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Damage of chromosomal proteins during isolation of chromatin and a chromatin-bound protease from germinated pea cotyledon
Plant Science Letters, 10 (1977) 361--366
361
© Elsevier/North-Holland Scientific Publishers Ltd.
DAMAGE OF CHROMOSOMAL PROTEINS DURING ISOLATION OF CHROMATIN AND A CHROMATIN-BOUND PROTEASE FROM GERMINATED PEA COTYLEDON
EIJI HIRASAWA, EIICHI TAKAHASHI and HIDEAKI MATSUMOTO
Department of Agricultural Chemistry, Kyoto University, Kyoto 606 (Japan) (Received April 18th, 1977) (Accepted August 25th, 1977)
SUMMARY
During the isolation of chromatin from germinated pea cotyledons, the HI histone was susceptible but the other histones were resistant to degradation as seen from the electrophoretical profile. The digestive damage of non-histone proteins appeared strongly in the region of large protein molecules. In order to avoid damage to chromosomal proteins, the shortening of time required for preparations before ultracentrifugation and the addition of sodium bisulfite were effective. After the dissociation of chromatin with 2 M NaC1, the reconstituted chromatin containing proteolytic activity caused the autolysis of the chromosomal proteins. This autolysis was inhibited by sodium bisulfite. The substrate specificity of chromatin-tightly bound protease, which preferred bovine serum albumin but protamine hardly, differed from that of cytoplasmic protease.
INTRODUCTION
In animal tissues, information concerning the proteolytic damage of chromatin has been increasing [1,2]. In a comparative study of chromosomal proteins in the different parts of pea seedlings, the electrOphoretical bands of nonhistone proteins in cotyledon were obscure when compared to those of bud [3]. A distinctly lower content of HI histone was observed in the cotyledons. A great store of proteolytic enzyme in germinating cotyledon may injure chromosomal proteins during preparation of chromatin and its fractionation. It is " well known in animal tissue that isolated chromatin contains a proteolytic enzyme, which differs from the cytoplasmic enzyme in many properties [4--9]. This finding suggested that it would be interesting to study the regulative roles of chromatin-associated protease and the need for careful handling in the fractionation of chromosomal proteins for the protection from proteolytic damage [6,10]. Recently, there has been increasing interest in chromosomal
362 proteins from higher plant, but much less care has been paid to their purity and handling compared to animal systems [11,12] ; no one to date has reported the proteolytic degradation of chromosomal proteins in plants. The present report describes the damage to chromosomal proteins by proteolytic enzymes, and discusses whether a chromatin-associated protease is present in pea cotyledons. MATERIALS AND METHODS
Plant material Pea seeds (Pisum sativum cv. Alaska) were germinated in vermiculite at 25°C in darkness for 4 day after swelling in running water for 1 day. The detached cotyledons were used throughout.
Preparation and dissociation of chromatin All operations were carried out at 1--5°C. The methods used for preparing and dissociating chromatin were essentially that of Bonnet et al. [ 13]. Except as otherwise noted, the methods used were as reported previously [3]. One change was that the first homogenate of cotyledons with grinding medium was centrifuged at 400 g for 5 min to eliminate starch and the subsequent supernatant was used for the isolation of chromatin. This improved considerably contamination by starch in the later steps. The dissociation of chromatin was completed by stirring for 1 h in the presence of 2 M NaC1, 5 M urea and 0.001 M MgC12, and dissociated chromatin (components) was gained in supernatant after centrifugation. Thus prepared, dissociated chromatin showed a satisfactory purity judging from optical density and chemical composition [3]. In the case of electrophoretical observation of chromosomal proteins, the time required prior to ultracentrifugation was varied in order to assess the timedependent chromatin degradation: namely the "slow procedure" (approx. 9 h) or the "rapid procedure" (approx. 4 h). Additionally, sodium bisulfite (final 10 mM NaHSO3 ) was added to every medium prior to histone extraction in the "rapid procedure" and this was named "rapid with NaHSO3 procedure"; the isolated chromatin from calf thymus contained a protease which was inhibited reversibly by NaHSO3 [6].
Preparation of histones and non-histone proteins The dissociated chromatin prepared by three different procedures was dialyzed against 0.01 M Tris--HC1 (pH 8.0) or the same buffer plus 10 mM NaHSO3. The dialyzed sample was extracted with 0.4 N H2SO4 in ice for 30 min to remove histones and the pellet collected by centrifugation was washed once with 0.4 N H2SO4. Both H2SO4 extracts were combined and added 4 vols. of ethanol to precipitate histones at -20°C. The precipitated histones were collected by centrifugation and solubilized in 0.9 N acetic acid containing 15% sucrose. The pellet containing non-histone proteins after 0.4 N H2SO4 extract was followed to rinse briefly with 0.01 M TriskHC1
363 (pH 8.0) and solubilized in 0.1% SDS, 0.05 M Tris--HC1 (pH 8.0}. Each solubilized sample was then dialyzed against the appropriate solution which was introduced into the column of polyacrylamide gel.
Polyacrylamide gel electrophoresis of histones and non-histone proteins Electrophoresis was essentially that of Panyim and Chalkley for histones [14] and that of Shapiro et al. for non-histone proteins [ 1 5 ] . The only alteration for non-histone proteins was the loading of 0.1 ml of stacking gel containing 2.5% acrylamide, 0.6% bis and 18% sucrose on the t o p of separating gel to reduce streaking and curvature of protein bands by DNA [ 1 6 ] . Current applied was 2 mA per tube for the first hour, followed by 8 mA for 5 h in non-histone proteins electrophoresis.
Enzy me preparation The chromatin was dissociated in the presence of only 2 M NaC1 and centrifuged at 10 000 g for 15 min. The dissociated chromatin in supernatant was dialyzed against a deionized water which finally reached 0.14 M NaCI. Dialyzed chromatin thus prepared was used for chromatin autolysis. In the assay of substrate specificity, two proteolytic enzyme sources were prepared. One was tightly b o u n d chromatin protease. The above dialyzed chromatin was centrifuged and the precipitate of reconstituted chromatin was suspended in 0.01 M Tris--HC1 (pH 8.0). By this treatment, contaminated cytoplasmic materials if present in the chromatin would be largely eliminated in the precipitate after centrifugation of dissociated chromatin. It is also true that some o f non-histone proteins were lost in the supernatant after the centrifugation of reconstituted chromatin [ 17]. Therefore it could be said that enzyme activity found in the final suspension came originally from chromatin. The other was cytoplasmic protease. This was prepared by the complete dialysis, against 0.01 M Tris--HC1 (pH 8.0), of the supernatant excluding chromatin after the centrifugation (4 000 g) of tissue homogenate.
Enzyme assay The proteolytic enzyme activity was measured using a quantitative ninhydrin assay [7]. One millilitre of reaction mixture consisting of 0.1 ml of 0.5 M Tris--HC1 buffer (pH 8.0), 1 mg o f substrate and appropriate amount of enzyme was incubated at 37°C for 3 h with shaking, and stopped by putting the incubation tube on ice. Then 2.5 ml of ninhydrin reagent [7] was added and heated at 80°C for 10 min. After cooling, the tube was centrifuged at 4 000 g for 10 min and the absorbance of the supernatant measured at 506 nm. Sodium glutamate was used as a standard. Proteins were determined by the method of Lowry et al. [18] and DNA by the diphenylamine m e t h o d [19]. RESULTS AND DISCUSSION
Damage of chromosomal proteins Histones. As can be seen in Fig. 1, the H1 histone was susceptible to pro-
364
1 B
5
2
C
+.
>-
_
)+
Fig. 1. Degradative pattern of histones and non-histone proteins during isolation of chromatin as shown by densitometer scan of gels. A: "s}ow procedure"; B: "rapid procedure "; C: "rapid with NaHSO3 procedure" (see Materials and Methods).
teolytic attack while the other histones were resistant. The rate of degradation of H1 histone depended clearly on the isolation procedure. The longer the time required for isolation, the greater the degradation. The addition of NaHSO3 to the isolation medium was highly effective in reducing this degradation, indicating that proteolytic degradation of H1 histone could take place. The regular distribution or the stable nature of histones are widely accepted. However, differences in the relative amount of H1 histone have been reported in the different tissue of the same organism and even within a single tissue, as a function of the developmental or physiological situation [20]. Indeed we observed that H1 histone from the cotyledon was less than from the buds [3]. On the other hand, the proteolytic enzyme in chromatin from animal tissue almost prefers histones, especially HI histone [2,4,6--8]. These facts, as well as the present result, strongly suggests that some of the alterations reported in H1 histone under various experimental situations may arise from proteolytic activity which perfers HI especially. Non-histone proteins. The electrophoretic profile showed that the loss of bands appeared largely in the region of large protein molecules in the sample prepared by "slow" and even "rapid procedure" (Fig. 1 A,B). Again the best result was gained in the sample prepared by "rapid with NaHSO3 procedure". This indicated clearly that some of the large protein molecules, 1--5, were preferentially degraded during preparation. As to the extent of proteolytic damage, non-histone proteins seemed to be more susceptible to attack than the histones. When the number of molecular species of non-histone proteins so far
365
reported are compared between animal and plant, there seem to be fewer plant than animal species [21]. This might be partially due to the isolation method for chromatin, because most methods used with plant materials employ direct homogenization of the tissue which was more susceptible to the contamination of cytoplasmic materials than chromatin prepared from isolated nuclei in the case of animals. In addition, the autolysis of dissociated chromatin was much less than that of undissociated chromatin, suggesting that cytoplasmic proteolytic enzymes were still attached to chromatin even after two ultracentrifugations through 2 M sucrose {data not presented here). These findings suggest that nonhistone proteins, like histones, are also easily susceptible to the digestion by proteolytic activity during their preparation, and careful treatment should be applied when they are compared.
Autolysis o f chromatin and cytoplasmic or chromatin-associated proteolytic activity The results hitherto suggested that chromosomal proteins in pea cotyledon were degraded during their preparation. Therefore we investigated the proteolytic activity which was associated with chromatim The autolysis of dissociated chromatin progressed almost linearly up to 3 h at 37°C and 2.6 ~g NH2--N was formed in the chromatin equivalent to 1.5 mg DNA. This autolysis was reduced by 42% with 10 mM NaHSO3 and completely suppressed after heating of chromatin at 90°C for 10 min. This repressive effect of NaHSO3 was not observed with cytoplasmic protease; in fact, some acceleration was observed with some substrates (data not presented here). These results indicated that the autolysis of dissociated chromatin may be due to chromatin-associated protease. Therefore, we confirmed the presence of proteolytic activity which was tightly associated with chromatin by comparison of substrate specificity of cytoplasmic and chromatin associated enzyme {Table I). Cytoplasmic and TABLE I SUBSTRATE SPECIFICITY OF CYTOPLASMIC AND CHROMATIN-BOUND • PROTEASE T h e values are e x p r e s s e d as t h e average o f t w o s e p a r a t e e x p e r i m e n t s . Also, value due to a u t o l y s i s was s u b t r a c t e d f r o m e a c h assayed value.
Substrate
P r o t e o l y t i c activity (ug NH2--N f o r m e d / m g p r o t e i n ) Cytoplasmic
H i s t o n e f r o m calf t h y m u s Polylysine Bovine serum albumin Casein f r o m milk Protamine from salmine
1.32 0.87 0.77 0.76 1.68
Reconstituted chromatin
'
1.19 1.29 5.11 0.38 0.14
366
chromatin tightly associated enzyme were prepared as shown in Materials and Methods. The substrate specificity of reconstituted chromatin-associated protease differed from that of cytoplasmic protease, especially in relation to albumin and protamine. A reverse preference to these two substrates was shown by the t w o enzymes, although they degraded histones nearly equally. The high preference to albumin in chromatin-associated protease will be related to the drastic degradation of non-histone proteins, because they are both acidic protein in nature. From the substrate specificity and method for reconstitution of chromatin, it is difficult to accept that chromatin-associated protease is merely a result of cytoplasmic contamination. Therefore this indicated that plant chromatin also contained the bound protease as have been found in animal chromatin. A neutral protease from rat liver chromatin attacked histones and L-polylysine preferentially but had little effect on bovine albumin [ 8 ] . Accordingly, chromatin-bound protease from pea cotyledon may differ from the neutral one from rat liver and have a role for the development of basic metabolism during early germination. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13
14 15 16 17 18 19 20 21
N.K. Sarkar and A.L. Dounce, Arch. Biochem. Biophys., 92 (1961) 321. S. Panyim, R.H. Jensen and R. Chalkley, Biochim. Biophys. Acta, 160 (1968) 252. H. Matsumoto, E. Hirasawa and E. Takahashi, Plant Cell Physiol., 17 (1976) 955. M. Furlan and M. Jericijo, Biochim. Biophys. Acta, 147 (1967) 135. M. Furlan and M. Jericijo, Biochim. Biophys. Acta, 147 (1967) 145. J. Bartley and R. Chalkley, J. Biol. Chem., 245 (1970) 4286. J.I. Garrels, S.C.R. Elgin and J. Bonner, Biochem. Biophys. Res. Commun., 46 (1972) 545. M.T. Chong, W.T. Garrand and J. Bonner, Biochemistry, 13 (1974) 5128. T. Kurecki, B. Kowalska-Loth, K. Toczko and I. Chmielewska, FEBS Lett., 53 (1975) 313. D.B. Carter and C. Chae, Biochemistry, 15 (1976) 180. S. Spiker and R. Chalkley, Plant Physiol., 47 (1971) 342. L.C. Vanloon, A. Trewavas and K.S.R. Chapmann, Plant Physiol., 55 (1975) 288. J. Bonner, R. Chalkley, M. Dahmus, D. Fambrough, F. Fujimura, R.C. Huang, J. Huberman, R. Jensen, K. Marushige, H. Ohlenbusch, B.M. Olivera and J. Widholm, Isolation and characterization o f chromosomal nucleoproteins, in L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Vol. 12B, Academic Press, New York, 1968, p. 3. S. Panyim and R. Chalkley, Arch. Biochem. Biophys., 130 (1969) 337. A.L. Shapiro, E. Vinuela and J.V. Maizel, Biochem. Biophys. Res. Commun., 28 (1967) 815. M.C. Smith and C. Chae, Biochim. Biophys. Acta, 317 (1973) 10. T.Y. Wang and E.W. Johns, Arch. Biochem. Biophys., 124 (1968) 176. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. K. Burton, Biochem. J., 62 (1956) 315. D.M. Fambrough, F. Fujimura an~ J. Bonner, Biochemistry, 7 (1968) 575. T.C. Spelsberg and I.V. Sarkissian, Phytochemistry, 9 (1970) 1385.
361
© Elsevier/North-Holland Scientific Publishers Ltd.
DAMAGE OF CHROMOSOMAL PROTEINS DURING ISOLATION OF CHROMATIN AND A CHROMATIN-BOUND PROTEASE FROM GERMINATED PEA COTYLEDON
EIJI HIRASAWA, EIICHI TAKAHASHI and HIDEAKI MATSUMOTO
Department of Agricultural Chemistry, Kyoto University, Kyoto 606 (Japan) (Received April 18th, 1977) (Accepted August 25th, 1977)
SUMMARY
During the isolation of chromatin from germinated pea cotyledons, the HI histone was susceptible but the other histones were resistant to degradation as seen from the electrophoretical profile. The digestive damage of non-histone proteins appeared strongly in the region of large protein molecules. In order to avoid damage to chromosomal proteins, the shortening of time required for preparations before ultracentrifugation and the addition of sodium bisulfite were effective. After the dissociation of chromatin with 2 M NaC1, the reconstituted chromatin containing proteolytic activity caused the autolysis of the chromosomal proteins. This autolysis was inhibited by sodium bisulfite. The substrate specificity of chromatin-tightly bound protease, which preferred bovine serum albumin but protamine hardly, differed from that of cytoplasmic protease.
INTRODUCTION
In animal tissues, information concerning the proteolytic damage of chromatin has been increasing [1,2]. In a comparative study of chromosomal proteins in the different parts of pea seedlings, the electrOphoretical bands of nonhistone proteins in cotyledon were obscure when compared to those of bud [3]. A distinctly lower content of HI histone was observed in the cotyledons. A great store of proteolytic enzyme in germinating cotyledon may injure chromosomal proteins during preparation of chromatin and its fractionation. It is " well known in animal tissue that isolated chromatin contains a proteolytic enzyme, which differs from the cytoplasmic enzyme in many properties [4--9]. This finding suggested that it would be interesting to study the regulative roles of chromatin-associated protease and the need for careful handling in the fractionation of chromosomal proteins for the protection from proteolytic damage [6,10]. Recently, there has been increasing interest in chromosomal
362 proteins from higher plant, but much less care has been paid to their purity and handling compared to animal systems [11,12] ; no one to date has reported the proteolytic degradation of chromosomal proteins in plants. The present report describes the damage to chromosomal proteins by proteolytic enzymes, and discusses whether a chromatin-associated protease is present in pea cotyledons. MATERIALS AND METHODS
Plant material Pea seeds (Pisum sativum cv. Alaska) were germinated in vermiculite at 25°C in darkness for 4 day after swelling in running water for 1 day. The detached cotyledons were used throughout.
Preparation and dissociation of chromatin All operations were carried out at 1--5°C. The methods used for preparing and dissociating chromatin were essentially that of Bonnet et al. [ 13]. Except as otherwise noted, the methods used were as reported previously [3]. One change was that the first homogenate of cotyledons with grinding medium was centrifuged at 400 g for 5 min to eliminate starch and the subsequent supernatant was used for the isolation of chromatin. This improved considerably contamination by starch in the later steps. The dissociation of chromatin was completed by stirring for 1 h in the presence of 2 M NaC1, 5 M urea and 0.001 M MgC12, and dissociated chromatin (components) was gained in supernatant after centrifugation. Thus prepared, dissociated chromatin showed a satisfactory purity judging from optical density and chemical composition [3]. In the case of electrophoretical observation of chromosomal proteins, the time required prior to ultracentrifugation was varied in order to assess the timedependent chromatin degradation: namely the "slow procedure" (approx. 9 h) or the "rapid procedure" (approx. 4 h). Additionally, sodium bisulfite (final 10 mM NaHSO3 ) was added to every medium prior to histone extraction in the "rapid procedure" and this was named "rapid with NaHSO3 procedure"; the isolated chromatin from calf thymus contained a protease which was inhibited reversibly by NaHSO3 [6].
Preparation of histones and non-histone proteins The dissociated chromatin prepared by three different procedures was dialyzed against 0.01 M Tris--HC1 (pH 8.0) or the same buffer plus 10 mM NaHSO3. The dialyzed sample was extracted with 0.4 N H2SO4 in ice for 30 min to remove histones and the pellet collected by centrifugation was washed once with 0.4 N H2SO4. Both H2SO4 extracts were combined and added 4 vols. of ethanol to precipitate histones at -20°C. The precipitated histones were collected by centrifugation and solubilized in 0.9 N acetic acid containing 15% sucrose. The pellet containing non-histone proteins after 0.4 N H2SO4 extract was followed to rinse briefly with 0.01 M TriskHC1
363 (pH 8.0) and solubilized in 0.1% SDS, 0.05 M Tris--HC1 (pH 8.0}. Each solubilized sample was then dialyzed against the appropriate solution which was introduced into the column of polyacrylamide gel.
Polyacrylamide gel electrophoresis of histones and non-histone proteins Electrophoresis was essentially that of Panyim and Chalkley for histones [14] and that of Shapiro et al. for non-histone proteins [ 1 5 ] . The only alteration for non-histone proteins was the loading of 0.1 ml of stacking gel containing 2.5% acrylamide, 0.6% bis and 18% sucrose on the t o p of separating gel to reduce streaking and curvature of protein bands by DNA [ 1 6 ] . Current applied was 2 mA per tube for the first hour, followed by 8 mA for 5 h in non-histone proteins electrophoresis.
Enzy me preparation The chromatin was dissociated in the presence of only 2 M NaC1 and centrifuged at 10 000 g for 15 min. The dissociated chromatin in supernatant was dialyzed against a deionized water which finally reached 0.14 M NaCI. Dialyzed chromatin thus prepared was used for chromatin autolysis. In the assay of substrate specificity, two proteolytic enzyme sources were prepared. One was tightly b o u n d chromatin protease. The above dialyzed chromatin was centrifuged and the precipitate of reconstituted chromatin was suspended in 0.01 M Tris--HC1 (pH 8.0). By this treatment, contaminated cytoplasmic materials if present in the chromatin would be largely eliminated in the precipitate after centrifugation of dissociated chromatin. It is also true that some o f non-histone proteins were lost in the supernatant after the centrifugation of reconstituted chromatin [ 17]. Therefore it could be said that enzyme activity found in the final suspension came originally from chromatin. The other was cytoplasmic protease. This was prepared by the complete dialysis, against 0.01 M Tris--HC1 (pH 8.0), of the supernatant excluding chromatin after the centrifugation (4 000 g) of tissue homogenate.
Enzyme assay The proteolytic enzyme activity was measured using a quantitative ninhydrin assay [7]. One millilitre of reaction mixture consisting of 0.1 ml of 0.5 M Tris--HC1 buffer (pH 8.0), 1 mg o f substrate and appropriate amount of enzyme was incubated at 37°C for 3 h with shaking, and stopped by putting the incubation tube on ice. Then 2.5 ml of ninhydrin reagent [7] was added and heated at 80°C for 10 min. After cooling, the tube was centrifuged at 4 000 g for 10 min and the absorbance of the supernatant measured at 506 nm. Sodium glutamate was used as a standard. Proteins were determined by the method of Lowry et al. [18] and DNA by the diphenylamine m e t h o d [19]. RESULTS AND DISCUSSION
Damage of chromosomal proteins Histones. As can be seen in Fig. 1, the H1 histone was susceptible to pro-
364
1 B
5
2
C
+.
>-
_
)+
Fig. 1. Degradative pattern of histones and non-histone proteins during isolation of chromatin as shown by densitometer scan of gels. A: "s}ow procedure"; B: "rapid procedure "; C: "rapid with NaHSO3 procedure" (see Materials and Methods).
teolytic attack while the other histones were resistant. The rate of degradation of H1 histone depended clearly on the isolation procedure. The longer the time required for isolation, the greater the degradation. The addition of NaHSO3 to the isolation medium was highly effective in reducing this degradation, indicating that proteolytic degradation of H1 histone could take place. The regular distribution or the stable nature of histones are widely accepted. However, differences in the relative amount of H1 histone have been reported in the different tissue of the same organism and even within a single tissue, as a function of the developmental or physiological situation [20]. Indeed we observed that H1 histone from the cotyledon was less than from the buds [3]. On the other hand, the proteolytic enzyme in chromatin from animal tissue almost prefers histones, especially HI histone [2,4,6--8]. These facts, as well as the present result, strongly suggests that some of the alterations reported in H1 histone under various experimental situations may arise from proteolytic activity which perfers HI especially. Non-histone proteins. The electrophoretic profile showed that the loss of bands appeared largely in the region of large protein molecules in the sample prepared by "slow" and even "rapid procedure" (Fig. 1 A,B). Again the best result was gained in the sample prepared by "rapid with NaHSO3 procedure". This indicated clearly that some of the large protein molecules, 1--5, were preferentially degraded during preparation. As to the extent of proteolytic damage, non-histone proteins seemed to be more susceptible to attack than the histones. When the number of molecular species of non-histone proteins so far
365
reported are compared between animal and plant, there seem to be fewer plant than animal species [21]. This might be partially due to the isolation method for chromatin, because most methods used with plant materials employ direct homogenization of the tissue which was more susceptible to the contamination of cytoplasmic materials than chromatin prepared from isolated nuclei in the case of animals. In addition, the autolysis of dissociated chromatin was much less than that of undissociated chromatin, suggesting that cytoplasmic proteolytic enzymes were still attached to chromatin even after two ultracentrifugations through 2 M sucrose {data not presented here). These findings suggest that nonhistone proteins, like histones, are also easily susceptible to the digestion by proteolytic activity during their preparation, and careful treatment should be applied when they are compared.
Autolysis o f chromatin and cytoplasmic or chromatin-associated proteolytic activity The results hitherto suggested that chromosomal proteins in pea cotyledon were degraded during their preparation. Therefore we investigated the proteolytic activity which was associated with chromatim The autolysis of dissociated chromatin progressed almost linearly up to 3 h at 37°C and 2.6 ~g NH2--N was formed in the chromatin equivalent to 1.5 mg DNA. This autolysis was reduced by 42% with 10 mM NaHSO3 and completely suppressed after heating of chromatin at 90°C for 10 min. This repressive effect of NaHSO3 was not observed with cytoplasmic protease; in fact, some acceleration was observed with some substrates (data not presented here). These results indicated that the autolysis of dissociated chromatin may be due to chromatin-associated protease. Therefore, we confirmed the presence of proteolytic activity which was tightly associated with chromatin by comparison of substrate specificity of cytoplasmic and chromatin associated enzyme {Table I). Cytoplasmic and TABLE I SUBSTRATE SPECIFICITY OF CYTOPLASMIC AND CHROMATIN-BOUND • PROTEASE T h e values are e x p r e s s e d as t h e average o f t w o s e p a r a t e e x p e r i m e n t s . Also, value due to a u t o l y s i s was s u b t r a c t e d f r o m e a c h assayed value.
Substrate
P r o t e o l y t i c activity (ug NH2--N f o r m e d / m g p r o t e i n ) Cytoplasmic
H i s t o n e f r o m calf t h y m u s Polylysine Bovine serum albumin Casein f r o m milk Protamine from salmine
1.32 0.87 0.77 0.76 1.68
Reconstituted chromatin
'
1.19 1.29 5.11 0.38 0.14
366
chromatin tightly associated enzyme were prepared as shown in Materials and Methods. The substrate specificity of reconstituted chromatin-associated protease differed from that of cytoplasmic protease, especially in relation to albumin and protamine. A reverse preference to these two substrates was shown by the t w o enzymes, although they degraded histones nearly equally. The high preference to albumin in chromatin-associated protease will be related to the drastic degradation of non-histone proteins, because they are both acidic protein in nature. From the substrate specificity and method for reconstitution of chromatin, it is difficult to accept that chromatin-associated protease is merely a result of cytoplasmic contamination. Therefore this indicated that plant chromatin also contained the bound protease as have been found in animal chromatin. A neutral protease from rat liver chromatin attacked histones and L-polylysine preferentially but had little effect on bovine albumin [ 8 ] . Accordingly, chromatin-bound protease from pea cotyledon may differ from the neutral one from rat liver and have a role for the development of basic metabolism during early germination. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13
14 15 16 17 18 19 20 21
N.K. Sarkar and A.L. Dounce, Arch. Biochem. Biophys., 92 (1961) 321. S. Panyim, R.H. Jensen and R. Chalkley, Biochim. Biophys. Acta, 160 (1968) 252. H. Matsumoto, E. Hirasawa and E. Takahashi, Plant Cell Physiol., 17 (1976) 955. M. Furlan and M. Jericijo, Biochim. Biophys. Acta, 147 (1967) 135. M. Furlan and M. Jericijo, Biochim. Biophys. Acta, 147 (1967) 145. J. Bartley and R. Chalkley, J. Biol. Chem., 245 (1970) 4286. J.I. Garrels, S.C.R. Elgin and J. Bonner, Biochem. Biophys. Res. Commun., 46 (1972) 545. M.T. Chong, W.T. Garrand and J. Bonner, Biochemistry, 13 (1974) 5128. T. Kurecki, B. Kowalska-Loth, K. Toczko and I. Chmielewska, FEBS Lett., 53 (1975) 313. D.B. Carter and C. Chae, Biochemistry, 15 (1976) 180. S. Spiker and R. Chalkley, Plant Physiol., 47 (1971) 342. L.C. Vanloon, A. Trewavas and K.S.R. Chapmann, Plant Physiol., 55 (1975) 288. J. Bonner, R. Chalkley, M. Dahmus, D. Fambrough, F. Fujimura, R.C. Huang, J. Huberman, R. Jensen, K. Marushige, H. Ohlenbusch, B.M. Olivera and J. Widholm, Isolation and characterization o f chromosomal nucleoproteins, in L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Vol. 12B, Academic Press, New York, 1968, p. 3. S. Panyim and R. Chalkley, Arch. Biochem. Biophys., 130 (1969) 337. A.L. Shapiro, E. Vinuela and J.V. Maizel, Biochem. Biophys. Res. Commun., 28 (1967) 815. M.C. Smith and C. Chae, Biochim. Biophys. Acta, 317 (1973) 10. T.Y. Wang and E.W. Johns, Arch. Biochem. Biophys., 124 (1968) 176. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. K. Burton, Biochem. J., 62 (1956) 315. D.M. Fambrough, F. Fujimura an~ J. Bonner, Biochemistry, 7 (1968) 575. T.C. Spelsberg and I.V. Sarkissian, Phytochemistry, 9 (1970) 1385.