancreatic RNase which had been inactivated by a modification at the active site (20). This suggests that the binding site of the inhibitor on the RNase is different from the active site of the enzyme. This is also the case in our experiment where a complete activation of the latent RNase could be observed without dissociation of the complex. The inhibitor may have attached to a sit’e on the enzyme other than active site and some subsequent structural changes which cause the inactivation of the enzyme may occur at the active site. Activation of the latent RNase with the release of the enzyme is observed only under rather severe conditions such as the treatment with 6 ALurea or 0.1 M H,SO+ Therefore, it can be considered that the release of the enzyme must be difficult in vim. On the other hand, the activation without release of the enzyme is observed by the treatment with SH reagents of rather low concentration. Metal ions such as Cu2+ and Zn2+ which react with SH groups in protein may play some roles on the activation without inducing the dissociation of the complex as a
AND
ITS
INHIBITOR
result of a possible condensation parts of the cell (21, 22).
77 at some
REFERENCES 1. ROTH, J. S., Biochink. Biophys. Acta 21, 34 (1956). 2. SHORTM.IN, K., Biochim. Biophys. Acta 61, 37 (1961). 3. ~~~~~~~~~~~~~~ Y., M.*sE, K., x-m SUZUNI, Y. Ezperientia 23, 526 (1967). 4. IMRIE, R. C., AND HUTCHISON, W. C., Biochim. Biophys. Acta 108, 106 (1965). 5. GIRIJ.~, N. S., AND SHEEXI~~IS~IN, A., Biochenk. J. 98, 562 (1966). 6. EICHEL, H. J., AND FIGUEROA, E. hl., BiOchi?fk. Biophys. Acta 61, 216 (1961). 7. RO~INO~ITCH, M. It., AND SCREE~NY, L. M., J. Biol. Chem. 243, 3441 (1968). 8. SHORTMSN, K., Biochim. Biophys. Acla 66, 88 (1962). G., .IND BLOE,W. S., REZELM.IN, 9. Bow, MENIX~L, H., Biochem. S. 96, 15~ (1965). P. V., HAMMOND, W. S., AND 10. NORTTHUP, VI.~, M. F. L., Proc. Xat. Acad. Sci. U.S.A 67, 273 (1967). G. R., AND SCH.ICHTER, II., Can. b. 11. L.I~FORD, Biochem. 46, 144 (1907). G., *\ND v.~x POTTER, R., Proc. 12. BLOBEL, Abut. Acad. Sci. U.S.A. 56, 1283 (1966). 13. HYMEW, W. C., .\ND KUFF, E. I~., Biochenk. Biophys. Res. Commun. 16, 506 (1964). A., .IND BUTCH, H., Riochem. 14. CH.IKR.IV~ITY, Pharmacol. 16, 1711 (1967). XI. M., .~ND BERTRAY, 15. HILZ, H., OLDERKOP, B., Hoppe-Seyler’s 2. Physiol. Chem. 349, 1475 (1968). II., ilr&. Biochem. 16. GOTO, S., .XND MIZUNO, Biophys. 146, 64 (1971). N. J., FAI~R, 17. LO~VRY, 0. H., ROSEBHOUGH, R. J., J. Biol. Chem. A., AND K.%ND.ILL, 183, 265 (1951). 18. PETERSON, E. A., AND SOILER, H. A., Methods Enzymol. 6, (1962). It., Biochem. J. 91, 222 (1964). 19. ANDREWS, I)., Biochem. J. 20. ROTH, J. S., AND HURLEY, 101, 112 (1966). 21. H.~RRISON, D. G., Ails LONG, C., J. Physiol. London 199, 367 (1968). G. A., AND REIS~, M. M., Biochim. 22. MORRILL, Biophys. Acta 179, 43 (1969).