Basic Res. Cardiol. 71, 27-35 (1976)
Department of Clinical Biochemistry, Banting Institute, Faculty o] Medicine, University o] Toronto, Toronto, Ontario, M5H IL5 (Canada)
Acetylation of nuclear proteins in the isolated perfused rat heart Acetylierung yon Kernproteinen beim isolierten perfundierten Rattenherzen P. B. T a y l o r
a n d C. C. L i e w
With 2 figures and 4 tables (Received December i1, 1974}
Summary Female rat hearts were maintained by retrograde perfusion under a hydrostatic pressure of 75 cm HeO. The perfused h e a r t had a coronary flow of 8-9 ml/min and a heart rate of 220-240 beats/min. Recirculation of ['~H]acctate (2 pCi/ml perfusate) for 20 min was sufficient to label nuclear proteins. Total nuclear proteins were separated into three major classes: (1) 0.14 M NaC1 soluble nucleoplasmic proteins, (2) nucleohistones and (3) nonhistone residual proteins. Approximately 88-90% of the {:~H}acetate incorporated was found in the nucleohistone fraction. Polyacrylamide-urea gel electrophoresis of the histones indicated that fraction f, was not acetylated while f:, and f'.'al were highly acetylated, containing 75% of the total histone radioactivity. Fractio,~ f~l,+f:a~ was moderately acetylated contributing 20-25 % of the radioactivity. Nucleohistones isolated from myocardial cells showed the same percentage distribution of {'~H}acetate in the histone fractions as the whole heart. Acid hydrolysis followed by steam distillation released more than 95% of the acetyl groups from the two major nucleoproteins. These data suggest that the isolated perfused heart may provide a model system to study covalent modification of nucleoproteins under controlled physiological and biochemical conditions.
T h e isolated p e r f u s e d r a t h e a r t has b e e n used for y e a r s as a m o d e l s y s t e m to s t u d y i n t e r m e d i a r y m e t a b o l i s m , p r o t e i n synthesis, m e m b r a n e t r a n s p o r t a n d n u c l e i c acid s y n t h e s i s (3, 16-20, 25, 26). As c o v a l e n t modii)cal i o n of n u c l e a r a n d r i b o s o m a l p r o t e i n s has b e e n s h o w n to be closely associated w i t h the r e g u l a t i o n of gene a c t i v i t y a n d p r o t e i n s y n t h e s i s (1, 7, 8, 10, 11, 13, 22, 23), w e i n t e n d to establish a model s y s t e m of a n i n t a c t o r g a n to s t u d y the i n c o r p o r a t i o n of {~H}acetate into n u c l e a r proteins. T h e isolated p e r f u s e d h e a r t seems to p r o v i d e a n u n i q u e s y s t e m for the s t u d i e s of c o v a l e n t modification of n u c l e a r p r o t e i n s by e l i m i n a t i n g factors which m a y h a v e r e s u l t e d f r o m i n t e r a c t i o n w i t h other organs. Besides, it gives m o r e c o n t r o l l e d c o n d i t i o n s to label p r o t e i n with a defined p e r fusate. T h e f o l l o w i n g r e p o r t p r e s e n t s e v i d e n c e t h a t a n isolated p e r f u s e d rat p r e p a r a t i o n c a n be used to s t u d y the a c e t y l a t i o n of n u c l e a r histones a n d n o n - h i s t o n e r e s i d u a l proteins. 598
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Basic Research in Cardiology, Vol. 71, No. 1 (1976)
Materials and methods Materials P u r o m y c i n dihydrochloride was purchased from Nutritional Biochemical C o r p o r a t i o n , C l e v e l a n d , Ohio. {3H}acetate s o d i u m salt w i t h a specific a c t i v i t y of 3.2 Ci/m m o l e was o b t a i n e d f r o m A m e r s h a m / S e a r l e C o r p o r a tion, A r l i n g t o n Heights, Illinois. C o l l a g e n a s e (type I) a n d h y a l u r o n i d a s e (type I) w e r e p u r c h a s e d f r o m W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n , F r e e hold, N e w J e r s e y a n d S i g m a C h e m i c a l C o m p a n y , St. Louis, Missouri. R e a g e n t g r a d e chemicals w e r e u s e d t h r o u g h o u t these studies.
Method of perfusion Female Wistar strain rats weighing 220-250 g were used for all experiments. The experimental procedure was essentially as described by Morgan et al. (16). The hearts ~were perfused with 60 ml of Krebs-Henseleit bicarbonate buffer (9) containing 1.2 mM KH2PO4, 25 mM NaHCO3, 118 mM NaC1, 4.7 mM KC1, 2.5 mM CaC12, 1.2 mM MgSO 4 and 1~/0 glucose 0.5 mM Na-EDTA u n d e r a hydrostatic pressure of 75 cm HeO gassed with O,,:CO._,(95:5 %) at 37~ C. The first 10 ml of perfusate was collected in a graduate cylinder and discarded. Recirculation of the r e m a i n i n g 50 ml of buffer was continued for 5 to 60 m i n after the addition of {3H}acetate. For every cycle the perfusate was filtered through a millipore filter (45 # diameter) which was placed on a coarse sintered glass filter. This step was an attempt to eliminate possible bacterial contamination (4). Under these conditions the hearts could be perfused for at least one hour and had a coronay flow of 8-9 ml/min as well as heart rate of 220-240 beats/min as described by others (24).
Preparation o] nuclei By routine, hearts were frozen in liquid nitrogen after perfusion and s t o r e d at - 7 0 ~ C. F i v e p e r f u s e d h e a r t s w e r e pooled, a n d h o m o g e n i z e d in five v o l u m e s (w/v) of ice-cold m e d i u m NS (10) c o n t a i n i n g 0.25 M sucrose, 20 m M Tris. HC1 (pH 7.5), 5 m M MgC1, 100 m M KC1 a n d 40 m M NaC1. T h e s u b s e q u e n t steps i n t h e i s o l a t i o n of n u c l e i are t h e s a m e as p r e v i o u s l y d e s c r i b e d (12).
Fractionation of nuclear proteins T h e e x t r a c t i o n of n u c l e a r p r o t e i n s w a s c a r r i e d o u t at 0-4 ~ C as p r e v i o u s l y d e s c r i b e d (13, 27). T h e 0.14 M NaC1 e x t r a c t i o n s of p r o t e i n f r o m the i s o l a t e d n u c l e i w e r e p r e c i p i t a t e d w i t h 10 ~ T C A (final c o n c e n t r a t i o n ) o v e r n i g h t i n t h e cold. T h e p r o t e i n p r e c i p i t a t e w a s r e c o v e r e d b y c e n t r i f u g a tion, w a s h e d w i t h c h l o r o f o r m - m e t h a n o l (1:1), a n d dissolved i n 0.1 N NaOH. H i s t o n e s w e r e e x t r a c t e d b y 2.0 m l of 0.25 N HCI a n d p r e c i p i t a t e d o v e r n i g h t at r o o m t e m p e r a t u r e w i t h 10 vol of acetone. T h e h i s t o n e s w e r e r e c o v e r e d b y c e n t r i f u g a t i o n a n d d i s s o l v e d i n 1.0 m l of 0.01 N HC1. T h e n o n h i s t o n e r e s i d u a l p e l l e t w a s first e x p o s e d to c h l o r o f o r m - m e t h a n o l to r e m o v e lipids a n d p h o s p h o l i p i d s a n d t h e n e x t r a c t e d o v e r n i g h t at 37 ~ C i n 1.0 m l of 1 % s o d i u m d o d e c y l s u l f a t e (SDS). T h e c l e a r s u p e r n a t a n t w a s o b t a i n e d b y c e r i t r i f u g a t i o n at 17,500 • g for 15 m i n a n d u s e d as n o n h i s t o n e residual proteins.
Polyacrylamide gel electrophoresis H i s t o n e s w e r e f r a c t i o n a t e d b y t h e m e t h o d of P a n y i m a n d Chalkley (21). I n some e x p e r i m e n t s for the d e t e r m i n a t i o n of the p e r c e n t d i s t r i b u t i o n of
Taylor and Liew, Acetylation of nuclear proteins
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{3H}acetate i n c o r p o r a t i o n into specific h i s t o n e f r a c t i o n s , i n d i v i d u a l s t a i n e d gel slices w e r e e x t r a c t e d w i t h 0.5 ml d i m e t h y l s u l f o x i d e at 40 ~ C. T h i s s t e p r e m o v e d t h e d y e as w e l l as e l i m i n a t e d v a r i a b l e q u e n c h i n g f r o m t h e h i s t o n e f r a c t i o n s . T h e d e s t a i n e d gel slices which c o n t a i n e d t h e r a d i o a c t i v i t y w e r e t h e n d i s s o l v e d in 30 % h y d r o g e n p e r o x i d e p r i o r to t h e r a d i o a c t i v i t y d e t e r m i n a t i o n (27). T h e o p t i c a l d e n s i t y of t h e p r o t e i n c o n c e n t r a t i o n w a s m e a s u r e d a t 615 .m as d e s c r i b e d p r e v i o u s l y b y Wangh et al. (29).
Isolation of myocardial cells T h e s e p a r a t i o n of m y o c a r d i a l cells f r o m o t h e r cell t y p e s g e n e r a l l y f o l l o w e d t h e p r o c e d u r e of Berry et al. (2). T h e h e a r t s w e r e first p e r f u s e d w i t h {SH)acetate as d e s c r i b e d a b o v e a n d t h e n t r a n s f e r r e d to a p a r a l l e l s y s t e m c o n t a i n i n g 50 m l of c a l c i u m - f r e e Krebs-HenseIeit b i c a r b o n a t e b u f fer. T h e first 5 m l of t h e m e d i u m w a s d i s c a r d e d a n d 5 ml of f r e s h e n z y m e m e d i u m w a s a d d e d to g i v e a final c o n c e n t r a t i o n of 0 . 1 % c o l l a g e n a s e a n d 0.2 % h y a l u r o n i d a s e . T h e h e a r t s w e r e t h e n p e r f u s e d for 20 m i n in a closed r e c i r c u l a t i n g s y s t e m u n d e r a h y d r o s t a t i c p r e s s u r e of 75 cm H20 g a s s e d w i t h O2:CO ~ (95:5 ~ A f t e r 20 m i n of p e r f u s i o n t h e h e a r t s w e r e t r a n s f e r r e d to a b e a k e r , g e n t l y m i n c e d w i t h scissors a n d s u s p e n d e d in 20 m l of e n z y m e m e d i u m . T h e t i s s u e s u s p e n s i o n w a s t r a n s f e r r e d to an Erlenmeyer flask a n d s h a k e n in a w a t e r b a t h for 15 m i n at 37 ~ C. T h e s u s p e n s i o n w a s t h e n filtered t h r o u g h 4 l a y e r s of s u r g i c a l g a u z e a n d c e n t r i f u g e d for 2 m i n at 50 X g. T h e s u p e r n a t a n t w a s a s p i r a t e d a n d t h e p e l l e t s u s p e n d e d in 2 ml of c a l c i u m - f r e e Krebs' buffer. T h e cells w e r e l a y e r e d o v e r 20 ml of 0.32 M sucrose in Krebs' c a l c i u m - f r e e m e d i u m a n d c e n t r i f u g e d for 2 m i n at 50 • g. T h i s s t e p w a s i n c l u d e d in an a t t e m p t to r e m o v e m o s t of t h e c o l l a g e n a s e a n d h y a l u r o n i d a s e in t h e cell suspension. Y i e l d s of 25-27 % of the m y o c a r d i u m w e r e o b t a i n e d w h e n m y o c a r d i a l cells w e r e c a l c u l a t e d as T C A - p r e c i p i t a b l e dry weight. Observation by phase contrast microscopy indicated that the muscle cells w e r e r e l a t i v e l y p u r e w i t h at l e a s t 5 0 - 6 0 % b e i n g m o r p h o l o g i c a l l y i n t a c t . W h e n t h e cells w e r e b r o u g h t to r o o m t e m p e r a t u r e a b o u t 50 % of t h e i n t a c t cells h a d s p o n t a n e o u s c o n t r a c t i l i t y . T h e i s o l a t e d m y o c a r d i a l cells w e r e v e r y s i m i l a r to t h o s e of Berry et al. (2). No c l a i m was m a d e t h a t t h e m o r p h o l o g i c a l s t u d i e s d i f f e r e n t i a t e m u s c l e a n d c o n n e c t i v e tissue cells. O u r p r i m a r y p u r p o s e w a s to p r e p a r e cell f r a c t i o n s of e n r i c h e d h e a r t m u s c l e cells.
Determination of protein and radioactivity P r o t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d b y t h e m e t h o d of Lowry et al. (14), u s i n g c r y s t a l l i n e b o v i n e s e r u m a l b u m i n as a s t a n d a r d . T h e r a d i o a c t i v i t y of n u c l e a r p r o t e i n s w a s m e a s u r e d in a l i q u i d s c i n t i l l a t i o n c o u n t e r ( N u c l e a r C h i c a g o m o d e l 4868) w i t h an efficiency for {3H} of 25-30 ~ Results
Incorporation of {3H}acetate into nuclear proteins H e a r t s w e r e p e r f u s e d w i t h (3H}acetate for p e r i o d s of 5 to 60 min. T h e r e s u l t s a r e s h o w n in Fig. 1. T h e i n c o r p o r a t i o n of a c e t a t e into n u c l e i a n d
Basic Research in Cardiology, Vol. 71, No. 1 (1976)
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50
5
Z w
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ag
Z 040
i'
4
a.
~30
U
~
Z
% -20 X
2
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1
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0
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~
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0
I
I
I
I
I
5
10
20
30
60
PERFUSION
TIME
(MIN.)
Fig. 1. Incorporation of {3H}acetate into nuclear proteins. Hearts were perfused w i t h {'~H}acetate (1 /~Ci/ml) p e r f u s a t e for 5, 10, 15, 30 and 60 min. F i v e h e a r t s w e r e pooled to isolate nuclei. The specific activities of n u c l e a r p r o t e i n s (0-0) and n u c l e o h i s t o n e s (o-o) w e r e d e t e r m i n e d as d e s c r i b e d in the M e t h o d section. histone fractions rapidly increased during the first 20 rain of perfusion and reached a maximum between 20 and 30 min. It was found that there was a reduction in acetate incorporation into nuclear proteins after 60 rain of perfusion. From these experiments a constant perfusion time of 20 rain was selected to examine the effect of increased concentration of acetate on the incorporation into different nuclear protein fractions.
E f f e c t of {3H}acetate c o n c e n t r a t i o n on i n c o r p o r a t i o n into n u c l e a r p r o t e i n s T h e i n c o r p o r a t i o n of { 3 H } a c e t a t e i n t o 0.14 M NaC1 s o l u b l e p r o t e i n s , histones and nonhistone residual proteins, was investigated by using d i f f e r e n t c o n c e n t r a t i o n s of { 3 H } a c e t a t e f o r 20 m i n . T a b l e 1 s h o w s t h e t o t a l Tab. I. Total radioactivity
in fractionated
nuclear proteins
Fractions
{3H} acetate concentration 1 ]~Ci/ml 2 ~Ci/ml
4/tCi/ml
Histones 14. M NaC1 Residual proteins
21,616 283 1,746
57,872 708 5,194
55,136 876 4,308
Five hearts were perfused for 20 min at each concentration and then frozen in liquid nitrogen. Nuclei were isolated and the protein fractions separated as described under Method. Aliquots (50 i~I) from each fraction were removed for determination of radioactivity (cpm). The values indicate the total activity in each fraction.
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r a d i o a c t i v i t y of {3H}acetate at d i f f e r e n t c o n c e n t r a t i o n s t h a t was inc o r p o r a t e d into each n u c l e a r p r o t e i n f r a c t i o n . It w a s f o u n d t h a t w h e n t h e c o n c e n t r a t i o n of {3H)acetate in t h e p e r f u s a t e w a s i n c r e a s e d f r o m 2 to 4 ~ C i / m l , t h e r e w a s no significant i n c r e a s e in t h e a m o u n t of a c e t a t e incorporated.
Fractionation of histones H i s t o n e s o b t a i n e d f r o m 20 m i n {SH}acetate l a b e l l e d p e r f u s e d h e a r t s w e r e a n a l y z e d b y p o l y a c r y l a m i d e - u r e a gel e l e c t r o p h o r e s i s (21). F o u r m a j o r f r a c t i o n s w e r e i d e n t i f i e d a n d e x p r e s s e d as counts p e r m i n p e r o p t i c a l d e n s i t y of 615,m as s h o w n in Fig. 2. T h e p e r c e n t d i s t r i b u t i o n of {3H}acetate i n c o r p o r a t e d into t h e h i s t o n e f r a c t i o n s w a s also d e t e r m i n e d . T h e s l o w e s t m o v i n g b a n d fl i n c o r p o r a t e d a n e g l i g i b l e a m o u n t of {~H}acetate w h i l e f r a c t i o n s f~ a n d f2~l w e r e h i g h l y a c e t y l a t e d , a c c o u n t i n g for 75 % of t h e t o t a l h i s t o n e r a d i o a c t i v i t y . T h e f r a c t i o n t h a t c o n t a i n e d h i s t o n e s f,.,h q- f2:~.~ w a s m o d e r a t e l y a c e t y l a t e d w i t h a b o u t 20-25 % of t h e t o t a l a c t i v i t y . In a n a t t e m p t to d e t e r m i n e t h e a c e t y l a t i o n of n u c l e o h i s t o n e s f r o m one cell type, m u s c l e cells w e r e i s o l a t e d f r o m p e r f u s e d h e a r t l a b e l l e d w i t h {SH}acetate for 20 min. T h e n u c l e i w e r e s u b s e q u e n t l y i s o l a t e d f r o m t h e m u s c l e cells. Tab. 2 c o m p a r e s t h e t o t a l r a d i o a c t i v i t y a n d p e r c e n t d i s t r i b u -
Fig. 2. Fractionation of histones. Nucleohistone (30 /~g) isolated from {'~H}acetate labelled perfused hearts was applied to polyacrylamide-urea gels. After electro~ phoresis, the gels were destained electrophoretically and fractions f,, f~, (f.,i, -t- f.2a.2)and f2a, were identified. Each fraction was cut from the gel, solubilized in H.,O.., and then dissolved in 15 ml Aquasol for radioactivity determination.
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Tab. 2. Distribution of {nit} acetate in fractionatcd histories Fraction
~b 9 ~a~ ~1
Normal perfused hearts cpm ~o of total
Myocardial cells cpm
% of total
Simulated perfused hearts cpm ~o of total
11 365 127 147
5 189 53 90
2 56 16 27
10 194 50 98
2 56 20 23
3 55 14 28
30 ~g of heart histones from each condition were analyzed by polyacrylamideurea gel electrophoresis. The gels were stained with Amido Black and destained electrophoretically. The identified histone bands were sliced and their radioactivity determined as described in the Method section. t i o n of each h i s t o n e f r a c t i o n for w h o l e h e a r t s , i s o l a t e d m y o c a r d i a l cells a n d w h o l e h e a r t s p e r f u s e d u n d e r i d e n t i c a l conditions, so as to s i m u l a t e t h e p r o c e d u r e s for s e p a r a t i o n of m u s c l e cells. T h e r e s u l t s f r o m these e x p e r i m e n t s r e v e a l s e v e r a l aspects of h i s t o n e a c e t y l a t i o n . First, u n d e r o u r c o n d i t i o n s for t h e l a b e l l i n g a n d i s o l a t i o n of m y o c a r d i a l cells t h e t o t a l a c t i v i t y for each h i s t o n e f r a c t i o n w a s a p p r o x i m a t e l y r e d u c e d b y 50 ~ w h e n c o m p a r e d to w h o l e h e a r t n u c l e o h i s t o n e s . S e c o n d l y , the p e r c e n t d i s t r i b u t i o n of i n c o r p o r a t e d a c e t y l g r o u p s b e t w e e n t h e m a j o r h i s t o n e f r a c t i o n s i n t h e i s o l a t e d m y o c a r d i a l cell w a s s i m i l a r to the w h o l e h e a r t . T h i r d , if w h o l e h e a r t s a r e p u l s e l a b e l l e d a n d t h e n p e r f u s e d to s i m u l a t e t h e s e p a r a t i o n of m u s c l e cells, t h e i n c o r p o r a t i o n of {3H}acetate into w h o l e h e a r t n u c l e o h i s t o n e s is s i m i l a r to the i s o l a t e d m y o c a r d i a l cells. T h e s e e x p e r i m e n t s i n d i c a t e t h a t t h e a c e t y l a t i o n of n u c l e o h i s t o n e s i n m u s c l e cells is s i m i l a r to t h a t i n t h e i n t a c t m y o c a r d i u m .
E f f e c t of p u r o m y c i n on a c e t a t e i n c o r p o r a t i o n T h e effect of p u r o m y c i n o n {3H}acetate i n c o r p o r a t i o n i n t o h e a r t n u c l e o p r o t e i n s is p r e s e n t e d i n Tab. 3. H e a r t s w e r e p e r f u s e d for 10 rain w i t h p u r o m y c i n at b o t h 50 a n d 75/~g/ml p e r f u s a t e , f o l l o w i n g 20 m i n {all}acetate p u l s e l a b e l l i n g . P u r o m y c i n w h i c h is k n o w n to dissociate h e a r t p o l y r i b o s o m e s (17) as w e l l as to block p r o t e i n b i o s y n t h e s i s at t h e t r a n s l a t i o n a l l e v e l (11) failed to i n h i b i t the i n c o r p o r a t i o n of a c e t a t e into n u c l e o p r o t e i n s . T h e e x p e r i m e n t s s u g g e s t t h a t the i n c o r p o r a t i o n of a c e t a t e i n t o n u c l e o p r o t e i n s is i n d e p e n d e n t of p r o t e i n s y n t h e s i s . Tab. 3. Effect of puromycin on the incorporation of {3H}acetate into nuclear proteins Treatment
Total nuclei
Histoncs
Residual proteins
Control Puromycin
10,658 10,857
43,792 44,344
5,671 5,483
Hearts were perlused with puromycin. The control group was given identical conditions except that the hearts were perfused without puromycin. The results are expressed as cpm/mg protein.
Taylor and Liew, Acetylation of nuclear proteins
33
Identification of acetyl groups The presence of radioactive acetyl groups in nuclear proteins was identified by acid hydrolysis and steam distillation as described previously (13, 27). As shown in Tab. 4, over 95% of the {'~H}label was released from the nucleohistone and nonhistone residual proteins by such treatment, indicating that the acetate was mainly incorporated as an acetyl group. Tab. 4. Release of {all}acetate from histories, 0.14 M NaCI solubh~ and r(,sidual proteins
Before hydrolysis After hydrolysis Released ~ Released
0.14 M N ~ C 1
Histon(,s
460 85 375 81
12,840 69 12,771 99
Residual proteins 2,160 63 2,097 97
Hearts were perfused with {SH}acetate (2 /~Ci/ml perfusate) for 20 min. Histones, 0.14 M NaC1 soluble and residual proteins were extracted from the isolated nuclei and used for acid hydrolysis as described in the text. Discussion
A relationship between acetylation of histones and RNA synthesis has been demonstrated in regenerating liver (23), hormone replacement studies (5, 28) and cell culture systems (6, 22). It is postulated that post-synthetic acetylation of nucleohistones increases template activity by weakening the DNA-histone interaction through charge neutralization. Previous reports from this laboratory have demonstrated that aldosterone administration to adrenalectomized rats stimulated the acetylation of heart nucleohistones prior to an increase in RNA polymerase activity and RNA synthesis (12, 13). However, studies that follow in r i v e incorporation of isotope into proteins are usually subject to neural and hormonal variations that may mask some biochemical changes. In addition, the pool size of radioactive precursors may change under different physiological conditions. In order to alleviate some of these problems we have used an isolated perfused heart system to examine the acetylation of nucleoproteins. The incorporation of {3H}acetate into whole nuclei and histones occurs rapidly, reaching a peak between 20-30 min of perfusion. The decline of {all}acetate incorporation into nuclear proteins that occurred after perfusion for 20-30 min could be due to the rapid utilization of the {'~H}acetate in the perfusate. However, these data suggest that the acetylation process is enzymatic and are in good agreement with experiments using isolated nuclei (1) and the acetylation of histones by partially purified nuclear histone-acetyltransferase (15). We have also examined the effect of varying concentrations of {3H}acetate on the incorporation into nucleoproteins and found that 2 #Ci/ml buffer gave near maximal incorporation into the different protein fractions. Increasing the concentration to 4 pCi/ml perfusate only slightly enhanced the amount of {3H/acetate incorporated into the
34
.Basic Research in Cardiology, Vol. 71, No. 1 (1976)
0.14 1V~NaC1 soluble, h i s t o n e s a n d r e s i d u a l p r o t e i n s . A t all c o n c e n t r a t i o n s of (3H}acetate used for t h e i n c o r p o r a t i o n studies, a p p r o x i m a t e l y 90 % of t h e r a d i o a c t i v i t y was f o u n d i n t h e h i s t o n e f r a c t i o n w h i l e o n l y a b o u t 7-8 % of {3H}acetate w a s i n c o r p o r a t e d i n t o n o n - h i s t o n e r e s i d u a l p r o t e i n s . W h e n n u c l e o h i s t o n e s w e r e s e p a r a t e d i n t o f o u r m a j o r classes b y p o l y a c r y l a m i d e u r e a gel e l e c t r o p h o r e s i s t h e specific a c t i v i t i e s of t h e a r g i n i n e - r i c h f~ a n d f2~1 w e r e t h e highest. F r a c t i o n f~b-~-f2a2 w a s m o d e r a t e l y a c e t y l a t e d w h i l e t h e l y s i n e - r i c h f r a c t i o n fl w a s b a r e l y detected. I n a n a t t e m p t to r e d u c e p o s s i b l e c o n t a m i n a t i o n b y n o n - m u s c l e cells, h e a r t cells w e r e s e p a r a t e d f r o m t h e i n t a c t m y o c a r d i u m f r o m w h i c h w h o l e n u c l e i w e r e i s o l a t e d a n d n u c l e a r p r o t e i n s e x t r a c t e d . T h e i n c o r p o r a t i o n of a c e t a t e i n t o 0.14 M NaC1 soluble, h i s t o n e s a n d r e s i d u a l p r o t e i n s w a s cons i d e r a b l y r e d u c e d w h e n c o m p a r e d to w h o l e h e a r t s (Tab. 2). H o w e v e r , w h e n h e a r t s w e r e l a b e l l e d for 20 m i n a n d t h e n p e r f u s e d u n d e r c o n d i t i o n s t h a t s i m u l a t e t h e i s o l a t i o n of m y o c a r d i a l cells t h e a m o u n t of {SH}acetate i n c o r p o r a t e d into t h e 0.14 M N a C l - s o l u b l e , h i s t o n e s a n d r e s i d u a l p r o t e i n s was s i m i l a r to t h e i s o l a t e d m y o c a r d i a l cells. T h e s e e x p e r i m e n t s i n d i c a t e t h a t t h e site of a c e t y l a t i o n is e q u a l l y a c t i v e i n t h e e n r i c h e d f r a c t i o n of t h e c o n t r a c t i n g m u s c l e cells. It is c e r t a i n l y possible t h a t a c e t y l a t i o n of n u c l e a r p r o t e i n s also occurs in the o t h e r cell types. I n g e n e r a l , t h e p o s t s y n t h e t i c c o v a l e n t m o d i f i c a t i o n of n u c l e a r p r o t e i n s has a close t e m p o r a l a s s o c i a t i o n w i t h a n i n c r e a s e i n R N A s y n t h e s i s i n a n u m b e r of biological s y s t e m s (13, 23, 28). O u r e x p e r i m e n t s h a v e a t t e m p t e d to d e m o n s t r a t e t h e possible u s e f u l n e s s of t h e i s o l a t e d p e r f u s e d r a t h e a r t to s t u d y c o v a l e n t m o d i f i c a t i o n s of n u c l e a r p r o t e i n s . T h i s s t u d y m a y also p r o v i d e some b i o c h e m i c a l basis for s t u d y i n g changes i n r e s p o n s e d u e to the p h y s i o l o g i c a l s t i m u l i ; e.g., w o r k - l o a d , b i o c h e m i c a l e n v i r o n m e n t s or pharmacological interventions. Acknowledgements
We are very grateful to Professor H. E. Morgan of the Milton S. Hershey Medical School, Penn. for introducing the isolated heart perfusion method and to Professor A. G. Gornall for his generous support as well as advice throughout the studies. P. B. Taylor is a postdoctoral fellow of the Ontario Heart F o u n d a tion. The technical assistance of Mrs. L. Suigu is appreciated. Zusammenfassung
Bei weiblichen Ratten wurde das Herz retrograd perfundiert mit einem hydrostatischen Druck von 55 cm H20. Die perfundierten Herzen zeigten einen KoronardurchfluB yon 8-9 ml/min u n d eine Herzfrequenz von 220-240 Schl~gen/ min. Mit rezirkulierendem {3H}-Acetat (2 ,uCi/ml Perfusat) w u r d e n innerhalb 20 rain die Kernproteine markiert. Die Nucleoproteine w u r d e n in drei gro~en Klassen isoliert: 1. 0,14 M NaCl-15sliche Proteine des Kernplasmas, 2. Nucleohistone u n d 3. nichthiston-~hnliche Restproteine. Nahezu 88-90 ~/0 des eingebauten {~i.i}-Acetate erschien in der Nucleohiston-Fraktion. Polyacrylamid-HarnstoffGelelektrophorese t r e n n t e die Histone auf in eine nichtacetylierte Fraktion fl u n d in stark acetylierte F r a k t i o n e n f~ u n d f,2al mit 75 % der gesamten Histonradioaktivit~t. Die Fraktion feb ~- fea~ war schw~cher acetyliert mit 20-25 % der Radioaktivit~t. Die aus Myokardzellen isolierten Nucleohistone besal3en die gleiche prozentuale Verteilung des {3H)-Acetats in den Histonfraktionen wie die Pr~parationen aus ganzen Herzen. Saure Hydrolyse u n d Wasserdampf-
Taylor and Liew, Acetylation of nuclear proteins
35
d e s t i l l a t i o n s e t z t e n m e h r a l s 95 % d e r A c e t y l g r u p p e n a u s d e n b e i d c n H a u p t f r a k t i o n e n d e r N u c l e o p r o t e i n e f r e i . D i e s e E r g e b n i s s e w e i s e n d a r a u f h i n , daft d a s isolierte, perfundierte Herz als Modell dienen kann zur Untersuchung yon kovalenten Modifizierung von Nucleoproteinen unter definierten physiologischen und biochemischen Bedingungen.
References 1. Allfrey, V. G., V. C. Littau a n d A. E. Mirsky, P r o c . N a t l . A c a d . Sci U.S.A.,
49, 414-421 (1963). - 2. Berry, M. N., D. S. Friend a n d J. Scheuer, C i r c u l a t . Ites. 26, 679-687 (1970). - 3. Bleehen, N. M. a n d R. B. Fisher, J. P h y s i o l . 123, 260-276 (1954). - 4. Franburg, B. L. a n d B. I. Pogner, B i o c h i m . B i o p h y s . A c t a 182, 577-579 (1969). - 5. Gornall, A. G. a n d C. C. Liew, A d v . Enz. R e g . 12, 267-285 (1974) - 6. Hnilica, L. S., M. E. McCture a n d T. C. Spelsberg, H i s t o n e B i o s y n t h e s i s a n d t h e C e l l C y c l e , p. 188-204 (1971), i n : H i s t o n e s a n d N u c l e o h i s t o n e , e d D. M. P. P h i l lips. - 7. Johnson, A. W., J. A. Wilhelm a n d L. S. Hnitica, B i o c h i m . B i o p h y s . A c t a 295, 150-158 (1973). - 8. Kabat, D., B i o c h e m . 9, 4160-4175 (1970). - 9. Krebs, H. A. a n d K. Henseleit, Z. H o p p e - S e y l Z. 120, 33 (1932). - 10. Liew, C. C. a n d A. G. Gornall, J. Biol. C h e m . 248, 977-983 (1973). - I1. Licw, C. C., G. W. Haslett a n d V. G. Allfrey, N a t u r e 226, 414-417 (1970). - 12. Liew, C. C., D. K. L i u a n d A. G. Gornalt, E n d o c r i n o l . 90, 488-495 (1972). - 13. Liew, C. C., D. Suria a n d A. G. Gornall, E n d o c r i n o l . 93, 1025-1034 (1973). - 14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr a n d R. J. Randalt, J. Biol. C h e m . 193, 265-275 (1951). - 15. Luc, P. F., A. G. Gornall a n d C. C. Liew, C a n . J. B i o c h e m . 51, 1177-1194 (1973) - 16. Morgan, H. E., M. J. Henderson, D. M. Regen a n d C. R. Park, J. Biol. C h e m . 236, 253-261 (1961). - 17. Morgan, H. E., D. C. N. Earl, A. Broadus, E. B. Wo[pert, K. E. Giger a n d L. S. Jefferson, J. Biol. C h e m . 246, 2152-2162 (1971). - 18. Morgan, H. E., L. S. Jefferson, E. D. Wolpert a n d D. E. Rannels, J. Biol. C h e m . 246, 2163-2170 (1971). - 19. Neely, J. R., R. H. B o w m a n a n d H. E. Morgan, A m e r . J. P h y s i o l . 216, 804-811 (1969). - 20. Opie, L. G., J. C. Shipp, J. R. Evans a n d B. Leboeaf, A m e r . J. P h y s i o l . 203, 839-843 (1962). - 21. Panyim, S. a n d R. Chalklell, A r c h . B i o c h e m . B i o p h y s . 130, 337-346 (1969). - 22. Pogo, B. G. T., V. G. Alllrey a n d A. E. Mirsky, P r o c . N a t l . A c a d . Sci. U . S . A . 55, 805-872 (1966). - 23. Pogo, B. G. T., A. O. Pogo, V. G. Altfrey a n d A. E. Mirsky, P r o c . N a t l . A c a d . Sci. U . S . A . 59, 1337-1344 (1968). - 24. Ross, B. D., P e r f u s i o n t e c h n i q u e s in B i o c h e m i s t r y . P a r t 2 H e a r t a n d S k e l e t a l M u s c l e , 271 (1972). - 25. Schreiber, S. S., M. Oratz, C. Evans, E. Silver a n d M. A. Rothschild, A m e r . J. P h y s i o l . 215, 1250-1259 (1968). - 26, Sender, P. M. a n d P. J. Garlick, B i o c h e m . J. 132, 603-608 (1973). - 27. Suria, D. a n d C. C. Liew, B i o c h e m . J. 137, 355-362 (1974). - 28. Trachewski, D. a n d S. Lawrence, P r o c . Soc. E x p t l . Biol. a n d M e d . 141, 14-20 (1972). - 29. Wangh, L., A. Ruiz-Carrillo a n d V. G. Allfrey, A r c h . B i o c h e m . B i o p h y s . 150, 44-58 (1972), Authors' address: D r . C. C. Liew, D e p t . o f C l i n i c a l B i o c h e m i s t r y , B a n t i n g I n s t i t u t e , 100 C o l l e g e S t r e e t , T o r o n t o , O n t a r i o , C a n a d a , M 5 H 1L5
Department of Clinical Biochemistry, Banting Institute, Faculty o] Medicine, University o] Toronto, Toronto, Ontario, M5H IL5 (Canada)
Acetylation of nuclear proteins in the isolated perfused rat heart Acetylierung yon Kernproteinen beim isolierten perfundierten Rattenherzen P. B. T a y l o r
a n d C. C. L i e w
With 2 figures and 4 tables (Received December i1, 1974}
Summary Female rat hearts were maintained by retrograde perfusion under a hydrostatic pressure of 75 cm HeO. The perfused h e a r t had a coronary flow of 8-9 ml/min and a heart rate of 220-240 beats/min. Recirculation of ['~H]acctate (2 pCi/ml perfusate) for 20 min was sufficient to label nuclear proteins. Total nuclear proteins were separated into three major classes: (1) 0.14 M NaC1 soluble nucleoplasmic proteins, (2) nucleohistones and (3) nonhistone residual proteins. Approximately 88-90% of the {:~H}acetate incorporated was found in the nucleohistone fraction. Polyacrylamide-urea gel electrophoresis of the histones indicated that fraction f, was not acetylated while f:, and f'.'al were highly acetylated, containing 75% of the total histone radioactivity. Fractio,~ f~l,+f:a~ was moderately acetylated contributing 20-25 % of the radioactivity. Nucleohistones isolated from myocardial cells showed the same percentage distribution of {'~H}acetate in the histone fractions as the whole heart. Acid hydrolysis followed by steam distillation released more than 95% of the acetyl groups from the two major nucleoproteins. These data suggest that the isolated perfused heart may provide a model system to study covalent modification of nucleoproteins under controlled physiological and biochemical conditions.
T h e isolated p e r f u s e d r a t h e a r t has b e e n used for y e a r s as a m o d e l s y s t e m to s t u d y i n t e r m e d i a r y m e t a b o l i s m , p r o t e i n synthesis, m e m b r a n e t r a n s p o r t a n d n u c l e i c acid s y n t h e s i s (3, 16-20, 25, 26). As c o v a l e n t modii)cal i o n of n u c l e a r a n d r i b o s o m a l p r o t e i n s has b e e n s h o w n to be closely associated w i t h the r e g u l a t i o n of gene a c t i v i t y a n d p r o t e i n s y n t h e s i s (1, 7, 8, 10, 11, 13, 22, 23), w e i n t e n d to establish a model s y s t e m of a n i n t a c t o r g a n to s t u d y the i n c o r p o r a t i o n of {~H}acetate into n u c l e a r proteins. T h e isolated p e r f u s e d h e a r t seems to p r o v i d e a n u n i q u e s y s t e m for the s t u d i e s of c o v a l e n t modification of n u c l e a r p r o t e i n s by e l i m i n a t i n g factors which m a y h a v e r e s u l t e d f r o m i n t e r a c t i o n w i t h other organs. Besides, it gives m o r e c o n t r o l l e d c o n d i t i o n s to label p r o t e i n with a defined p e r fusate. T h e f o l l o w i n g r e p o r t p r e s e n t s e v i d e n c e t h a t a n isolated p e r f u s e d rat p r e p a r a t i o n c a n be used to s t u d y the a c e t y l a t i o n of n u c l e a r histones a n d n o n - h i s t o n e r e s i d u a l proteins. 598
28
Basic Research in Cardiology, Vol. 71, No. 1 (1976)
Materials and methods Materials P u r o m y c i n dihydrochloride was purchased from Nutritional Biochemical C o r p o r a t i o n , C l e v e l a n d , Ohio. {3H}acetate s o d i u m salt w i t h a specific a c t i v i t y of 3.2 Ci/m m o l e was o b t a i n e d f r o m A m e r s h a m / S e a r l e C o r p o r a tion, A r l i n g t o n Heights, Illinois. C o l l a g e n a s e (type I) a n d h y a l u r o n i d a s e (type I) w e r e p u r c h a s e d f r o m W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n , F r e e hold, N e w J e r s e y a n d S i g m a C h e m i c a l C o m p a n y , St. Louis, Missouri. R e a g e n t g r a d e chemicals w e r e u s e d t h r o u g h o u t these studies.
Method of perfusion Female Wistar strain rats weighing 220-250 g were used for all experiments. The experimental procedure was essentially as described by Morgan et al. (16). The hearts ~were perfused with 60 ml of Krebs-Henseleit bicarbonate buffer (9) containing 1.2 mM KH2PO4, 25 mM NaHCO3, 118 mM NaC1, 4.7 mM KC1, 2.5 mM CaC12, 1.2 mM MgSO 4 and 1~/0 glucose 0.5 mM Na-EDTA u n d e r a hydrostatic pressure of 75 cm HeO gassed with O,,:CO._,(95:5 %) at 37~ C. The first 10 ml of perfusate was collected in a graduate cylinder and discarded. Recirculation of the r e m a i n i n g 50 ml of buffer was continued for 5 to 60 m i n after the addition of {3H}acetate. For every cycle the perfusate was filtered through a millipore filter (45 # diameter) which was placed on a coarse sintered glass filter. This step was an attempt to eliminate possible bacterial contamination (4). Under these conditions the hearts could be perfused for at least one hour and had a coronay flow of 8-9 ml/min as well as heart rate of 220-240 beats/min as described by others (24).
Preparation o] nuclei By routine, hearts were frozen in liquid nitrogen after perfusion and s t o r e d at - 7 0 ~ C. F i v e p e r f u s e d h e a r t s w e r e pooled, a n d h o m o g e n i z e d in five v o l u m e s (w/v) of ice-cold m e d i u m NS (10) c o n t a i n i n g 0.25 M sucrose, 20 m M Tris. HC1 (pH 7.5), 5 m M MgC1, 100 m M KC1 a n d 40 m M NaC1. T h e s u b s e q u e n t steps i n t h e i s o l a t i o n of n u c l e i are t h e s a m e as p r e v i o u s l y d e s c r i b e d (12).
Fractionation of nuclear proteins T h e e x t r a c t i o n of n u c l e a r p r o t e i n s w a s c a r r i e d o u t at 0-4 ~ C as p r e v i o u s l y d e s c r i b e d (13, 27). T h e 0.14 M NaC1 e x t r a c t i o n s of p r o t e i n f r o m the i s o l a t e d n u c l e i w e r e p r e c i p i t a t e d w i t h 10 ~ T C A (final c o n c e n t r a t i o n ) o v e r n i g h t i n t h e cold. T h e p r o t e i n p r e c i p i t a t e w a s r e c o v e r e d b y c e n t r i f u g a tion, w a s h e d w i t h c h l o r o f o r m - m e t h a n o l (1:1), a n d dissolved i n 0.1 N NaOH. H i s t o n e s w e r e e x t r a c t e d b y 2.0 m l of 0.25 N HCI a n d p r e c i p i t a t e d o v e r n i g h t at r o o m t e m p e r a t u r e w i t h 10 vol of acetone. T h e h i s t o n e s w e r e r e c o v e r e d b y c e n t r i f u g a t i o n a n d d i s s o l v e d i n 1.0 m l of 0.01 N HC1. T h e n o n h i s t o n e r e s i d u a l p e l l e t w a s first e x p o s e d to c h l o r o f o r m - m e t h a n o l to r e m o v e lipids a n d p h o s p h o l i p i d s a n d t h e n e x t r a c t e d o v e r n i g h t at 37 ~ C i n 1.0 m l of 1 % s o d i u m d o d e c y l s u l f a t e (SDS). T h e c l e a r s u p e r n a t a n t w a s o b t a i n e d b y c e r i t r i f u g a t i o n at 17,500 • g for 15 m i n a n d u s e d as n o n h i s t o n e residual proteins.
Polyacrylamide gel electrophoresis H i s t o n e s w e r e f r a c t i o n a t e d b y t h e m e t h o d of P a n y i m a n d Chalkley (21). I n some e x p e r i m e n t s for the d e t e r m i n a t i o n of the p e r c e n t d i s t r i b u t i o n of
Taylor and Liew, Acetylation of nuclear proteins
29
{3H}acetate i n c o r p o r a t i o n into specific h i s t o n e f r a c t i o n s , i n d i v i d u a l s t a i n e d gel slices w e r e e x t r a c t e d w i t h 0.5 ml d i m e t h y l s u l f o x i d e at 40 ~ C. T h i s s t e p r e m o v e d t h e d y e as w e l l as e l i m i n a t e d v a r i a b l e q u e n c h i n g f r o m t h e h i s t o n e f r a c t i o n s . T h e d e s t a i n e d gel slices which c o n t a i n e d t h e r a d i o a c t i v i t y w e r e t h e n d i s s o l v e d in 30 % h y d r o g e n p e r o x i d e p r i o r to t h e r a d i o a c t i v i t y d e t e r m i n a t i o n (27). T h e o p t i c a l d e n s i t y of t h e p r o t e i n c o n c e n t r a t i o n w a s m e a s u r e d a t 615 .m as d e s c r i b e d p r e v i o u s l y b y Wangh et al. (29).
Isolation of myocardial cells T h e s e p a r a t i o n of m y o c a r d i a l cells f r o m o t h e r cell t y p e s g e n e r a l l y f o l l o w e d t h e p r o c e d u r e of Berry et al. (2). T h e h e a r t s w e r e first p e r f u s e d w i t h {SH)acetate as d e s c r i b e d a b o v e a n d t h e n t r a n s f e r r e d to a p a r a l l e l s y s t e m c o n t a i n i n g 50 m l of c a l c i u m - f r e e Krebs-HenseIeit b i c a r b o n a t e b u f fer. T h e first 5 m l of t h e m e d i u m w a s d i s c a r d e d a n d 5 ml of f r e s h e n z y m e m e d i u m w a s a d d e d to g i v e a final c o n c e n t r a t i o n of 0 . 1 % c o l l a g e n a s e a n d 0.2 % h y a l u r o n i d a s e . T h e h e a r t s w e r e t h e n p e r f u s e d for 20 m i n in a closed r e c i r c u l a t i n g s y s t e m u n d e r a h y d r o s t a t i c p r e s s u r e of 75 cm H20 g a s s e d w i t h O2:CO ~ (95:5 ~ A f t e r 20 m i n of p e r f u s i o n t h e h e a r t s w e r e t r a n s f e r r e d to a b e a k e r , g e n t l y m i n c e d w i t h scissors a n d s u s p e n d e d in 20 m l of e n z y m e m e d i u m . T h e t i s s u e s u s p e n s i o n w a s t r a n s f e r r e d to an Erlenmeyer flask a n d s h a k e n in a w a t e r b a t h for 15 m i n at 37 ~ C. T h e s u s p e n s i o n w a s t h e n filtered t h r o u g h 4 l a y e r s of s u r g i c a l g a u z e a n d c e n t r i f u g e d for 2 m i n at 50 X g. T h e s u p e r n a t a n t w a s a s p i r a t e d a n d t h e p e l l e t s u s p e n d e d in 2 ml of c a l c i u m - f r e e Krebs' buffer. T h e cells w e r e l a y e r e d o v e r 20 ml of 0.32 M sucrose in Krebs' c a l c i u m - f r e e m e d i u m a n d c e n t r i f u g e d for 2 m i n at 50 • g. T h i s s t e p w a s i n c l u d e d in an a t t e m p t to r e m o v e m o s t of t h e c o l l a g e n a s e a n d h y a l u r o n i d a s e in t h e cell suspension. Y i e l d s of 25-27 % of the m y o c a r d i u m w e r e o b t a i n e d w h e n m y o c a r d i a l cells w e r e c a l c u l a t e d as T C A - p r e c i p i t a b l e dry weight. Observation by phase contrast microscopy indicated that the muscle cells w e r e r e l a t i v e l y p u r e w i t h at l e a s t 5 0 - 6 0 % b e i n g m o r p h o l o g i c a l l y i n t a c t . W h e n t h e cells w e r e b r o u g h t to r o o m t e m p e r a t u r e a b o u t 50 % of t h e i n t a c t cells h a d s p o n t a n e o u s c o n t r a c t i l i t y . T h e i s o l a t e d m y o c a r d i a l cells w e r e v e r y s i m i l a r to t h o s e of Berry et al. (2). No c l a i m was m a d e t h a t t h e m o r p h o l o g i c a l s t u d i e s d i f f e r e n t i a t e m u s c l e a n d c o n n e c t i v e tissue cells. O u r p r i m a r y p u r p o s e w a s to p r e p a r e cell f r a c t i o n s of e n r i c h e d h e a r t m u s c l e cells.
Determination of protein and radioactivity P r o t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d b y t h e m e t h o d of Lowry et al. (14), u s i n g c r y s t a l l i n e b o v i n e s e r u m a l b u m i n as a s t a n d a r d . T h e r a d i o a c t i v i t y of n u c l e a r p r o t e i n s w a s m e a s u r e d in a l i q u i d s c i n t i l l a t i o n c o u n t e r ( N u c l e a r C h i c a g o m o d e l 4868) w i t h an efficiency for {3H} of 25-30 ~ Results
Incorporation of {3H}acetate into nuclear proteins H e a r t s w e r e p e r f u s e d w i t h (3H}acetate for p e r i o d s of 5 to 60 min. T h e r e s u l t s a r e s h o w n in Fig. 1. T h e i n c o r p o r a t i o n of a c e t a t e into n u c l e i a n d
Basic Research in Cardiology, Vol. 71, No. 1 (1976)
30
50
5
Z w
la.i
ag
Z 040
i'
4
a.
~30
U
~
Z
% -20 X
2
v 10
1
•
0
d
~
o
%
0
I
I
I
I
I
5
10
20
30
60
PERFUSION
TIME
(MIN.)
Fig. 1. Incorporation of {3H}acetate into nuclear proteins. Hearts were perfused w i t h {'~H}acetate (1 /~Ci/ml) p e r f u s a t e for 5, 10, 15, 30 and 60 min. F i v e h e a r t s w e r e pooled to isolate nuclei. The specific activities of n u c l e a r p r o t e i n s (0-0) and n u c l e o h i s t o n e s (o-o) w e r e d e t e r m i n e d as d e s c r i b e d in the M e t h o d section. histone fractions rapidly increased during the first 20 rain of perfusion and reached a maximum between 20 and 30 min. It was found that there was a reduction in acetate incorporation into nuclear proteins after 60 rain of perfusion. From these experiments a constant perfusion time of 20 rain was selected to examine the effect of increased concentration of acetate on the incorporation into different nuclear protein fractions.
E f f e c t of {3H}acetate c o n c e n t r a t i o n on i n c o r p o r a t i o n into n u c l e a r p r o t e i n s T h e i n c o r p o r a t i o n of { 3 H } a c e t a t e i n t o 0.14 M NaC1 s o l u b l e p r o t e i n s , histones and nonhistone residual proteins, was investigated by using d i f f e r e n t c o n c e n t r a t i o n s of { 3 H } a c e t a t e f o r 20 m i n . T a b l e 1 s h o w s t h e t o t a l Tab. I. Total radioactivity
in fractionated
nuclear proteins
Fractions
{3H} acetate concentration 1 ]~Ci/ml 2 ~Ci/ml
4/tCi/ml
Histones 14. M NaC1 Residual proteins
21,616 283 1,746
57,872 708 5,194
55,136 876 4,308
Five hearts were perfused for 20 min at each concentration and then frozen in liquid nitrogen. Nuclei were isolated and the protein fractions separated as described under Method. Aliquots (50 i~I) from each fraction were removed for determination of radioactivity (cpm). The values indicate the total activity in each fraction.
Taylor and Liew, Acetylation o] nuclear proteins
31
r a d i o a c t i v i t y of {3H}acetate at d i f f e r e n t c o n c e n t r a t i o n s t h a t was inc o r p o r a t e d into each n u c l e a r p r o t e i n f r a c t i o n . It w a s f o u n d t h a t w h e n t h e c o n c e n t r a t i o n of {3H)acetate in t h e p e r f u s a t e w a s i n c r e a s e d f r o m 2 to 4 ~ C i / m l , t h e r e w a s no significant i n c r e a s e in t h e a m o u n t of a c e t a t e incorporated.
Fractionation of histones H i s t o n e s o b t a i n e d f r o m 20 m i n {SH}acetate l a b e l l e d p e r f u s e d h e a r t s w e r e a n a l y z e d b y p o l y a c r y l a m i d e - u r e a gel e l e c t r o p h o r e s i s (21). F o u r m a j o r f r a c t i o n s w e r e i d e n t i f i e d a n d e x p r e s s e d as counts p e r m i n p e r o p t i c a l d e n s i t y of 615,m as s h o w n in Fig. 2. T h e p e r c e n t d i s t r i b u t i o n of {3H}acetate i n c o r p o r a t e d into t h e h i s t o n e f r a c t i o n s w a s also d e t e r m i n e d . T h e s l o w e s t m o v i n g b a n d fl i n c o r p o r a t e d a n e g l i g i b l e a m o u n t of {~H}acetate w h i l e f r a c t i o n s f~ a n d f2~l w e r e h i g h l y a c e t y l a t e d , a c c o u n t i n g for 75 % of t h e t o t a l h i s t o n e r a d i o a c t i v i t y . T h e f r a c t i o n t h a t c o n t a i n e d h i s t o n e s f,.,h q- f2:~.~ w a s m o d e r a t e l y a c e t y l a t e d w i t h a b o u t 20-25 % of t h e t o t a l a c t i v i t y . In a n a t t e m p t to d e t e r m i n e t h e a c e t y l a t i o n of n u c l e o h i s t o n e s f r o m one cell type, m u s c l e cells w e r e i s o l a t e d f r o m p e r f u s e d h e a r t l a b e l l e d w i t h {SH}acetate for 20 min. T h e n u c l e i w e r e s u b s e q u e n t l y i s o l a t e d f r o m t h e m u s c l e cells. Tab. 2 c o m p a r e s t h e t o t a l r a d i o a c t i v i t y a n d p e r c e n t d i s t r i b u -
Fig. 2. Fractionation of histones. Nucleohistone (30 /~g) isolated from {'~H}acetate labelled perfused hearts was applied to polyacrylamide-urea gels. After electro~ phoresis, the gels were destained electrophoretically and fractions f,, f~, (f.,i, -t- f.2a.2)and f2a, were identified. Each fraction was cut from the gel, solubilized in H.,O.., and then dissolved in 15 ml Aquasol for radioactivity determination.
Basic Research in Cardiology, Vol. 71, No. 1 (1976)
32
Tab. 2. Distribution of {nit} acetate in fractionatcd histories Fraction
~b 9 ~a~ ~1
Normal perfused hearts cpm ~o of total
Myocardial cells cpm
% of total
Simulated perfused hearts cpm ~o of total
11 365 127 147
5 189 53 90
2 56 16 27
10 194 50 98
2 56 20 23
3 55 14 28
30 ~g of heart histones from each condition were analyzed by polyacrylamideurea gel electrophoresis. The gels were stained with Amido Black and destained electrophoretically. The identified histone bands were sliced and their radioactivity determined as described in the Method section. t i o n of each h i s t o n e f r a c t i o n for w h o l e h e a r t s , i s o l a t e d m y o c a r d i a l cells a n d w h o l e h e a r t s p e r f u s e d u n d e r i d e n t i c a l conditions, so as to s i m u l a t e t h e p r o c e d u r e s for s e p a r a t i o n of m u s c l e cells. T h e r e s u l t s f r o m these e x p e r i m e n t s r e v e a l s e v e r a l aspects of h i s t o n e a c e t y l a t i o n . First, u n d e r o u r c o n d i t i o n s for t h e l a b e l l i n g a n d i s o l a t i o n of m y o c a r d i a l cells t h e t o t a l a c t i v i t y for each h i s t o n e f r a c t i o n w a s a p p r o x i m a t e l y r e d u c e d b y 50 ~ w h e n c o m p a r e d to w h o l e h e a r t n u c l e o h i s t o n e s . S e c o n d l y , the p e r c e n t d i s t r i b u t i o n of i n c o r p o r a t e d a c e t y l g r o u p s b e t w e e n t h e m a j o r h i s t o n e f r a c t i o n s i n t h e i s o l a t e d m y o c a r d i a l cell w a s s i m i l a r to the w h o l e h e a r t . T h i r d , if w h o l e h e a r t s a r e p u l s e l a b e l l e d a n d t h e n p e r f u s e d to s i m u l a t e t h e s e p a r a t i o n of m u s c l e cells, t h e i n c o r p o r a t i o n of {3H}acetate into w h o l e h e a r t n u c l e o h i s t o n e s is s i m i l a r to the i s o l a t e d m y o c a r d i a l cells. T h e s e e x p e r i m e n t s i n d i c a t e t h a t t h e a c e t y l a t i o n of n u c l e o h i s t o n e s i n m u s c l e cells is s i m i l a r to t h a t i n t h e i n t a c t m y o c a r d i u m .
E f f e c t of p u r o m y c i n on a c e t a t e i n c o r p o r a t i o n T h e effect of p u r o m y c i n o n {3H}acetate i n c o r p o r a t i o n i n t o h e a r t n u c l e o p r o t e i n s is p r e s e n t e d i n Tab. 3. H e a r t s w e r e p e r f u s e d for 10 rain w i t h p u r o m y c i n at b o t h 50 a n d 75/~g/ml p e r f u s a t e , f o l l o w i n g 20 m i n {all}acetate p u l s e l a b e l l i n g . P u r o m y c i n w h i c h is k n o w n to dissociate h e a r t p o l y r i b o s o m e s (17) as w e l l as to block p r o t e i n b i o s y n t h e s i s at t h e t r a n s l a t i o n a l l e v e l (11) failed to i n h i b i t the i n c o r p o r a t i o n of a c e t a t e into n u c l e o p r o t e i n s . T h e e x p e r i m e n t s s u g g e s t t h a t the i n c o r p o r a t i o n of a c e t a t e i n t o n u c l e o p r o t e i n s is i n d e p e n d e n t of p r o t e i n s y n t h e s i s . Tab. 3. Effect of puromycin on the incorporation of {3H}acetate into nuclear proteins Treatment
Total nuclei
Histoncs
Residual proteins
Control Puromycin
10,658 10,857
43,792 44,344
5,671 5,483
Hearts were perlused with puromycin. The control group was given identical conditions except that the hearts were perfused without puromycin. The results are expressed as cpm/mg protein.
Taylor and Liew, Acetylation of nuclear proteins
33
Identification of acetyl groups The presence of radioactive acetyl groups in nuclear proteins was identified by acid hydrolysis and steam distillation as described previously (13, 27). As shown in Tab. 4, over 95% of the {'~H}label was released from the nucleohistone and nonhistone residual proteins by such treatment, indicating that the acetate was mainly incorporated as an acetyl group. Tab. 4. Release of {all}acetate from histories, 0.14 M NaCI solubh~ and r(,sidual proteins
Before hydrolysis After hydrolysis Released ~ Released
0.14 M N ~ C 1
Histon(,s
460 85 375 81
12,840 69 12,771 99
Residual proteins 2,160 63 2,097 97
Hearts were perfused with {SH}acetate (2 /~Ci/ml perfusate) for 20 min. Histones, 0.14 M NaC1 soluble and residual proteins were extracted from the isolated nuclei and used for acid hydrolysis as described in the text. Discussion
A relationship between acetylation of histones and RNA synthesis has been demonstrated in regenerating liver (23), hormone replacement studies (5, 28) and cell culture systems (6, 22). It is postulated that post-synthetic acetylation of nucleohistones increases template activity by weakening the DNA-histone interaction through charge neutralization. Previous reports from this laboratory have demonstrated that aldosterone administration to adrenalectomized rats stimulated the acetylation of heart nucleohistones prior to an increase in RNA polymerase activity and RNA synthesis (12, 13). However, studies that follow in r i v e incorporation of isotope into proteins are usually subject to neural and hormonal variations that may mask some biochemical changes. In addition, the pool size of radioactive precursors may change under different physiological conditions. In order to alleviate some of these problems we have used an isolated perfused heart system to examine the acetylation of nucleoproteins. The incorporation of {3H}acetate into whole nuclei and histones occurs rapidly, reaching a peak between 20-30 min of perfusion. The decline of {all}acetate incorporation into nuclear proteins that occurred after perfusion for 20-30 min could be due to the rapid utilization of the {'~H}acetate in the perfusate. However, these data suggest that the acetylation process is enzymatic and are in good agreement with experiments using isolated nuclei (1) and the acetylation of histones by partially purified nuclear histone-acetyltransferase (15). We have also examined the effect of varying concentrations of {3H}acetate on the incorporation into nucleoproteins and found that 2 #Ci/ml buffer gave near maximal incorporation into the different protein fractions. Increasing the concentration to 4 pCi/ml perfusate only slightly enhanced the amount of {3H/acetate incorporated into the
34
.Basic Research in Cardiology, Vol. 71, No. 1 (1976)
0.14 1V~NaC1 soluble, h i s t o n e s a n d r e s i d u a l p r o t e i n s . A t all c o n c e n t r a t i o n s of (3H}acetate used for t h e i n c o r p o r a t i o n studies, a p p r o x i m a t e l y 90 % of t h e r a d i o a c t i v i t y was f o u n d i n t h e h i s t o n e f r a c t i o n w h i l e o n l y a b o u t 7-8 % of {3H}acetate w a s i n c o r p o r a t e d i n t o n o n - h i s t o n e r e s i d u a l p r o t e i n s . W h e n n u c l e o h i s t o n e s w e r e s e p a r a t e d i n t o f o u r m a j o r classes b y p o l y a c r y l a m i d e u r e a gel e l e c t r o p h o r e s i s t h e specific a c t i v i t i e s of t h e a r g i n i n e - r i c h f~ a n d f2~1 w e r e t h e highest. F r a c t i o n f~b-~-f2a2 w a s m o d e r a t e l y a c e t y l a t e d w h i l e t h e l y s i n e - r i c h f r a c t i o n fl w a s b a r e l y detected. I n a n a t t e m p t to r e d u c e p o s s i b l e c o n t a m i n a t i o n b y n o n - m u s c l e cells, h e a r t cells w e r e s e p a r a t e d f r o m t h e i n t a c t m y o c a r d i u m f r o m w h i c h w h o l e n u c l e i w e r e i s o l a t e d a n d n u c l e a r p r o t e i n s e x t r a c t e d . T h e i n c o r p o r a t i o n of a c e t a t e i n t o 0.14 M NaC1 soluble, h i s t o n e s a n d r e s i d u a l p r o t e i n s w a s cons i d e r a b l y r e d u c e d w h e n c o m p a r e d to w h o l e h e a r t s (Tab. 2). H o w e v e r , w h e n h e a r t s w e r e l a b e l l e d for 20 m i n a n d t h e n p e r f u s e d u n d e r c o n d i t i o n s t h a t s i m u l a t e t h e i s o l a t i o n of m y o c a r d i a l cells t h e a m o u n t of {SH}acetate i n c o r p o r a t e d into t h e 0.14 M N a C l - s o l u b l e , h i s t o n e s a n d r e s i d u a l p r o t e i n s was s i m i l a r to t h e i s o l a t e d m y o c a r d i a l cells. T h e s e e x p e r i m e n t s i n d i c a t e t h a t t h e site of a c e t y l a t i o n is e q u a l l y a c t i v e i n t h e e n r i c h e d f r a c t i o n of t h e c o n t r a c t i n g m u s c l e cells. It is c e r t a i n l y possible t h a t a c e t y l a t i o n of n u c l e a r p r o t e i n s also occurs in the o t h e r cell types. I n g e n e r a l , t h e p o s t s y n t h e t i c c o v a l e n t m o d i f i c a t i o n of n u c l e a r p r o t e i n s has a close t e m p o r a l a s s o c i a t i o n w i t h a n i n c r e a s e i n R N A s y n t h e s i s i n a n u m b e r of biological s y s t e m s (13, 23, 28). O u r e x p e r i m e n t s h a v e a t t e m p t e d to d e m o n s t r a t e t h e possible u s e f u l n e s s of t h e i s o l a t e d p e r f u s e d r a t h e a r t to s t u d y c o v a l e n t m o d i f i c a t i o n s of n u c l e a r p r o t e i n s . T h i s s t u d y m a y also p r o v i d e some b i o c h e m i c a l basis for s t u d y i n g changes i n r e s p o n s e d u e to the p h y s i o l o g i c a l s t i m u l i ; e.g., w o r k - l o a d , b i o c h e m i c a l e n v i r o n m e n t s or pharmacological interventions. Acknowledgements
We are very grateful to Professor H. E. Morgan of the Milton S. Hershey Medical School, Penn. for introducing the isolated heart perfusion method and to Professor A. G. Gornall for his generous support as well as advice throughout the studies. P. B. Taylor is a postdoctoral fellow of the Ontario Heart F o u n d a tion. The technical assistance of Mrs. L. Suigu is appreciated. Zusammenfassung
Bei weiblichen Ratten wurde das Herz retrograd perfundiert mit einem hydrostatischen Druck von 55 cm H20. Die perfundierten Herzen zeigten einen KoronardurchfluB yon 8-9 ml/min u n d eine Herzfrequenz von 220-240 Schl~gen/ min. Mit rezirkulierendem {3H}-Acetat (2 ,uCi/ml Perfusat) w u r d e n innerhalb 20 rain die Kernproteine markiert. Die Nucleoproteine w u r d e n in drei gro~en Klassen isoliert: 1. 0,14 M NaCl-15sliche Proteine des Kernplasmas, 2. Nucleohistone u n d 3. nichthiston-~hnliche Restproteine. Nahezu 88-90 ~/0 des eingebauten {~i.i}-Acetate erschien in der Nucleohiston-Fraktion. Polyacrylamid-HarnstoffGelelektrophorese t r e n n t e die Histone auf in eine nichtacetylierte Fraktion fl u n d in stark acetylierte F r a k t i o n e n f~ u n d f,2al mit 75 % der gesamten Histonradioaktivit~t. Die Fraktion feb ~- fea~ war schw~cher acetyliert mit 20-25 % der Radioaktivit~t. Die aus Myokardzellen isolierten Nucleohistone besal3en die gleiche prozentuale Verteilung des {3H)-Acetats in den Histonfraktionen wie die Pr~parationen aus ganzen Herzen. Saure Hydrolyse u n d Wasserdampf-
Taylor and Liew, Acetylation of nuclear proteins
35
d e s t i l l a t i o n s e t z t e n m e h r a l s 95 % d e r A c e t y l g r u p p e n a u s d e n b e i d c n H a u p t f r a k t i o n e n d e r N u c l e o p r o t e i n e f r e i . D i e s e E r g e b n i s s e w e i s e n d a r a u f h i n , daft d a s isolierte, perfundierte Herz als Modell dienen kann zur Untersuchung yon kovalenten Modifizierung von Nucleoproteinen unter definierten physiologischen und biochemischen Bedingungen.
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