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The distribution of ribonucleases in corn, cucumber, and soybean seedlings effects of isolation media
311
BIOCHIMICA ET BIOPHYSICA ACTA
Short communications SC 7068
The distribution of ribonucleases in corn, cucumber, and soybean seedlings Effects of isolation media RNAases from apple 1, wheat s, and corn 3 have been separated centrifugally into a soluble enzyme and an enzyme located on the particulate matter, characterized b y pH optima near 5 and 6, respectively. The RNAases on pea microsomes and ribonucleoprotein particles were also found to differa. Two RNAases in corn were isolated by chromatography on CM-cellulose, but the centrifugal separation of the two RNAases was not complete 3. The location of RNAase activity is of importance because it may be intimately associated with plant developmentX,5,e. Corn (Zea mays, L.) and cucumber (Cucumis sativus, L.) seeds were germinated for 3 days in the dark at 28 ° on absorbent paper supported by gravel and wet by lO -4 M CaCI~. Soybeans (Glycine max, L., Merr.) were germinated in Krum*, an expanded volcanic glass. The roots of corn and the whole seedling minus cotyledon of cucumber and soybeans were harvested. The isolation media are given in Table I. Sucrose is commonly used for isolation of mitochondria and microsomes. The SPE medium** disperses microsomes and enhances the respiratory activity of mitochondria 7. 0.5 M KC1 removes the RNAase from particulate matter of corn 3. The seedlings were homogenized in four parts of medium for I rain at high speed in a Vir-Tis homogenizer and squeezed through cheesecloth to give the "crude" homogenate. This was centrifuged successively at 8 o o × g for IO rain (to precipitate cellular debris and nuclei), at 40 ooo × g for 30 min (to precipitate mitochondria), and at 114 ooo × g for 9 ° rain (to precipitate microsomes). The mitochondria and microsomes were dispersed in o.oi M sodium citrate (pH 6.0). The successive supernatant solutions and the mitochondria and microsomes were assayed for protein s and RNAase 3. The protein and RNAase of the debris were calculated by difference. The protein extracted (Table I) was affected only slightly by the medium. T K stimulated cucumber RNAase. The EDTA carried from the SPE*** and SE media to the TABLE I EXTRACTION OF PROTEIN AND RNAASE FROM PLANT TISSUE Isolation Medium Symbol
S ST STK
Composition
Protein (mg/ml) Corn Cucumber
RNAase (units/ml)
Soybeans
Corn Cucumber
Soybeans
SE
0. 5 M sucro se 0. 5 M s u c r o s e + o . o 2 5 M T r i s - H C 1 (pH 7-5) 0.5 M s u c r o s e + o , o 2 5 M T r i s - H C 1 (pH 7.5) + o . 5 N[ KC1 0.o25 M T r i s - H C l (pH 7.5) + 0 . 5 M KC1 o.5Msucrose+o.o67Mpotassiumphosphate (pH 7 . o ) + o . o o 5 M E D T A 0. 5 M s u c r o s e + o . I M E D T A (pH 7.3)
* anent ** **"
T r a d e n a m e s are g i v e n as p a r t of t h e e x a c t e x p e r i m e n t a l c o n d i t i o n s a n d n o t a s a n endorseof p r o d u c t s o v e r t h o s e of o t h e r m a n u f a c t u r e r s . F o r t h e c o m p o s i t i o n of t h e v a r i o u s m e d i a see T a b l e I. F o r t h e e x p l a n a t i o n of t h i s a b b r e v i a t i o n s see T a b l e I.
TK SPE
I.I 5 1.2I
i ,oo 0.99
1.34 1.41
44 48
16.4 I5-4
7 .6 7.4
1.4o 1.21 1.28
0.95 0.89 1.o2
I.z 8 1-43 1.43
54 47 35
I6.4 23 48
6.6 7.9 6.6
1.25
0.96
1.31
38
48
7.8
Biochim.
Biophys.
A c t a , 68 (1963) 311-313
312
SHORT COMMUNICATIONS
assay was enough to account for the inhibition of corn RNAase and the stimulatior~ of cucumber RNAase. No consistent variation in soybean RNAase was noted. Increasing the ionic strength of the medium shifted the protein in corn from the debris and mitochondria to the microsomes and soluble fraction (Fig. I). The shift of protein to the soluble fraction also occurred in cucumbers, but was not apparent in soybeans. PROTEIN CUCUMBER
CORN
125| I00 I-
ST --
TK
SOYBEANS
S
7B
i
SO
.
S
STK SPE
S
STK SPE
S
STK SPE
Fig. I. D i s t r i b u t i o n ~f p r o t e i n as affected b y isolation m e d i u m . (Media are g i v e n in T a b l e I.) T h e a c t i v i t y of t h e c r u d e h o m o g e n a t e e q u a l s i o o %. H a t c h m a r k s , debris; clear, m i t o c h o n d r i a ; b l a c k , m i c r o s o m e s ; d o t t e d , soluble.
The RNAase activities did not vary in proportion to the protein content (Fig. 2). In corn the debris and mitochondria contained most of the RNAase in S medium, while the microsomes contained appreciable RNAase onlyin the ST medium. In higher-ionicstrength media the RNAase was almost completely soluble. Cucumber RNAase was found in both mitochondria and microsomes in S medium, with a shift to the soluble CORN
150
~NAASE CUCUMBER
SOYBEANS
liUOliHiDliliWl DIi |00
% 75 50
25 0
-25
S
STK SPE
S
N
STKr/ASPE
S
STK
SPE. J
Fig. 2. D i s t r i b u t i o n of R N A a s e as affected b y isolation m e d i u m . E x p l a n a t i o n s a m e as for Fig. I.
fraction in the other media. Soybeans were marked by a high content of RNAase in the microsomes in S and ST media, but the shift to the soluble fraction also occurred. In several cases the activity of the separate fractions was greater than the activity of the original homogenate. This m a y have been caused b y separation of the enzyme from an inhibitor or by an effect of particulate matter on the assay. Where the debris-free supernatant solution was more active than the crude homogenate, the activity of the debris is represented as negative. The three species, grown and treated alike, showed large differences in the distribution of RNAase, especially in the mitochondria and microsomes from the S and ST media. Sucrose is plainly unsatisfactory for corn because much RNAase is lost b y low-speed centrifugation. The high-ionic-strength media make it possible to discard much inactive protein b y high-speed centrifugation in studies of total RNAase s. EDTA should be used with caution, for it m a y either inhibit or stimulate RNAase. The specific activity of the microsomes (in ST for corn and in S for cucumber and Biochim. Biophys. Acta, 68 (I963) 3 I I - 3 1 3
313
SHORT COMMUNICATIONS
soybean) was higher than that of any mitochondrial fraction. RNAase has been found to be associated with ribosomes4,9-12. In media of low ionic strength, the ribosomes may be precipitated with associated membranes in the mitochondrial fraction, whereas in media of intermediate strength, the ribosomes are isolated in the microsomal fraction. At high ionic strengths the ribosomes would be dissociated so that they would not precipitate zS. An alternate explanation is that the RNAase is merely adsorbed onto surfaces and is released into solution if the ionic strength is increased.
Crops Research Division, Agricultural Research Service, U.S. Department o/ Agriculture, Agronomy Department, University o/Illinois, Urbana, Ill. (U.S.A.)
CURTIS M. WILSON JACK C. SHANNON"
x ][3. KESSLER AND 1~. ENGELBERG, Biochim. Biophys. Aeta, 55 (1962) 7 o. = S, MATSUSHITA, Mere. Res. Inst. Food Sci., Kyoto Univ., 17 (1959) 29. * C. M. WILSON, Biochim. Biophys. Aaa, 68 (1963) 177. S. MATSUSHITA ANn F. ISUKI, Biochim. Biophys. Acta, 4 ° (196o) 358. * G. ~ . BARKER AND T. DOUGLAS, Nature, 188 (196o) 9436 L. LEDOUX, P. GALAND AND 1~.. HUART, Biockim. Biophys. Acta, 55 (1962) 97. H. A. LUND, A. E. VATTER AND J. B. HANSON, J. Biophys. Biochem. Cytol., 4 (I958) 87. s O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J . Biol. Chem., 193 (1951) 265. 9 S. R. DXCKMAN AND G. A. MORRILL, Ann. N.Y. Acad. Sci., 8I (I959) 585 • a0 D. ELSON, Biochira. Biophys. Acta, 36 (1959) 372. al S. M~TSUSHIXA, Mere. Res. Inst. Food Sci., Kyoto Univ., I8 (I959) 8. z~ y . TASHRO, J. Biockem. Tokyo, 45 (I958) 937. as p. O. P. T s ' o , J. BONNER AND J. VINOGRAD, Biochim. Biophys. Acta, 3 ° (1958) 57 o.
Received October z2th, 1962 " P r e s e n t a d d r e s s : D e p a r t m e n t of B o t a n y a n d P l a n t P a t h o l o g y , P u r d u e U n i v e r s i t y , L a f a y e t t e , Ind. (U.S.A,).
Biochim. Biophys. Acta, 68 (1963) 311-313 SC 7063
Post-treotment cross-linking of D N A reocted with polyfunctionol olkyloting ogents When salmon-sperm nucleoprotein is exposed to the action of polyfunctional nitrogen mustards, an intermolecular cross-linking of DNA is observed, converting the DNA to an insoluble gel1,2. The work reported here has shown that covalent intelanolecular bonds are formed when DNA treated with polyfunctional alkylating agents in dilute aqueous solution is subsequently converted to the solid state. A peculiar feature of this effect is that the cross-linking takes place in the absence of any free alkylating agent. The nitrogen and sulphur mustards react predominantly with N- 7 of guanines,4. Difunctional compounds were found to form two types of products, one of them being an alkylated guanine and the other consisting of two guanine residues joined by an alkyl chain. For steric reasons the di-guaninyl derivatives were supposed to be formed from two opposite guanine residues forming part of the sequence GpC in each strand ~, thus producing an intramolecular inter-strand cross-link 6. According to this assumption the di-guaninyl derivative should always be less frequent than th e mono-guaninyl derivative and its total amount in exhaustively alkylated DNA should approach 25 % of the total guanine, i.e. the approximate frequency of the sequence GpC. Biockim. Biophys. Aeta, 68 (I963) 3 1 3 - 3 1 6
BIOCHIMICA ET BIOPHYSICA ACTA
Short communications SC 7068
The distribution of ribonucleases in corn, cucumber, and soybean seedlings Effects of isolation media RNAases from apple 1, wheat s, and corn 3 have been separated centrifugally into a soluble enzyme and an enzyme located on the particulate matter, characterized b y pH optima near 5 and 6, respectively. The RNAases on pea microsomes and ribonucleoprotein particles were also found to differa. Two RNAases in corn were isolated by chromatography on CM-cellulose, but the centrifugal separation of the two RNAases was not complete 3. The location of RNAase activity is of importance because it may be intimately associated with plant developmentX,5,e. Corn (Zea mays, L.) and cucumber (Cucumis sativus, L.) seeds were germinated for 3 days in the dark at 28 ° on absorbent paper supported by gravel and wet by lO -4 M CaCI~. Soybeans (Glycine max, L., Merr.) were germinated in Krum*, an expanded volcanic glass. The roots of corn and the whole seedling minus cotyledon of cucumber and soybeans were harvested. The isolation media are given in Table I. Sucrose is commonly used for isolation of mitochondria and microsomes. The SPE medium** disperses microsomes and enhances the respiratory activity of mitochondria 7. 0.5 M KC1 removes the RNAase from particulate matter of corn 3. The seedlings were homogenized in four parts of medium for I rain at high speed in a Vir-Tis homogenizer and squeezed through cheesecloth to give the "crude" homogenate. This was centrifuged successively at 8 o o × g for IO rain (to precipitate cellular debris and nuclei), at 40 ooo × g for 30 min (to precipitate mitochondria), and at 114 ooo × g for 9 ° rain (to precipitate microsomes). The mitochondria and microsomes were dispersed in o.oi M sodium citrate (pH 6.0). The successive supernatant solutions and the mitochondria and microsomes were assayed for protein s and RNAase 3. The protein and RNAase of the debris were calculated by difference. The protein extracted (Table I) was affected only slightly by the medium. T K stimulated cucumber RNAase. The EDTA carried from the SPE*** and SE media to the TABLE I EXTRACTION OF PROTEIN AND RNAASE FROM PLANT TISSUE Isolation Medium Symbol
S ST STK
Composition
Protein (mg/ml) Corn Cucumber
RNAase (units/ml)
Soybeans
Corn Cucumber
Soybeans
SE
0. 5 M sucro se 0. 5 M s u c r o s e + o . o 2 5 M T r i s - H C 1 (pH 7-5) 0.5 M s u c r o s e + o , o 2 5 M T r i s - H C 1 (pH 7.5) + o . 5 N[ KC1 0.o25 M T r i s - H C l (pH 7.5) + 0 . 5 M KC1 o.5Msucrose+o.o67Mpotassiumphosphate (pH 7 . o ) + o . o o 5 M E D T A 0. 5 M s u c r o s e + o . I M E D T A (pH 7.3)
* anent ** **"
T r a d e n a m e s are g i v e n as p a r t of t h e e x a c t e x p e r i m e n t a l c o n d i t i o n s a n d n o t a s a n endorseof p r o d u c t s o v e r t h o s e of o t h e r m a n u f a c t u r e r s . F o r t h e c o m p o s i t i o n of t h e v a r i o u s m e d i a see T a b l e I. F o r t h e e x p l a n a t i o n of t h i s a b b r e v i a t i o n s see T a b l e I.
TK SPE
I.I 5 1.2I
i ,oo 0.99
1.34 1.41
44 48
16.4 I5-4
7 .6 7.4
1.4o 1.21 1.28
0.95 0.89 1.o2
I.z 8 1-43 1.43
54 47 35
I6.4 23 48
6.6 7.9 6.6
1.25
0.96
1.31
38
48
7.8
Biochim.
Biophys.
A c t a , 68 (1963) 311-313
312
SHORT COMMUNICATIONS
assay was enough to account for the inhibition of corn RNAase and the stimulatior~ of cucumber RNAase. No consistent variation in soybean RNAase was noted. Increasing the ionic strength of the medium shifted the protein in corn from the debris and mitochondria to the microsomes and soluble fraction (Fig. I). The shift of protein to the soluble fraction also occurred in cucumbers, but was not apparent in soybeans. PROTEIN CUCUMBER
CORN
125| I00 I-
ST --
TK
SOYBEANS
S
7B
i
SO
.
S
STK SPE
S
STK SPE
S
STK SPE
Fig. I. D i s t r i b u t i o n ~f p r o t e i n as affected b y isolation m e d i u m . (Media are g i v e n in T a b l e I.) T h e a c t i v i t y of t h e c r u d e h o m o g e n a t e e q u a l s i o o %. H a t c h m a r k s , debris; clear, m i t o c h o n d r i a ; b l a c k , m i c r o s o m e s ; d o t t e d , soluble.
The RNAase activities did not vary in proportion to the protein content (Fig. 2). In corn the debris and mitochondria contained most of the RNAase in S medium, while the microsomes contained appreciable RNAase onlyin the ST medium. In higher-ionicstrength media the RNAase was almost completely soluble. Cucumber RNAase was found in both mitochondria and microsomes in S medium, with a shift to the soluble CORN
150
~NAASE CUCUMBER
SOYBEANS
liUOliHiDliliWl DIi |00
% 75 50
25 0
-25
S
STK SPE
S
N
STKr/ASPE
S
STK
SPE. J
Fig. 2. D i s t r i b u t i o n of R N A a s e as affected b y isolation m e d i u m . E x p l a n a t i o n s a m e as for Fig. I.
fraction in the other media. Soybeans were marked by a high content of RNAase in the microsomes in S and ST media, but the shift to the soluble fraction also occurred. In several cases the activity of the separate fractions was greater than the activity of the original homogenate. This m a y have been caused b y separation of the enzyme from an inhibitor or by an effect of particulate matter on the assay. Where the debris-free supernatant solution was more active than the crude homogenate, the activity of the debris is represented as negative. The three species, grown and treated alike, showed large differences in the distribution of RNAase, especially in the mitochondria and microsomes from the S and ST media. Sucrose is plainly unsatisfactory for corn because much RNAase is lost b y low-speed centrifugation. The high-ionic-strength media make it possible to discard much inactive protein b y high-speed centrifugation in studies of total RNAase s. EDTA should be used with caution, for it m a y either inhibit or stimulate RNAase. The specific activity of the microsomes (in ST for corn and in S for cucumber and Biochim. Biophys. Acta, 68 (I963) 3 I I - 3 1 3
313
SHORT COMMUNICATIONS
soybean) was higher than that of any mitochondrial fraction. RNAase has been found to be associated with ribosomes4,9-12. In media of low ionic strength, the ribosomes may be precipitated with associated membranes in the mitochondrial fraction, whereas in media of intermediate strength, the ribosomes are isolated in the microsomal fraction. At high ionic strengths the ribosomes would be dissociated so that they would not precipitate zS. An alternate explanation is that the RNAase is merely adsorbed onto surfaces and is released into solution if the ionic strength is increased.
Crops Research Division, Agricultural Research Service, U.S. Department o/ Agriculture, Agronomy Department, University o/Illinois, Urbana, Ill. (U.S.A.)
CURTIS M. WILSON JACK C. SHANNON"
x ][3. KESSLER AND 1~. ENGELBERG, Biochim. Biophys. Aeta, 55 (1962) 7 o. = S, MATSUSHITA, Mere. Res. Inst. Food Sci., Kyoto Univ., 17 (1959) 29. * C. M. WILSON, Biochim. Biophys. Aaa, 68 (1963) 177. S. MATSUSHITA ANn F. ISUKI, Biochim. Biophys. Acta, 4 ° (196o) 358. * G. ~ . BARKER AND T. DOUGLAS, Nature, 188 (196o) 9436 L. LEDOUX, P. GALAND AND 1~.. HUART, Biockim. Biophys. Acta, 55 (1962) 97. H. A. LUND, A. E. VATTER AND J. B. HANSON, J. Biophys. Biochem. Cytol., 4 (I958) 87. s O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J . Biol. Chem., 193 (1951) 265. 9 S. R. DXCKMAN AND G. A. MORRILL, Ann. N.Y. Acad. Sci., 8I (I959) 585 • a0 D. ELSON, Biochira. Biophys. Acta, 36 (1959) 372. al S. M~TSUSHIXA, Mere. Res. Inst. Food Sci., Kyoto Univ., I8 (I959) 8. z~ y . TASHRO, J. Biockem. Tokyo, 45 (I958) 937. as p. O. P. T s ' o , J. BONNER AND J. VINOGRAD, Biochim. Biophys. Acta, 3 ° (1958) 57 o.
Received October z2th, 1962 " P r e s e n t a d d r e s s : D e p a r t m e n t of B o t a n y a n d P l a n t P a t h o l o g y , P u r d u e U n i v e r s i t y , L a f a y e t t e , Ind. (U.S.A,).
Biochim. Biophys. Acta, 68 (1963) 311-313 SC 7063
Post-treotment cross-linking of D N A reocted with polyfunctionol olkyloting ogents When salmon-sperm nucleoprotein is exposed to the action of polyfunctional nitrogen mustards, an intermolecular cross-linking of DNA is observed, converting the DNA to an insoluble gel1,2. The work reported here has shown that covalent intelanolecular bonds are formed when DNA treated with polyfunctional alkylating agents in dilute aqueous solution is subsequently converted to the solid state. A peculiar feature of this effect is that the cross-linking takes place in the absence of any free alkylating agent. The nitrogen and sulphur mustards react predominantly with N- 7 of guanines,4. Difunctional compounds were found to form two types of products, one of them being an alkylated guanine and the other consisting of two guanine residues joined by an alkyl chain. For steric reasons the di-guaninyl derivatives were supposed to be formed from two opposite guanine residues forming part of the sequence GpC in each strand ~, thus producing an intramolecular inter-strand cross-link 6. According to this assumption the di-guaninyl derivative should always be less frequent than th e mono-guaninyl derivative and its total amount in exhaustively alkylated DNA should approach 25 % of the total guanine, i.e. the approximate frequency of the sequence GpC. Biockim. Biophys. Aeta, 68 (I963) 3 1 3 - 3 1 6