Exp. Geront. Vol. 14, pp. 101-107. © Pergamon Press Ltd. 1979. Printed in Great Britain.
0531-5565/79/0601-0101502.00/0
NO AGE-DEPENDENT OXIDATION OF H3 HISTONE DONALD
B. CARTER*
Department of Biochemistry and Nutrition, University of North Carolina, Chapel Hill, NC 27514, U.S.A.
(Received I October 1978) INTRODUCTION CHANGES in disulfide b o n d i n g in c h r o m o s o m a l proteins have been postulated as a m ech an ism to explain a decrease in t e m p l a t e activity o f c h r o m a t i n with age (Phytila and Sherman, 1968; Z h e l a b o v s k a y a a n d Berdyshev, 1972; R y a n an d Cristofalo, 1975) and the age associated increase in the t h e r m a l stability o f c h r o m a t i n ( K u r t z et al., 1974). Th e cysteine o f Ha histone is present in interphase c h r o m a t i n in reduced f o r m (Panyim et al., 1971). H o w e v e r , S a d o p o g a l a n d B o n n e r (1970) reported that H3 in m et ap h ase c h r o m o s o m e s is present in oxidized dimeric form. Tas (1976) has reported that c h r o m a t i n fi'om rat liver and blowfly enters a m o r e c o m p a c t state as a function o f age and that the co m p act n ess was due in part to an increased n u m b e r o f disulfide b o n d s in older chromatin. In view o f the role o f Ha histone in c h r o m a t i n structure ( K o r n b e r g and T h o m a s , 1974) it is o f interest to investigate the possible cross-linking between Ha and Ha and between Ha and n o n h i s t o n e proteins by disulfide bridges d u r i n g aging. MATERIALS AND METHODS
Animals All rats used in this experiment were adult male albino rats of the Dublin Disease Resistant strain (Sprague-Da,~ley derived). The animals were born and raised in our animal colony; maintained in airconditioned quarters (room temperature 22-24°C and relative humidity 50-60~). There was an automatically controlled light-dark cycle (6 a.m.-6 p.m.-6 a.m.). They were housed two to a cage and allowed ad lib. food (Purina Laboratory Chow) and water. Over a 4 yr period 82-87~ of the animals reached 36 months of age with a 50~ mean survival expectancy of 40 months. Isolation of nuclei and chromatin Brains and livers were excised immediately after killing and placed in ice-cold 0.25 M sucrose-3 mM MgCla-10 mM potassium phosphate (pH 5'8) (Solution A) with a volume-mass of tissue ratio of 3 : 1. The tissue was homogenized with a Potter-Elvehjem type homogenizer with motor driven teflon pestle. The homogenate was centrifuged at 1000 g for 20 rain, and the pellet was suspended in 7.10 vol. (vol./wt of tissue) of 2.3 M sucrose-l'5 mM CaCI2-10 mM Tris (pH 6"8). The suspension was then filtered through 4 layers of cheesecloth and centrifuged for 45 min at 45,000 g. The pelleted nuclei were suspended in 1 Triton X-100-So~ution A and centrifuged at 1000 g for 10 min. The nuclei were washed in this solution once more. The Triton-treated nuclei were successively washed with the following solutions two times each : 0.075 M NaCI-0'024 M EDTA (pH 7-0), 50 mM Tris pH 7"9, 10 mM Tris pH 7'9, as described by Huang and Huang (1969). The final chromatin was suspended in ice-cold water and sheared lightly at 20 V for 60 s in a Sorval Omnimixer. All of the solutions used were flushed with nitrogen gas for several hours before use. N-ethylmaleimide ( NEM) labeling of chromathl Chromatin was adjusted to a concentration of 0-22 mg/ml DNA in 1 ~ SDS-10 mM KPO4, pH 7"0 (Solution B) and dialyzed in the dark 15 h at 25°. One milliliter of non-reduced chromatin was then incubated with 20 tal of I0 mM [aH]NEM (specific activity 10-2 Ci/mmol) for 9 h at 25°. Excess NEM was removed by exhaustive dialysis against Solution B. To prepare labeled reduced chromatin, an aliquot of the non-reduced chromatin was made 2 mM in 13-mercaptoethanol in Solution B, iilcubated 15 h and *Present address: National Institute of Environmental Health and Sciences, Research Triangle Park, North Carolina, U.S.A. 101
102
DONALD B. CARTER
dialyzed to 0.02 mM 13-mercaptoethanol concentration in Solution B. A 10 fold excess of [aH]NEM over 13-mercaptoethanol was then added to the chromatin solution and incubated 9 h at 25°. Excess NEM was again removed by exhaustive dialysis against Solution B. Disc gel electrophoresis Histones were analyzed on 15 ~ polyacrylamide gels (pH 3-2) according to the method of Panyim and Chalkley (1969). SDS gel electrophoresis was carried out as described before (Smith and Chae, 1973).
RESULTS A N D D I S C U S S I O N Chromatin isolated from the brain and liver of Sprague-Dawley Dublin Disease Resistant rats contains 1.1 mg histone per mg DNA. The histone/DNA ratio shows no appreciable age dependent variation. As shown in Table 1, there are small changes in the total protein/ D N A ratio from 3 month to 8 month chromatin but after 8 months the ratio stabilizes for liver and decreases slightly for brain. TABLE 1. P R O T E I N / D N A RATIO FOR WHOLE RAT BRAIN AND RAT LIVER CHROMATIN
Age (months) 3 8 18 33
Brain 2"55 :k 0"1" 2'21 2"26 2'02
Liver 2"24 -~ 0"12" 1"91 2"05 1"95
*Standard deviation determined by 6 independent experiments on 3 month chromatin. Remaining values were repeated 2 times and averaged for chromatin isolated from 10 pooled rat brains or 3 pooled livers from the indicated age groups. Histones were extracted from the chromatin as described before (Carter and Chae, 1975) and separated by the acid-urea polyacrylamide gel electrophoresis system reported by Panyim and Chalkley (1969). Histones were prepared for electrophoresis under reducing and non-reducing conditions. F r o m Fig. 1 it is apparent that histones prepared under nonreducing conditions show no detectable oxidized H3 histone when compared to control histones which have been oxidized by hydrogen peroxide. Oxidized H3 migrates slower than Ha histone during acid-urea gel electrophoresis (Panyim et al., 1971a). Identical results were also obtained with histones from brain, liver and testis of 3 month, 8 month and 18 month rats when histones were extracted from freshly prepared chromatin. When histones are extracted from frozen chromatin stored in air, there is generally a variable amount of H3 oxidation apparent on acid-urea gels. We have also determined the amount of the reduced cysteine in Hz histone by labeling dissociated chromatin with [SH]NEM. The results of [3H]NEM uptake by the chromatin dissociated in SDS is shown in Fig. 2. In the 7.5 ~ SDS polyacrylamide gel system, histone Ha migrates with an electrophoretic mobility of 0.83. When chromatin is acid-extracted to remove histones and then incubated with [aH]NEM, very little label is found in the Ha region of the gel, as shown in Fig. 2. This indicates that there are no major non-histone proteins capable of binding [aH]NEM which migrate with Ha histone in the 7 . 5 ~ SDS gel system. Accordingly one may interpret the quantity of label in the Ha region of the gel as being due to Hs histone uptake of [aH]NEM. There were small variations in the extent of labeling of chromatin in different experiments and in the total recovery of radioactivity in H8 histone after SDS gel electrophoresis. Therefore, we determined the per cent of total radioactivity of N E M in Ha histone after
NOAGE-DEPENDENTOXIDATIONOF H3 HISTONE
103
H2B 3
I
H3
HI
12A H4
4
OXH3
FIG. 1. Densitometric tracings of acid-urea gels described in Materials and Methods for brain and liver histones from DDR rats 33 months old. Tracings 1 and 2 are for histones prepared for electrophoresis with and without 1 ~ [3-mercaptoethanol from brain. Tracings 3 and 4 are for histones prepared for electrophoresis with and without 1 ~ [3-mercaptoethanol from liver. Tracing 5 is a control showing the presence of oxidized H3 prepared by oxidizing liver histories with 0.01 ~ hydrogen peroxide. electrophoresis. Also in order to determine the a m o u n t o f the reduced cysteine in Ha histone, the labeling o f H3 histone by [aH]NEM was compared between the chromatin before and after the reduction o f chromatin sample with 13-mercaptoethanol. When the peak position for H3 histone is integrated and c o m p a r e d to total recovered radioactivity in each gel, the a m o u n t o f label uptake in Ha is found to decrease with age after 8-18 m o n t h s (Fig. 3 and Table 2). The decrease in label uptake occurs in both non-reduced and reduced
104
DONALD B. CARTER
I0
s o
e'-
O.I
0.2
O.g
0.4
0.5
0.6
0.7
0,8
0.9
t.O
FIG. 2. Thiol content of acid extracted chromatin and whole chromatin control as determined by [SH]NEM labeling. Chromatin acid extracted by 0.4 N H2SOt, as described in Materials and Methods, was dissociated in l ~ SDS-10 mM KPO4, pH 7'0 and labeled with [3H]NEM. The percentage of total counts recovered is shown for each slice on the ordinate. The abscissa shows the fraction of the distance migrated by bromophenol blue of each slice. ( C - - - - ©) Acid extracted chromatin ( x - - - - × ) Control chromatin c h r o m a t i n a n d therefore does not represent a n y changes in oxidation of H 8 during aging. These data are consistent with the results presented showing that oxidized H 3 does n o t exist in detectable quantities in the liver a n d brain c h r o m a t i n of D D R rats (see Fig. 1). Also there is no age-dependent reduction of the a m o u n t of H z histone, as we have shown before (Carter a n d Chae, 1975). O n several occasions we have seen age-dependent reduction of the N E M labeling of H z histone in the n o n - r e d u c e d c h r o m a t i n c o m p a r e d to reduced c h r o m a t i n ( u n p u b l i s h e d observations). This observation appears to be due to experimental artifact. We have taken several precautions to avoid the possible artifact in the labeling of c h r o m o s o m a l proteins with [SH]NEM. We flushed all of the solutions used with nitrogen gas to remove oxygen a n d the c h r o m a t i n was labeled with [SH]NEM on the same day as it was prepared. TABLE 2.
QUANTITY OF
[OH]NEM
Age (months) 3 8 18 33
Ha HISTONE ON SDS GELS
LABEL INCORPORATED INTO
NR* brain 24 29 26"4 20-1
R~ (%) 27 25"2 23"8 18.9
AS ~oo OF TOTAL RECOVERED COUNTS
NR* liver 39-7 32 -:~ 23.5
Rt (%) 35 36"9 25 25
NR* = not reduced before incubation of [SH]NEM and chromatin. Rt = reduced before incubation of ISH]NEM and chromatin. ~ = sample lost.
NO AGE-DEPENDENT OXIDATION OF
Ha HISTONE
105
14
12
10
- -
8
0.1
0,2
0,3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
28 --
24
20
8
~-
16
12
8
OJ
02
0,3
C4
0.3
13¢
0.7
0.~
0.9
1,0
FIG. 3. Labeling of rat liver chromosomal proteins with [3H]NEM. Chromatin dissociated in SDS was labeled with [SH]NEM and chromosomal proteins were fractionated by SDS gel electrophoresis, as described in Materials and Methods. (A) Liver chromatin reduced by 13-mercaptoethanol before labeling. (B) Liver chromatin not reduced. (• • ) , 3 months; ( 0 0 ) , 8 months; ( x × ), 33 months.
106
DONALD a . CARTER
Nonhistone chromosomal proteins are also labeled by [aH]NEM, and SDS gel electrophoresis of the chromatin labeled with [aH]NEM before and after the reduction with [3mercaptoethanol show no significant age dependent qualitative changes in the amount of reduced cysteine for both liver and brain. However, the decrease with age in relative labeling of H a with respect to nonhistone proteins is significant. The amount of label in H a relative to total label recovered on the gels drops by 30 % for both reduced brain and liver chromatin from 3 to 33 month animals. There are several possible explanations for these results: (a) the quantity of nonHa cysteine present in SDS-dissociated and dialyzed chromatin increases relative to a fixed quantity of Ha cysteine, implying that the quantity of cysteine in nonhistone chromatin proteins increases with age; (b) the [aH]NEM reacts with moieties in addition to cysteine which increase in concentration in aging chromatin (Brewer and Riehm, 1967; Sharpless and Flavin, 1966; Smyth et al., 1964). Under our selected conditions, however, we would expect [aH]NEM to react almost exclusively with the cysteine moieties; (c) the dissociating conditions of 1% SDS-10 m M K P O 4, pH 7.0 are not sufficiently stringent to completely disaggregate old chromatin relative to young chromatin. It has been reported that the number of disulfide bonds in rat liver chromatin increases from 20 day to 9 month old rats (Tas, 1976). When chromatin is dissociated in 1% SDS and labeled as described in Methods, no age related trend in the chromatin thiol/thiol d- disulfide ratio is apparent in the age groups used in this experiment (Table 3). Thus it cannot be argued on the basis of these results that any increase in the compactness of interphase chromatin is due to an increase in the number of disulfide bonds in chromatin. TABLE 3. T H I O L / T H I O L @ DISULFIDE* RATIO FOR RAT BRAIN AND LIVER CHROMATIN AS A FUNCTION OF AGE
Age (months) 3 8 18 33
Brain 0"708 0-68 0'88 0'69
Liver 0'793 0'75 0'85 0"70
*Fractions of unreduced and reduced chromatin 0"05 ml were assayed after excess [SH]NEM had been removed by exhaustive dialysis. Typical count rates for brain and liver chromatin (0-22 mg DNA/ml) are 5000-8000 counts per minute in 0'05 ml when labeled as described in Materials and Methods. SUMMARY Histones were extracted from chromatin isolated from the brain, liver and testis of Dublin Disease Resistant Sprague-Dawley rats and prepared for electrophoresis under reducing and non-reducing conditions. Histories prepared under non-reducing conditions show no detectable age related changes in oxidized Ha histone. The quantity of reduced cysteine in H3 histone relative to total chromatin thiol was determined by labeling SDS dissociated chromatin with [SH]NEM. Equivalent decrements in [aH]NEM incorporation in H3 histone, relative to total [aH]NEM incorporation in chromatin, occur in reduced and non-reduced preparations with age. In addition, no significant age related changes were found in the thiol/thiol ÷ disulfide ratio, when rat brain and liver chromatin were dissociated as described. Hence, any change in the content of disulfide bridges with age is proportional to the change in thiol content. Acknowledgements--This study was supported by a grant from the National Institute of Aging, National Institutes of Health (AG00103) awarded to Dr. Chi-Bom Chae. The author is grateful to Dr. Chi-Bom Chae for advice and consultation during the course of this study.
NO AGE-DEPENDENTOXIDATIONOF H a HISTONE REFERENCES BREWER, C. F. and RIEHM, J. P. (1967) Analyt. Biochem. 18, 248.
CARTER,D. B. and CHAE, C.-B. (1975) J. Geront. 30, 28. HUANG, R. C. and HUANG, P. C. (1969) J. molec. Biol. 39, 365. KORNBERG, R. D. and THOMAS,J. O. (1974) Science 184, 868. KURTZ, D. I., RUSSELL,A. P. and SINEX, F. M. (1974) Mech. Ageing Dev. 3, 37. PANYIM, S. and CHALKLEY,R. (1969) Arch Biochem. Biophys. 130, 337. PANYIM, S., SOMMER,K. R. and CHALKLEY,R. (1971) Biocl, emistry 10, 3911, PHYTILA, M. and SHERMAN,F. 0968) Biochem. biophys. Res. Commun. 31, 340. RYAN, J. M. and CRISTOFALO,V. J. (1975) Expl Cell Res. 90, 456. SADOPOGAL,A. and BONNER, J. (1970) Biochim. biophyz'. Acta 207, 227. SHARPLESS, N. E. and FLAV1N, M. (1966) Biochemistry 5, 2963. StunT-t, M. C. and CHAE, C.-B. (1973) Biochim. biophys. Acta 317, 10. SMYTH, D. G., BLUMENFELD,O. O. and KONIGSBERG,W. (1964) Biochem. J. 91, 589. TAS, S. (1976) Exp. Geront. l l , 17. ZHELABOVSKAYA,S. and BERDYSHEV,G. (1972) Exp. Geront. 7, 313.
107
0531-5565/79/0601-0101502.00/0
NO AGE-DEPENDENT OXIDATION OF H3 HISTONE DONALD
B. CARTER*
Department of Biochemistry and Nutrition, University of North Carolina, Chapel Hill, NC 27514, U.S.A.
(Received I October 1978) INTRODUCTION CHANGES in disulfide b o n d i n g in c h r o m o s o m a l proteins have been postulated as a m ech an ism to explain a decrease in t e m p l a t e activity o f c h r o m a t i n with age (Phytila and Sherman, 1968; Z h e l a b o v s k a y a a n d Berdyshev, 1972; R y a n an d Cristofalo, 1975) and the age associated increase in the t h e r m a l stability o f c h r o m a t i n ( K u r t z et al., 1974). Th e cysteine o f Ha histone is present in interphase c h r o m a t i n in reduced f o r m (Panyim et al., 1971). H o w e v e r , S a d o p o g a l a n d B o n n e r (1970) reported that H3 in m et ap h ase c h r o m o s o m e s is present in oxidized dimeric form. Tas (1976) has reported that c h r o m a t i n fi'om rat liver and blowfly enters a m o r e c o m p a c t state as a function o f age and that the co m p act n ess was due in part to an increased n u m b e r o f disulfide b o n d s in older chromatin. In view o f the role o f Ha histone in c h r o m a t i n structure ( K o r n b e r g and T h o m a s , 1974) it is o f interest to investigate the possible cross-linking between Ha and Ha and between Ha and n o n h i s t o n e proteins by disulfide bridges d u r i n g aging. MATERIALS AND METHODS
Animals All rats used in this experiment were adult male albino rats of the Dublin Disease Resistant strain (Sprague-Da,~ley derived). The animals were born and raised in our animal colony; maintained in airconditioned quarters (room temperature 22-24°C and relative humidity 50-60~). There was an automatically controlled light-dark cycle (6 a.m.-6 p.m.-6 a.m.). They were housed two to a cage and allowed ad lib. food (Purina Laboratory Chow) and water. Over a 4 yr period 82-87~ of the animals reached 36 months of age with a 50~ mean survival expectancy of 40 months. Isolation of nuclei and chromatin Brains and livers were excised immediately after killing and placed in ice-cold 0.25 M sucrose-3 mM MgCla-10 mM potassium phosphate (pH 5'8) (Solution A) with a volume-mass of tissue ratio of 3 : 1. The tissue was homogenized with a Potter-Elvehjem type homogenizer with motor driven teflon pestle. The homogenate was centrifuged at 1000 g for 20 rain, and the pellet was suspended in 7.10 vol. (vol./wt of tissue) of 2.3 M sucrose-l'5 mM CaCI2-10 mM Tris (pH 6"8). The suspension was then filtered through 4 layers of cheesecloth and centrifuged for 45 min at 45,000 g. The pelleted nuclei were suspended in 1 Triton X-100-So~ution A and centrifuged at 1000 g for 10 min. The nuclei were washed in this solution once more. The Triton-treated nuclei were successively washed with the following solutions two times each : 0.075 M NaCI-0'024 M EDTA (pH 7-0), 50 mM Tris pH 7"9, 10 mM Tris pH 7'9, as described by Huang and Huang (1969). The final chromatin was suspended in ice-cold water and sheared lightly at 20 V for 60 s in a Sorval Omnimixer. All of the solutions used were flushed with nitrogen gas for several hours before use. N-ethylmaleimide ( NEM) labeling of chromathl Chromatin was adjusted to a concentration of 0-22 mg/ml DNA in 1 ~ SDS-10 mM KPO4, pH 7"0 (Solution B) and dialyzed in the dark 15 h at 25°. One milliliter of non-reduced chromatin was then incubated with 20 tal of I0 mM [aH]NEM (specific activity 10-2 Ci/mmol) for 9 h at 25°. Excess NEM was removed by exhaustive dialysis against Solution B. To prepare labeled reduced chromatin, an aliquot of the non-reduced chromatin was made 2 mM in 13-mercaptoethanol in Solution B, iilcubated 15 h and *Present address: National Institute of Environmental Health and Sciences, Research Triangle Park, North Carolina, U.S.A. 101
102
DONALD B. CARTER
dialyzed to 0.02 mM 13-mercaptoethanol concentration in Solution B. A 10 fold excess of [aH]NEM over 13-mercaptoethanol was then added to the chromatin solution and incubated 9 h at 25°. Excess NEM was again removed by exhaustive dialysis against Solution B. Disc gel electrophoresis Histones were analyzed on 15 ~ polyacrylamide gels (pH 3-2) according to the method of Panyim and Chalkley (1969). SDS gel electrophoresis was carried out as described before (Smith and Chae, 1973).
RESULTS A N D D I S C U S S I O N Chromatin isolated from the brain and liver of Sprague-Dawley Dublin Disease Resistant rats contains 1.1 mg histone per mg DNA. The histone/DNA ratio shows no appreciable age dependent variation. As shown in Table 1, there are small changes in the total protein/ D N A ratio from 3 month to 8 month chromatin but after 8 months the ratio stabilizes for liver and decreases slightly for brain. TABLE 1. P R O T E I N / D N A RATIO FOR WHOLE RAT BRAIN AND RAT LIVER CHROMATIN
Age (months) 3 8 18 33
Brain 2"55 :k 0"1" 2'21 2"26 2'02
Liver 2"24 -~ 0"12" 1"91 2"05 1"95
*Standard deviation determined by 6 independent experiments on 3 month chromatin. Remaining values were repeated 2 times and averaged for chromatin isolated from 10 pooled rat brains or 3 pooled livers from the indicated age groups. Histones were extracted from the chromatin as described before (Carter and Chae, 1975) and separated by the acid-urea polyacrylamide gel electrophoresis system reported by Panyim and Chalkley (1969). Histones were prepared for electrophoresis under reducing and non-reducing conditions. F r o m Fig. 1 it is apparent that histones prepared under nonreducing conditions show no detectable oxidized H3 histone when compared to control histones which have been oxidized by hydrogen peroxide. Oxidized H3 migrates slower than Ha histone during acid-urea gel electrophoresis (Panyim et al., 1971a). Identical results were also obtained with histones from brain, liver and testis of 3 month, 8 month and 18 month rats when histones were extracted from freshly prepared chromatin. When histones are extracted from frozen chromatin stored in air, there is generally a variable amount of H3 oxidation apparent on acid-urea gels. We have also determined the amount of the reduced cysteine in Hz histone by labeling dissociated chromatin with [SH]NEM. The results of [3H]NEM uptake by the chromatin dissociated in SDS is shown in Fig. 2. In the 7.5 ~ SDS polyacrylamide gel system, histone Ha migrates with an electrophoretic mobility of 0.83. When chromatin is acid-extracted to remove histones and then incubated with [aH]NEM, very little label is found in the Ha region of the gel, as shown in Fig. 2. This indicates that there are no major non-histone proteins capable of binding [aH]NEM which migrate with Ha histone in the 7 . 5 ~ SDS gel system. Accordingly one may interpret the quantity of label in the Ha region of the gel as being due to Hs histone uptake of [aH]NEM. There were small variations in the extent of labeling of chromatin in different experiments and in the total recovery of radioactivity in H8 histone after SDS gel electrophoresis. Therefore, we determined the per cent of total radioactivity of N E M in Ha histone after
NOAGE-DEPENDENTOXIDATIONOF H3 HISTONE
103
H2B 3
I
H3
HI
12A H4
4
OXH3
FIG. 1. Densitometric tracings of acid-urea gels described in Materials and Methods for brain and liver histones from DDR rats 33 months old. Tracings 1 and 2 are for histones prepared for electrophoresis with and without 1 ~ [3-mercaptoethanol from brain. Tracings 3 and 4 are for histones prepared for electrophoresis with and without 1 ~ [3-mercaptoethanol from liver. Tracing 5 is a control showing the presence of oxidized H3 prepared by oxidizing liver histories with 0.01 ~ hydrogen peroxide. electrophoresis. Also in order to determine the a m o u n t o f the reduced cysteine in Ha histone, the labeling o f H3 histone by [aH]NEM was compared between the chromatin before and after the reduction o f chromatin sample with 13-mercaptoethanol. When the peak position for H3 histone is integrated and c o m p a r e d to total recovered radioactivity in each gel, the a m o u n t o f label uptake in Ha is found to decrease with age after 8-18 m o n t h s (Fig. 3 and Table 2). The decrease in label uptake occurs in both non-reduced and reduced
104
DONALD B. CARTER
I0
s o
e'-
O.I
0.2
O.g
0.4
0.5
0.6
0.7
0,8
0.9
t.O
FIG. 2. Thiol content of acid extracted chromatin and whole chromatin control as determined by [SH]NEM labeling. Chromatin acid extracted by 0.4 N H2SOt, as described in Materials and Methods, was dissociated in l ~ SDS-10 mM KPO4, pH 7'0 and labeled with [3H]NEM. The percentage of total counts recovered is shown for each slice on the ordinate. The abscissa shows the fraction of the distance migrated by bromophenol blue of each slice. ( C - - - - ©) Acid extracted chromatin ( x - - - - × ) Control chromatin c h r o m a t i n a n d therefore does not represent a n y changes in oxidation of H 8 during aging. These data are consistent with the results presented showing that oxidized H 3 does n o t exist in detectable quantities in the liver a n d brain c h r o m a t i n of D D R rats (see Fig. 1). Also there is no age-dependent reduction of the a m o u n t of H z histone, as we have shown before (Carter a n d Chae, 1975). O n several occasions we have seen age-dependent reduction of the N E M labeling of H z histone in the n o n - r e d u c e d c h r o m a t i n c o m p a r e d to reduced c h r o m a t i n ( u n p u b l i s h e d observations). This observation appears to be due to experimental artifact. We have taken several precautions to avoid the possible artifact in the labeling of c h r o m o s o m a l proteins with [SH]NEM. We flushed all of the solutions used with nitrogen gas to remove oxygen a n d the c h r o m a t i n was labeled with [SH]NEM on the same day as it was prepared. TABLE 2.
QUANTITY OF
[OH]NEM
Age (months) 3 8 18 33
Ha HISTONE ON SDS GELS
LABEL INCORPORATED INTO
NR* brain 24 29 26"4 20-1
R~ (%) 27 25"2 23"8 18.9
AS ~oo OF TOTAL RECOVERED COUNTS
NR* liver 39-7 32 -:~ 23.5
Rt (%) 35 36"9 25 25
NR* = not reduced before incubation of [SH]NEM and chromatin. Rt = reduced before incubation of ISH]NEM and chromatin. ~ = sample lost.
NO AGE-DEPENDENT OXIDATION OF
Ha HISTONE
105
14
12
10
- -
8
0.1
0,2
0,3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
28 --
24
20
8
~-
16
12
8
OJ
02
0,3
C4
0.3
13¢
0.7
0.~
0.9
1,0
FIG. 3. Labeling of rat liver chromosomal proteins with [3H]NEM. Chromatin dissociated in SDS was labeled with [SH]NEM and chromosomal proteins were fractionated by SDS gel electrophoresis, as described in Materials and Methods. (A) Liver chromatin reduced by 13-mercaptoethanol before labeling. (B) Liver chromatin not reduced. (• • ) , 3 months; ( 0 0 ) , 8 months; ( x × ), 33 months.
106
DONALD a . CARTER
Nonhistone chromosomal proteins are also labeled by [aH]NEM, and SDS gel electrophoresis of the chromatin labeled with [aH]NEM before and after the reduction with [3mercaptoethanol show no significant age dependent qualitative changes in the amount of reduced cysteine for both liver and brain. However, the decrease with age in relative labeling of H a with respect to nonhistone proteins is significant. The amount of label in H a relative to total label recovered on the gels drops by 30 % for both reduced brain and liver chromatin from 3 to 33 month animals. There are several possible explanations for these results: (a) the quantity of nonHa cysteine present in SDS-dissociated and dialyzed chromatin increases relative to a fixed quantity of Ha cysteine, implying that the quantity of cysteine in nonhistone chromatin proteins increases with age; (b) the [aH]NEM reacts with moieties in addition to cysteine which increase in concentration in aging chromatin (Brewer and Riehm, 1967; Sharpless and Flavin, 1966; Smyth et al., 1964). Under our selected conditions, however, we would expect [aH]NEM to react almost exclusively with the cysteine moieties; (c) the dissociating conditions of 1% SDS-10 m M K P O 4, pH 7.0 are not sufficiently stringent to completely disaggregate old chromatin relative to young chromatin. It has been reported that the number of disulfide bonds in rat liver chromatin increases from 20 day to 9 month old rats (Tas, 1976). When chromatin is dissociated in 1% SDS and labeled as described in Methods, no age related trend in the chromatin thiol/thiol d- disulfide ratio is apparent in the age groups used in this experiment (Table 3). Thus it cannot be argued on the basis of these results that any increase in the compactness of interphase chromatin is due to an increase in the number of disulfide bonds in chromatin. TABLE 3. T H I O L / T H I O L @ DISULFIDE* RATIO FOR RAT BRAIN AND LIVER CHROMATIN AS A FUNCTION OF AGE
Age (months) 3 8 18 33
Brain 0"708 0-68 0'88 0'69
Liver 0'793 0'75 0'85 0"70
*Fractions of unreduced and reduced chromatin 0"05 ml were assayed after excess [SH]NEM had been removed by exhaustive dialysis. Typical count rates for brain and liver chromatin (0-22 mg DNA/ml) are 5000-8000 counts per minute in 0'05 ml when labeled as described in Materials and Methods. SUMMARY Histones were extracted from chromatin isolated from the brain, liver and testis of Dublin Disease Resistant Sprague-Dawley rats and prepared for electrophoresis under reducing and non-reducing conditions. Histories prepared under non-reducing conditions show no detectable age related changes in oxidized Ha histone. The quantity of reduced cysteine in H3 histone relative to total chromatin thiol was determined by labeling SDS dissociated chromatin with [SH]NEM. Equivalent decrements in [aH]NEM incorporation in H3 histone, relative to total [aH]NEM incorporation in chromatin, occur in reduced and non-reduced preparations with age. In addition, no significant age related changes were found in the thiol/thiol ÷ disulfide ratio, when rat brain and liver chromatin were dissociated as described. Hence, any change in the content of disulfide bridges with age is proportional to the change in thiol content. Acknowledgements--This study was supported by a grant from the National Institute of Aging, National Institutes of Health (AG00103) awarded to Dr. Chi-Bom Chae. The author is grateful to Dr. Chi-Bom Chae for advice and consultation during the course of this study.
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