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The binding of arsenazo III to cell components
317
Bmch~mlca et Bmphyswa Acta, 6 2 9 ( 1 9 8 0 ) 3 1 7 - - 3 2 7 © E l s e v m r / N o r t h - H o l l a n d Biomedical Press
BBA 29231
THE BINDING OF A R S E N A Z O III TO CELL COMPONENTS
T R O Y J B E E L E R *, A N G E L O S C H I B E C I * a n d A. M A R T O N O S I *
E.A. Do~sy Department of Biochemistry, St Louis University School of Medicine, St. Louts, MO 63104 (U.S A ) (Received September 14th, 1979)
Key words Arsenazo III binding, Ca2+ binding, Parvalbumin, Calcmm assay, (Rabbit skeletal muscle)
Summary The Ca 2÷ indicator, arsenazo III, binds to subcellular fractions of rabbit skeletal muscle with sufficient affinity that in living muscle containing 1--2 mM arsenazo III, the estimated free arsenazo III concentration is only 50--200 ~M; 80--90% of the b o u n d arsenazo III is associated with soluble proteins. The binding of arsenazo III to soluble proteins decreases the optical response of the dye to Ca2+; this is due to a decrease in the affinity of the protein-bound dye for Ca 2*. Approximately half of the b o u n d arsenazo III is released from the particulate fraction and soluble proteins upon addition of 5 mM Ca 2÷, suggesting that the C a ~ s e n a z o complex has lower affinity for the protein binding sites than the free dye. The Ca 2÷ binding to the soluble protein fraction of rabbit skeletal muscle is attributable largely to its parvalbumin content.
Introduction The widespread use of arsenazo III for measurement of cytoplasmic free [Ca 2÷] yielded valuable qualitative information a b o u t the regulatory role of Ca 2÷ in muscle [1], squid axon [2,3], aplysia neuron [4], and various subcellular systems [3,5,6]. The quantitative evaluation of the changes in cytoplasmic Ca 2÷ during physiological activity requires information about: (a) the interaction of arsenazo III with cell constituents, and (b) the influence of these interactions upon the response of the dye to free Ca 2÷. * Present address D e p a r t m e n t of Blochemlstry, State Umverstty of New York, Upstate Medmal Center, Syracuse, NY 13210, U.S.A.
318 The studms described in this report indicate massive bmdmg of arsenazo III to the subcellular organelles and soluble proteins of rabbit skeletal muscle with a decrease in the optical response of the dye to free calcium. For analytical purposes the effect can be minimized by increasing the arsenazo III concentration to levels where much of the dye remains free. Materials and Methods For the isolation of cell fractions, rabbit skeletal muscle was homogenized m 4 vols. 0.1 M KC1/10 mM imidazole (pH 7.0) for 2 min at 2°C. The crude myofibrillar fraction obtained by centnfugatlon at 775 × g for 15 mm was resuspended in 20 vols. homogemzing medium and washed three times by centrifugation at 775 × g for 15 min, followed by resuspenslon in the same medium. The crude mitochondrial fraction was obtained by centrifugatlon of the post-myofibrillar supernatant at 8000 × g for 20 min. The m i t o c h o n d n a were washed three times m 20 vols. homogenizing m e d m m by repeated centnfugatlon at 8000 × g for 20 min. For the isolation of microsomes the supernatant obtained after the removal of mitochondria was centrifuged at 36 000 × g for 1 h. The supernatant containing the soluble proteins was saved. The sediment was washed three times with the homogenizing medium by centnfugatlon at 78 000 X g for 1 h to yield the washed mmrosomal fraction. All particulate fractions were resuspended by gentle homogenization in 0.1 M KC1/10 mM imidazole (pH 7.0) to final concentrations of 3--30 mg protein/ml and were used for experiments within 1 day. The various fractions are expected to contain cross-contaminations and the term 'myofibrillar', ' m i t o c h o n d n a l ' , or 'mmrosomal' merely denotes the major component. The supernatant obtained after removal of microsomes was centrifuged again at 78 000 × g for 1 h. The essentially lipid-free soluble fraction was dialyzed against repeated changes of 60 vols. 0.1 M KC1/10 mM imidazole and 0.01% NaN3 for 24 h and was used immediately for experiments. Protein was determined according to Lowry et al. [7] with bovine serum albumin as standard. Arsenazo III, obtained from Aldnch Chemmal Co. (Milwaukee, WI) was purified before use according to Kendrick [8]. The binding of arsenazo III and 4SCa to particulate fractions was measured by centrifuge transport and to the soluble protems by equflibrmm dialysis under conditions described m the legends. The concentratmn of arsenazo III was determined in solutions containing 10 mM CaC12 by measuring the optical density at 568 nm using a molar extmction coefficmnt of 33 000. The absorption spectrum of arsenazo III was analyzed m an Aminco DW-2 spectrophotometer in the wavelength range of 350--730 nm, using control samples of identical composition w i t h o u t arsenazo III as reference. The Ca 2+ c o n t e n t of the various fractions was analyzed by atomic absorptmn s p e c t r o p h o t o m e t r y after precipitation o f the proteins with 2% trichloroacetm acid and 1% LaC13. Results
The binding o f arsenazo III to subcellular fractions o f rabb~t skeletal muscle The binding of arsenazo III to the varmus cell fractions was measured over
319 a range of free arsenazo III concentrations of 2--200 ~M, at Ca 2+ concentrations from less than 10-SM to 5 mM (Fig. 1). The binding of arsenazo III involves heterogeneous sets of binding sites w i t h o u t clear saturation up to 200 pM free arsenazo III concentration. The soluble proteins bound larger amounts of arsenazo III with greater affinity than the mitochondria, microsomes, or myofibrils. At an arsenazo III concentration o f 51 pM, 86.3% of the dye was bound to the soluble proteins (14.75 mg/ml). Even at total arsenazo III concentration as high as 609 ~M, 66% of the dye was protein-bound. Calcium in millimolar concentrations inhibited the binding o f arsenazo III, indicating that the Ca-arsenazo III complex has less affinity for the binding sites than the Mg2÷ and K ÷ complexes or the free acid. The data of Fig. 1 were used to estimate the relative concentrations of free and bound arsenazo III expected to occur in muscle cells at varying total arsenazo III concentrations (Tables I and II). In one series of experiments the Ca 2÷ concentration of the medium was maintained below 10 -s M with 1 mM EGTA in the presence of 0.1 M KC1 and 2 mM MgC12 (Table I). At total arsenazo III concentrations of 0.8 and 2.3 mM, respectively, an estimated
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FREE ARSENAZ0 nl, pM Fig. 1. Arsenazo III binding to subceHular fractions of rabbit skeletal muscle. T h e binding of arsenazo Ill to crude myofibrils (A), mitochondria (B), and microsomes (C) was measured b y centrifuge transport. T h e decrease in the absorbance of the supernatant after removal of the particulate elements by centrifugation was assayed at 568 n m in the presence of 10 m M CaCl 2. T h e dye binding to soluble proteins was analyzed b y equilibrium dialysis (D). T h e m e d i u m contained 0.1 M K C I 10 m M imidazole ( p H 7.0), 2 m M MgCl2, 10--400 /~M arsenazo III and 1 m M E G T A (e) or 5 m M CaCl 2 (o). T o samples labeled with open circles (o 7 neither E G T A nor CaCl 2 was added; the free C a 2+ concentration of these samples was 20.8 ~ M (A), 1 5 5 ~tM (C), a n d 2.06 juM (D), respectively. Protein concentrations were: 31 m g / m l (A); 3.3 m g / m l (B); 19 m g / m l (C); 14.75 m g / m l (D). Temperature: 4°C. In equilibrium dialysis experiments (D), 2 m l of soluble protein fraction were placed in dialysis tubing and dialyzed against 2 m l buffer (outside solution) for 4 8 h at 4°C. T h e binding of arsenazo III to proteins was calculated from the absorbance difference at 568 n m between the outside solutions and the inside solutions or protein-free control systems.
5 Total
199.7
108.6 2.1 12.2 76.8
1 2 3 4
Myofibrils Mitochondria Microsomes Soluble p r o t e i n s
Protein content (g/kg wet wt.)
Fraction
II. W I T H 5 m M CaCI 2
5 Total
199.7
108.6 2.1 12.2 76.8
1 2 3 4
My o fibrils Mitochondria Mierosomes Soluble p r o t e i n s
Protein content (gfkg wet wt.)
Fraction
I. W I T H 1 m M E G T A
total amount of protein.
20
20 20 20 20
Free (pM)
20
20 20 20 20
Free (~M)
The binding of arsenazo III was measured axe A r s e n a z o l l I c o n c e n t r a t i o n s . T h e f r e e b o u n d arsenazo III (/amol/kg muscle) was fraction per kg muscle. The total of bound
FI~ACTIONS OF RABBIT SKELETAL MUSCLE
1.62
0.13 -0.20 3.90
/zmol/g Protein
Bound
3.9
0.4 1.3 0.9 9.4
/amol/g protein
Bound
324.15
14.1 -2.4 307.6
/amol/kg muscle
781.1
45.6 2.7 10.9 721.8
plalol/kg muscle
94.21
4.1 <0.1 0.7 89.4
% of total (344.1 /aM)
97.5
5.7 0.3 1.4 90.1
% of total (801.1 /aM)
50
50 50 50 50
Free (pM)
50
50 50 50 50
Free (/aM)
2.48
0.27 -0.50 5.9
/~mol/g protein
Bound
5.5
0.75 4.0 1.5 13
Dmol/g Protein
Bound
469.2
29.3 -6.1 460.8
pmol/kg muscle
1106.3
81.5 8.3 18.3 998.3
/amol/kg muscle
90.83
5.4 <0.1 1.1 84.4
% of total (546.2 pM)
95.7
7.0 0.7 1.6 86.3
% of total (1156.3 /aM)
200
200 200 200 200
Free (pM)
200
200 200 200 200
Free (/aM)
5.43
0.98 -1.40 12.50
/~mol/g protein
Bound
10.89
2.02 8.6 2.7 24.8
/amol/g protein
Bound
1083.4
106.4 -17.1 960
~mol]kg muscle
2175.2
219.4 18.1 33.1 1904.6
/amol/kg muscle
84.4
8.3 <0.1 1.3 74.8
% of total (1283.5 ~M)
91.5
9.2 0.8 1.4 80.2
% of total (2375.2 pM)
in t h e p r e s e n c e o f 1 m M E G T A ( T a b l e I) o r 5 m M CaC12 ( T a b l e I I ) as d e s c r i b e d i n t h e l e g e n d t o Fig. 1. I n all cases, v a l u e s a r s e n a z o I I I c o n c e n t r a t i o n s ( 2 0 , 50, 1 0 0 a n d 2 0 0 / a M ) w e r e o b t a i n e d b y i n t e r p o l a t i o n f r o m Fig. 1. T h e c o n c e n t r a t i o n o f c a l c u l a t e d f o r e a c h f r a c t i o n as t h e p r o d u c t o f t h e a m o u n t o f a r s e n a z o ( / a m o l / g p r o t e i n ) a n d t h e a m o u n t o f p r o t e i n i n t h e arsenazo (/amol/g p r o t e i n ) was o b t a i n e d b y dividing the total c o n c e n t r a t i o n of b o u n d arsenazo III per k g muscle with the
THE BINDING OF ARSENAZO III TO SUBCELLULAR
T A B L E S I A N D II
t~
321 97.5% and 91.5% of the dye was b o u n d to the various cell constituents, primarily soluble proteins. In the presence of 5 mM CaC12, at a total d y e concentration as high as 1.2 mM (Table II), still more than 80% o f the dye was estimated to the bound. As microinjection of arsenazo III into muscle and other cells usually results in cellular dye concentrations less than 1 mM, the question arises whether the binding of the d y e to proteins interferes with its optical response to Ca 2÷.
The effect o f the soluble protein fraction o f rabbit skeletal muscle upon the absorption spectrum o f arsenazo III The absorption spectrum of arsenazo III was measured at varying total Ca 2+ concentrations with and without soluble proteins (Fig. 2). With 10 ~M arsenazo III at a soluble protein concentration of 14.7 mg/ml, the optical change caused by the addition of 12--446 pM Ca 2÷ is significantly reduced (Fig. 2B), compared with the absorbance change observed under similar conditions in the absence of soluble proteins (Fig. 2A). The effect of soluble proteins upon the Ca2+-induced absorbance change is less pronounced at 100 #M total arsenazo IIT concentration (Fig. 2C and D). Since at 5 mM CaC12 concentration a b o u t half of the total arsenazo III is still bound to soluble proteins,
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Fig. 2. T h e e f f e c t o f soluble p r o t e i n s o n t h e o p t i c a l r e s p o n s e o f a r s e n a z o I I I t o Ca 2+. T h e axsenazo I I I s p e c t r a w e r e m e a s u r e d a t 4 ° C in a n A m i n c o DW-2 s p e c t r o p h o t o m e t e r in a m e d i u m o f 0.1 M K C I / I O m M i m i d a z o l e p H 7 . 0 / 2 m M MgC12 in t h e a b s e n c e ( A and C) o r p r e s e n c e (B a n d D ) o f 1 4 . 7 5 m g soluble m u s c l e p r o t e i n s p e r m l a t a z s e n a z o I I I c o n c e n t r a t i o n s o f 10 /~M (A a n d B) or 1 0 0 / ~ M (C a n d D), r e s p e c t i v e l y . Ca 2+ was a d d e d t o final c o n c e n t r a t i o n s i n d i c a t e d in the figure. T h e s p e c t r a w e r e m e a s u r e d a g a i n s t b u f f e r (A a n d C) or p r o t e i n s o l u t i o n s (B a n d D) c o n t a i n i n g t h e s a m e a m o u n t of Ca 2+ as t h e c o m p l e t e s a m p l e s . T h e s a m p l e s c o n t a i n i n g 10 /~M a r s e n a z o I I I (A a n d B) w e r e a s s a y e d in c u v e t t e s o f I e m p a t h l e n g t h while t h o s e c o n t a i n i n g 1 0 0 / J M a r s e n a z o I I I in c u v e t t e s of 1 m m p a t h l e n g t h .
322
some o f the protein b o u n d dye apparently responds to Ca 2÷ with a spectrum change similar to t h a t of the free dye. T h e diminished response of arsenazo III to Ca 2÷ in the presence of soluble proteins is n o t explained by Ca 2÷ binding to proteins, since t he high affinity Ca 2÷ binding sites of t he soluble fraction are saturated at a free Ca 2÷ concentration o f less than 10 ~M (Fig. 3) and no significant additional Ca 2÷ binding was observed between 11 and 213 /IM free Ca 2÷ concentrations. Even at 0.5 mM free Ca 2÷ less than 5% of the total Ca 2÷ was b o u n d to proteins. In experiments where soluble proteins were preequilibrated at free Ca 2÷ c o n c e n t r a t i o n s o f 25, 50, and 100 pM by dialysis against repeated changes of solutions adjusted t o these Ca 2÷ concentrations, additions of arsenazo III still p r o d u c e d a reduced Ca 2÷ response com pa r ed with protein-free buffer solutions o f the same free Ca 2÷ levels. Under these conditions the total Ca 2÷ c o n t e n t of protein solutions was greater than the total Ca 2÷ c o n t e n t of the protein-free c o n t ro l samples. Ca 2÷ binding to soluble proteins contributes to the reduced arsenazo III response only u n d e r conditions where the high affinity Ca 2÷ binding sites of t h e proteins are n o t saturated with Ca 2÷. For example, after com pl et e removal o f b o u n d Ca 2÷ f r o m soluble proteins by t r e a t m e n t with skeletal muscle microsomes, in the presence o f ATP and Mg, addition o f 15--20 pM Ca 2÷ caused little change in arsenazo III absorption (Fig. 4) because most of the added Ca 2÷ is b o u n d to proteins. F u r t h e r increase in Ca 2÷ c o n c e n t r a t i o n was reflected in an increase in the difference absorbance o f arsenazo III, but the change was m u c h less than th a t observed in a protein-free buffer solution or with soluble proteins saturated with a b o u t 40--60 #M Ca 2÷. This difference is due t o the binding o f Ca 2÷ and arsenazo III t o the soluble proteins.
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BOUND Ca, umoles/g PROTEIN F i g . 3. C a l c i u m b i n d i n g t o r a b b i t s k e l e t a l m u s c l e s u b f r a c t i o n s . M y o f i b r i l l a r , m i t o c h o n d r i a l , m i c r o s o m a l , a n d s o l u b l e p r o t e i n f r a c t i o n s w e r e t a k e n u p in 0.1 M K C L 1 0 m M i m i d a z o l e (pI-I 7.0), 2 m M MgC12 to f i n a l p r o t e i n c o n c e n t r a t i o n s o f 1 8 . 0 6 g/l, 4 . 5 g/l, 1 8 . 3 g/l a n d 1 4 . 7 5 g/l, r e s p e c t i v e l y . T o 2-ml a l i q u o t s o f t h e s e s o l u t i o n s w a s a d d e d 4 5 C a t o f i n a l c o n c e n t r a t i o n s o f 0 - - 5 0 0 ~M (0.1 ~ C i / m l ) . A f t e r e q u i l i b r a tion for 2 h at 4°C, the myofibrils, mitochondria, and microsomes were removed by centrifugation at 1 7 0 0 × g, 8 0 0 0 X g, a n d 7 8 0 0 0 X g r e s p e c t i v e l y . A l i q u o t s o f t h e s u p e r n a t a n t s w e r e c o u n t e d t o o b t a i n t h e c o n c e n t r a t i o n o f f r e e 4 5 C a 2 + . T h e a m o u n t o f Ca 2+ b o u n d t o t h e p a r t i c u l a t e e l e m e n t s w a s c a l c u l a t e d f r o m the differenee b e t w e e n total 45Ca2+ a n d free 45Ca2+ c o n c e n t r a t i o n s . Calcium binding to the soluble p r o t e i n f r a c t i o n s w a s m e a s u r e d b y e q u i l i b r i u m d i a l y s i s a t 4 ° C . T h e i n i t i a l Ca 2+ c o n c e n t r a t i o n o f t h e fract i o n s w a s : 7 0 . 9 ~M ( m y o f i b r i l s ) , 3 0 . 8 # M ( m i t o c h o n d x i a ) , 1 7 6 #M ( m i c r o s o m e s ) a n d 21 /JM ( s o l u b l e p r o t e i n f r a c t i o n ) . S y m b o l s : o m y o f i b r i l s ; &, m i t o c h o n d r i a ; D m i e r o s o m e s ; m s o l u b l e p r o t e i n s .
323
.7 .6
1
0
200
300
ADDEDCa, pM Fig, 4. C a l c i u m t i t r a t i o n of t h e soluble p r o t e i n f r a c t i o n in t h e p r e s e n c e o f a r s e n a z o I I I . T h e soluble p r o t e i n f r a c t i o n c o n t a i n e d 1 1 . 5 gfl P r o t e i n along w i t h 0.1 M K C I / I O m M i m i d a z o l e , p H 7 . 0 / 5 m M K 2 o x a l a t e / 5 m M MgC12, 80 stM a r s e n a z o I I I a n d 0 . 2 5 g/! o f r a b b i t m u s c l e m i c r o s o m e . A d d i t i o n o f 1 m M A T P a t r o o m t e m p e r a t u r e l o w e r e d t h e free Ca 2+ c o n c e n t r a t i o n b e l o w 1 ~tM. T h e m i c r o s o m e s w e r e separ a t e d f r o m t h e soluble p r o t e i n s a t 5°C b y c e n t r i f u g a t i o n f o r 30 m i n a t 9 0 0 0 0 X g. T h e d i f f e r e n c e a b s o r b a n c e ( 6 6 0 - - 6 8 5 rim) of t h e s u p e r n a t a n t was m e a s t t r e d at a d d e d CaCI 2 c o n c e n t r a t i o n s o f 0 . 1 - - 3 0 0 / I M a t 4 ° C ( e ) . I n o t h e r s a m p l e s , e i t h e r t h e s a m e p r o c e d u r e was r e p e a t e d b u t w i t h 1 DM A 2 3 1 8 7 a d d e d t o i n h i b i t t h e r e m o v a l of Ca (o), o r t h e soluble P r o t e i n s w e r e o m i t t e d ( s ) .
The binding of C a 2+ to subcellular fractions The m a x i m u m a m o u n t of Ca 2+ bound to the high affinity sites of the soluble protein fraction was 1.3 pmol/g protein (Fig. 3 and Table III), which corresponds to about 99 gmol bound Ca per kg wet muscle weight (Table III). These sites are expected to be saturated in activated muscle with free cytoplasmic Ca 2+ concentrations in the vicinity of 10 -s M. Similar Ca 2+ titrations on the myofibrillar, mitochondrial and microsomal fractions gave 3.2, 4.2 and 5.2 gmol Ca per g protein for the high affinity sites, which corresponds to 367.2, 8.8 and 63.4 pmol Ca per kg wet muscle weight for the three fractions respectively (Table III). All measurements were performed in media containing 0.1 M KC1 and 2 mM MgC12 to minimize contribution by nonspecific cation binding sites. These observations imply that the total a m o u n t of Ca 2+ bound to all constituents in activated muscle may be close to 0.5 mM, and of this approx. one-fifth is bound to the soluble proteins. Even if the mitochondrial and microsomal binding sites are not equilibrated with Ca 2÷ during single spikes of activity (due to internal localization) 0.46 mM bound Ca/kg muscle remains associated with the myofibrils and soluble proteins. Characterization of the Ca2+-binding component of the soluble pro rein fraction Polyacrylamide gel electrophoresis of the soluble protein fraction at p H 8 . 3 in the absence of sodium dodecyl sulfate followed by equilibration of the gel with 4SCa2÷ and autoradiography permitted the identification of the Ca 2÷binding components.
324
TABLE III BINDING OF CALCIUM TO RABBIT SKELETAL MUSCLE SUBFRACTIONS T h e b i n d i n g o f Ca 2+ to r a b b i t s k e l e t a l m u s c l e s u b f r a c t i o n s w a s m e a s u r e d as d e s c r i b e d in t h e l e g e n d o f Fig. 3. T h e n u m b e r o f h i g h a f f i n i t y s i t e s w a s d e t e r m i n e d in e a c h s u b f r a c t i o n b y e x t r a p o l a t i o n o f t h e s t e e p l i n e a r p o r t i o n o f t h e S c a t c h a r d p l o t s g i v e n in F i g . 3. T h e c o n c e n t r a t i o n o f h i g h a f f i n i t y Ca 2+ b i n d i n g s i t e s p e r k g m u s c l e w a s c a l c u l a t e d as t h e p r o d u c t o f t h e n u m b e r o f s i t e s p e r g p r o t e i n a n d t h e p r o t e i n c o n t e n t of each fraction per kg wet weight. g protein/kg wet weight
Myofibrils * Mitochondria Microsomes Soluble protein
108 2.1 12.2 76.8
Total
199.7
Max. number o f sites A (~umol/g) 3.4 4.2 ~ 5.2 1.3 2 . 7 0 **
Ca b o u n d t o sites A (pmol]kg muscle) 367.2 8.8 63.4 99.8 539.20
* Likely to include some mitochondria and microsomes. ** 5 3 9 . 2 / 1 9 9 . 7
The major Ca2+-binding protein of the soluble fraction of muscle homogenate migrated to the same position as parvalbumin (Fig. 5), and Ca2+ or EGTA influenced the mobilities of authentic parvalbumin and of the Ca2+binding component of the soluble protein fraction to a similar extent. The electrophoretic mobility of troponin C was much faster than that of parvalbumin due to its greater negative charge. Troponin C and the Ca2+-binding protein of sarcoplasmic reticulum had greater electrophoretic mobility in the presence of 0.1 mM Ca2+ than in 1 mM EGTA. In contrast, the mobilities of the parvalbumin and of the Ca2+-binding component of the supernatant fraction ('myogen') were reduced by Ca 2+. On the basis of these observations it is plausible to assume that the Ca2+binding protein detected in the soluble fraction of muscle homogenate is parvalbumin.
Fig. 5. Identification of the Ca 2+ binding proteins of rabbit skeletal muscle on polyacrylamide gels. Electrophoresis was performed on quadruplicate polyacrylamide gel slabs ( 1 0 % acrylamide/0.27% N , N pmethylene-bisacrylamide in 0.375 M Tris-HCl buffer, p H 8.9). T h e electrophoresis buffer contained 25 m M Tris and 1 9 2 m M glycine, p H 8.3, with additions as indicated. For t w o sets of gels (labeled 'Ca') the samples were applied in 10 m M Tris-HCl buffer p H 7.0, containing 0.1 m M CaCl 2 and 1 0 % glycerol. O n e set of 'Ca' samples was stained with Coomassie blue (Fig. 5A), the other was equilibrated with 45C'a to identify the C a 2+ binding proteins as described below (Fig. 5B). For t w o sets of gels (labeled 'EGTA') the samples were applied in 10 m M Tris-HCl, p H 7.0/1 m M E G T A / 1 0 % glycerol. During electrophoresis the upper electrophoresis c h a m b e r also contained 1 m M E G T A . O n e set of E G T A samples was stained with Coomassie blue (Fig. 5 A ) the other was equilibrated with 4 S C a as described below (Fig. 5B). Equilibration with 45Ca. After electrophoresis the gels were rinsed with electrophoresis buffer and incubated for 2 h with constant shaking at z o o m temperature in electrophoresis buffer containing 1 ~ M 45CAC12 (1.47 mCi//~mol) and 2 % glycerol. In the case of E G T A gels the equilibration solution was changed to fresh solution after 1 h to reduce E G T A content. After brief rinsing in electrophoresis buffer containing 2 % glycerol, followed by water, the wet gels were transferred to glass plates, covered with Saran w r a p and exposed to K o d a k X R - 5 film for 12--18 h. Samples: Troponin C (25/~g); Ca binding protein ('C protein') of rabbit sascoplasmic reticulum (400 ~g), rabbit parvalbumin (50 ~g); rabbit skeletal muscle 78 0 0 0 X g supernatant ('myogen', 720/~g).
325
326
Discussion The following main observations were made: (1) The particulate and soluble fractions of rabbit skeletal muscle bind arsenazo III in unexpectedly large amounts, with the result that in muscle cells even at millimolar arsenazo III concentrations an estimated 80--90% of the dye is bound to cell constituents. Most of the bound arsenazo III is associated with soluble proteins. (2) At 10--20 #M arsenazo III concentrations (Fig. 2B) the absorbance response of the dye to Ca is drastically reduced by the soluble fraction of rabbit skeletal muscle. This effect is attributable in part to a decrease in free arsenazo III concentration caused by the binding of the dye to proteins. The estimated free arsenazo III concentration under these conditions is about 1 pM, which is below the dissociation constant of arsenazo III for Ca 2÷ [9]. In these systems the arsenazo III bound to the high affinity binding sites of proteins apparently forms Ca 2÷ complexes with smaller affinity than the free dye. As the arsenazo III concentration is increased to 100 #M (Fig. 2D) the effect of soluble proteins on the Ca 2÷ response of the dye is diminished. Under these conditions the free dye concentration is about 27 pM, which is close to the dissociation constant of Ca-arsenazo III complex [9]. Since 73% of the dye is still bound to proteins it is likely t h a t some of the arsenazo III bound to low affinity binding sites responds with absorbance change to Ca 2÷. In living cells where the concentration of proteins is higher than in the in vitro test system a comparable Ca response would require total arsenazo III concentration of about 1 mM (Tables I and II). It is clear t h a t quantitative evaluation of free ionized Ca 2÷ concentration in living muscle, based on analysis of arsenazo III spectra, must take into account the actual concentration of free arsenazo III in the cell, and a simple relationship between spectral change and free Ca 2÷ concentration is expected only with arsenazo III concentrations of 1 mM or greater. Baylor et al. [10] observed in intact frog muscle a dichroic c o m p o n e n t in the arsenazo III signal during a twitch, indicating a change in alignment of some of the dye molecules with respect to the fiber axis. The contribution of the dichroic c o m p o n e n t to the total absorption diminished as the arsenazo III concentration within the fiber was increased. The dichroic c o m p o n e n t of absorption presumably reflects the binding of the dye to the myofibrils or to some other oriented structure. According to our observations this is only a fraction of the total a m o u n t of bound dye within the cell. As the dye concentration is increased the dichroic contribution is expected to be diluted by increasing free dye concentrations, and by increase in the binding of the dye to soluble proteins (Fig. 1). (3) Ca 2÷ binding to the soluble protein fraction of skeletal muscle was earlier observed by Briggs and Fleischman [11]. The estimated concentration of the corresponding Ca 2÷ binding sites in rabbit skeletal muscle was 2 × 10 -4 M with a dissociation constant of 6.67 pM. The a m o u n t of parvalbumin in rabbit skeletal muscle is 0.6--1.1 g/kg wet weight, capable of binding 100--182 pmol Ca 2÷ per kg muscle with a dissociation constant close to 1 ~M [12]. The Ca 2÷
327 bmding capacity of parvalbumin in rabbit skeletal muscle is comparable to the Ca :÷ binding observed in the soluble fraction. Electrophoretic studies also support the conclusion that the principal Ca 2÷ binding component of the soluble protein fraction is parvalbumin. The calmodulin content of rabbit skeletal muscle is less than 30 mg/kg wet weight or about 1.76 ~mol/kg [13,14] and contributes little to the observed Ca 2÷ binding. Troponin C and myosin light chains are associated with the myofibrils [15] and do not participate in the Ca 2÷ binding of the soluble fraction. The total Ca 2÷ binding capacity of the high affinity binding sites in the various cell fractions is about 0.54 mmol/kg muscle. The Ca 2÷ bound to F actm is apparently nonexchangeable [16] and the accessibility of mitochondrial and mlcrosomal sites to cytoplasmic Ca 2÷ is uncertain. Discounting these contributions the saturation of the remaining cytosolic and myofibnllar Ca 2÷ bindmg sites requires the release of about 0.35 mmol Ca/kg muscle during each contraction cycle. Acknowledgements This work was supported by research grants AM 18117 and AM 26832 from NIH, PCM 7705393 from the National Science Foundation and a grant in aid from the Muscular Dystrophy Association. T.J.B. was the recipient of a postdoctoral fellowship from the Muscular Dystrophy Association. The collaboration of Miss Debbie Hermann is gratefully acknowledged. References 1 Mfledl, R , P~trker, I a n d S c h a l o w , G. ( 1 9 7 7 ) P r o c R S o c L o n d o n B 1 9 8 , 2 0 1 - - 2 1 0 2 D1Polo, R , R e q u e n a , J., B n n l e y , F . J . , M u l h n s , L . S . , S c a r p a , A . a n d T l f f e r t , T ( 1 9 7 6 ) J . G e n . P h y s i o l . 67,433---467 3 S c a r p a , A., B r i n l e y , F . J . , T l f f e r t , T a n d D u b y a k , G . R . ( 1 9 7 8 ) A n n u N.Y A c a d Scl 3 0 7 , 8 6 - - 1 1 2 4 Thomas, M.V and Gorman, A.C.F. (1977) Science 196, 531--533 5 W e i s s m a n , G., Collins, T , Evers, A . a n d D u n h a m , P ( 1 9 7 6 ) P r o c . N a t l . A c a d . Sci U . S . A . 7 3 , 5 1 0 - 514 6 R u s s e l l , J . T , Beeler, T. a n d M a r t o n o s l , A . ( 1 9 7 9 ) J . Biol. C h e m . 2 5 4 , 2 0 4 0 - - 2 0 4 6 7 L o w r y , O H . , R o s e b r o u g h , N . J , F a r r , A . L . a n d R a n d a l l , R J ( 1 9 5 1 ) J Biol. C h e m 1 9 3 , 2 6 5 - - 2 7 5 8 Kendrlck, N.C. (1976) Anal. Bloehem. 76, 487--501 9 Russell, J.T. and Maxtonosi, A. (1978) Blochlm. Blophys. Aeta 544,418--429 1 0 B a y l o r , S.M., C h a n d l e r , W . K a n d M a r s h a l l , M.W. ( 1 9 7 9 ) B l o p h y s . J . 2 5 , 1 4 1 a I I B n g g s , F . N . a n d F l e l s h m a n , M. ( 1 9 6 5 ) J G e n . P h y s l o l 4 9 , 1 3 1 - - 1 4 9 1 2 B l u m , H E , L e h l a , P., K o h l e r , L., S t e i n , E . A a n d F i s c h e r , E ~ I ( 1 9 7 7 ) J . Blol C h e m 2 5 2 , 2 8 3 4 - 2838 1 3 Yagl, K . , Y a z a w a , M., K a k m c h l , S., O h s h i m a , M. a n d U e n l s h l , K . ( 1 9 7 8 ) J . B1ol C h e m . 2 5 3 , 1 3 3 8 - 1340 1 4 N a l r n , A C. a n d P e r r y , S . V ( 1 9 7 9 ) B l o c h e m . J . 1 7 9 , 8 9 - - 9 7 15 Head, J.F. and Perry, S.V. (1974) Biochem. J 137,145--154 1 6 S t r z e l e c k a - G o l a s z e w s k a 0 H , J a k u b l a k , M. a n d D r a b l k o w s k ~ , W. ( 1 9 7 5 ) E u r . J . B i o c h e m . 5 5 , 2 2 1 - 230
Bmch~mlca et Bmphyswa Acta, 6 2 9 ( 1 9 8 0 ) 3 1 7 - - 3 2 7 © E l s e v m r / N o r t h - H o l l a n d Biomedical Press
BBA 29231
THE BINDING OF A R S E N A Z O III TO CELL COMPONENTS
T R O Y J B E E L E R *, A N G E L O S C H I B E C I * a n d A. M A R T O N O S I *
E.A. Do~sy Department of Biochemistry, St Louis University School of Medicine, St. Louts, MO 63104 (U.S A ) (Received September 14th, 1979)
Key words Arsenazo III binding, Ca2+ binding, Parvalbumin, Calcmm assay, (Rabbit skeletal muscle)
Summary The Ca 2÷ indicator, arsenazo III, binds to subcellular fractions of rabbit skeletal muscle with sufficient affinity that in living muscle containing 1--2 mM arsenazo III, the estimated free arsenazo III concentration is only 50--200 ~M; 80--90% of the b o u n d arsenazo III is associated with soluble proteins. The binding of arsenazo III to soluble proteins decreases the optical response of the dye to Ca2+; this is due to a decrease in the affinity of the protein-bound dye for Ca 2*. Approximately half of the b o u n d arsenazo III is released from the particulate fraction and soluble proteins upon addition of 5 mM Ca 2÷, suggesting that the C a ~ s e n a z o complex has lower affinity for the protein binding sites than the free dye. The Ca 2÷ binding to the soluble protein fraction of rabbit skeletal muscle is attributable largely to its parvalbumin content.
Introduction The widespread use of arsenazo III for measurement of cytoplasmic free [Ca 2÷] yielded valuable qualitative information a b o u t the regulatory role of Ca 2÷ in muscle [1], squid axon [2,3], aplysia neuron [4], and various subcellular systems [3,5,6]. The quantitative evaluation of the changes in cytoplasmic Ca 2÷ during physiological activity requires information about: (a) the interaction of arsenazo III with cell constituents, and (b) the influence of these interactions upon the response of the dye to free Ca 2÷. * Present address D e p a r t m e n t of Blochemlstry, State Umverstty of New York, Upstate Medmal Center, Syracuse, NY 13210, U.S.A.
318 The studms described in this report indicate massive bmdmg of arsenazo III to the subcellular organelles and soluble proteins of rabbit skeletal muscle with a decrease in the optical response of the dye to free calcium. For analytical purposes the effect can be minimized by increasing the arsenazo III concentration to levels where much of the dye remains free. Materials and Methods For the isolation of cell fractions, rabbit skeletal muscle was homogenized m 4 vols. 0.1 M KC1/10 mM imidazole (pH 7.0) for 2 min at 2°C. The crude myofibrillar fraction obtained by centnfugatlon at 775 × g for 15 mm was resuspended in 20 vols. homogemzing medium and washed three times by centrifugation at 775 × g for 15 min, followed by resuspenslon in the same medium. The crude mitochondrial fraction was obtained by centrifugatlon of the post-myofibrillar supernatant at 8000 × g for 20 min. The m i t o c h o n d n a were washed three times m 20 vols. homogenizing m e d m m by repeated centnfugatlon at 8000 × g for 20 min. For the isolation of microsomes the supernatant obtained after the removal of mitochondria was centrifuged at 36 000 × g for 1 h. The supernatant containing the soluble proteins was saved. The sediment was washed three times with the homogenizing medium by centnfugatlon at 78 000 X g for 1 h to yield the washed mmrosomal fraction. All particulate fractions were resuspended by gentle homogenization in 0.1 M KC1/10 mM imidazole (pH 7.0) to final concentrations of 3--30 mg protein/ml and were used for experiments within 1 day. The various fractions are expected to contain cross-contaminations and the term 'myofibrillar', ' m i t o c h o n d n a l ' , or 'mmrosomal' merely denotes the major component. The supernatant obtained after removal of microsomes was centrifuged again at 78 000 × g for 1 h. The essentially lipid-free soluble fraction was dialyzed against repeated changes of 60 vols. 0.1 M KC1/10 mM imidazole and 0.01% NaN3 for 24 h and was used immediately for experiments. Protein was determined according to Lowry et al. [7] with bovine serum albumin as standard. Arsenazo III, obtained from Aldnch Chemmal Co. (Milwaukee, WI) was purified before use according to Kendrick [8]. The binding of arsenazo III and 4SCa to particulate fractions was measured by centrifuge transport and to the soluble protems by equflibrmm dialysis under conditions described m the legends. The concentratmn of arsenazo III was determined in solutions containing 10 mM CaC12 by measuring the optical density at 568 nm using a molar extmction coefficmnt of 33 000. The absorption spectrum of arsenazo III was analyzed m an Aminco DW-2 spectrophotometer in the wavelength range of 350--730 nm, using control samples of identical composition w i t h o u t arsenazo III as reference. The Ca 2+ c o n t e n t of the various fractions was analyzed by atomic absorptmn s p e c t r o p h o t o m e t r y after precipitation o f the proteins with 2% trichloroacetm acid and 1% LaC13. Results
The binding o f arsenazo III to subcellular fractions o f rabb~t skeletal muscle The binding of arsenazo III to the varmus cell fractions was measured over
319 a range of free arsenazo III concentrations of 2--200 ~M, at Ca 2+ concentrations from less than 10-SM to 5 mM (Fig. 1). The binding of arsenazo III involves heterogeneous sets of binding sites w i t h o u t clear saturation up to 200 pM free arsenazo III concentration. The soluble proteins bound larger amounts of arsenazo III with greater affinity than the mitochondria, microsomes, or myofibrils. At an arsenazo III concentration o f 51 pM, 86.3% of the dye was bound to the soluble proteins (14.75 mg/ml). Even at total arsenazo III concentration as high as 609 ~M, 66% of the dye was protein-bound. Calcium in millimolar concentrations inhibited the binding o f arsenazo III, indicating that the Ca-arsenazo III complex has less affinity for the binding sites than the Mg2÷ and K ÷ complexes or the free acid. The data of Fig. 1 were used to estimate the relative concentrations of free and bound arsenazo III expected to occur in muscle cells at varying total arsenazo III concentrations (Tables I and II). In one series of experiments the Ca 2÷ concentration of the medium was maintained below 10 -s M with 1 mM EGTA in the presence of 0.1 M KC1 and 2 mM MgC12 (Table I). At total arsenazo III concentrations of 0.8 and 2.3 mM, respectively, an estimated
2.0
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FREE ARSENAZ0 nl, pM Fig. 1. Arsenazo III binding to subceHular fractions of rabbit skeletal muscle. T h e binding of arsenazo Ill to crude myofibrils (A), mitochondria (B), and microsomes (C) was measured b y centrifuge transport. T h e decrease in the absorbance of the supernatant after removal of the particulate elements by centrifugation was assayed at 568 n m in the presence of 10 m M CaCl 2. T h e dye binding to soluble proteins was analyzed b y equilibrium dialysis (D). T h e m e d i u m contained 0.1 M K C I 10 m M imidazole ( p H 7.0), 2 m M MgCl2, 10--400 /~M arsenazo III and 1 m M E G T A (e) or 5 m M CaCl 2 (o). T o samples labeled with open circles (o 7 neither E G T A nor CaCl 2 was added; the free C a 2+ concentration of these samples was 20.8 ~ M (A), 1 5 5 ~tM (C), a n d 2.06 juM (D), respectively. Protein concentrations were: 31 m g / m l (A); 3.3 m g / m l (B); 19 m g / m l (C); 14.75 m g / m l (D). Temperature: 4°C. In equilibrium dialysis experiments (D), 2 m l of soluble protein fraction were placed in dialysis tubing and dialyzed against 2 m l buffer (outside solution) for 4 8 h at 4°C. T h e binding of arsenazo III to proteins was calculated from the absorbance difference at 568 n m between the outside solutions and the inside solutions or protein-free control systems.
5 Total
199.7
108.6 2.1 12.2 76.8
1 2 3 4
Myofibrils Mitochondria Microsomes Soluble p r o t e i n s
Protein content (g/kg wet wt.)
Fraction
II. W I T H 5 m M CaCI 2
5 Total
199.7
108.6 2.1 12.2 76.8
1 2 3 4
My o fibrils Mitochondria Mierosomes Soluble p r o t e i n s
Protein content (gfkg wet wt.)
Fraction
I. W I T H 1 m M E G T A
total amount of protein.
20
20 20 20 20
Free (pM)
20
20 20 20 20
Free (~M)
The binding of arsenazo III was measured axe A r s e n a z o l l I c o n c e n t r a t i o n s . T h e f r e e b o u n d arsenazo III (/amol/kg muscle) was fraction per kg muscle. The total of bound
FI~ACTIONS OF RABBIT SKELETAL MUSCLE
1.62
0.13 -0.20 3.90
/zmol/g Protein
Bound
3.9
0.4 1.3 0.9 9.4
/amol/g protein
Bound
324.15
14.1 -2.4 307.6
/amol/kg muscle
781.1
45.6 2.7 10.9 721.8
plalol/kg muscle
94.21
4.1 <0.1 0.7 89.4
% of total (344.1 /aM)
97.5
5.7 0.3 1.4 90.1
% of total (801.1 /aM)
50
50 50 50 50
Free (pM)
50
50 50 50 50
Free (/aM)
2.48
0.27 -0.50 5.9
/~mol/g protein
Bound
5.5
0.75 4.0 1.5 13
Dmol/g Protein
Bound
469.2
29.3 -6.1 460.8
pmol/kg muscle
1106.3
81.5 8.3 18.3 998.3
/amol/kg muscle
90.83
5.4 <0.1 1.1 84.4
% of total (546.2 pM)
95.7
7.0 0.7 1.6 86.3
% of total (1156.3 /aM)
200
200 200 200 200
Free (pM)
200
200 200 200 200
Free (/aM)
5.43
0.98 -1.40 12.50
/~mol/g protein
Bound
10.89
2.02 8.6 2.7 24.8
/amol/g protein
Bound
1083.4
106.4 -17.1 960
~mol]kg muscle
2175.2
219.4 18.1 33.1 1904.6
/amol/kg muscle
84.4
8.3 <0.1 1.3 74.8
% of total (1283.5 ~M)
91.5
9.2 0.8 1.4 80.2
% of total (2375.2 pM)
in t h e p r e s e n c e o f 1 m M E G T A ( T a b l e I) o r 5 m M CaC12 ( T a b l e I I ) as d e s c r i b e d i n t h e l e g e n d t o Fig. 1. I n all cases, v a l u e s a r s e n a z o I I I c o n c e n t r a t i o n s ( 2 0 , 50, 1 0 0 a n d 2 0 0 / a M ) w e r e o b t a i n e d b y i n t e r p o l a t i o n f r o m Fig. 1. T h e c o n c e n t r a t i o n o f c a l c u l a t e d f o r e a c h f r a c t i o n as t h e p r o d u c t o f t h e a m o u n t o f a r s e n a z o ( / a m o l / g p r o t e i n ) a n d t h e a m o u n t o f p r o t e i n i n t h e arsenazo (/amol/g p r o t e i n ) was o b t a i n e d b y dividing the total c o n c e n t r a t i o n of b o u n d arsenazo III per k g muscle with the
THE BINDING OF ARSENAZO III TO SUBCELLULAR
T A B L E S I A N D II
t~
321 97.5% and 91.5% of the dye was b o u n d to the various cell constituents, primarily soluble proteins. In the presence of 5 mM CaC12, at a total d y e concentration as high as 1.2 mM (Table II), still more than 80% o f the dye was estimated to the bound. As microinjection of arsenazo III into muscle and other cells usually results in cellular dye concentrations less than 1 mM, the question arises whether the binding of the d y e to proteins interferes with its optical response to Ca 2÷.
The effect o f the soluble protein fraction o f rabbit skeletal muscle upon the absorption spectrum o f arsenazo III The absorption spectrum of arsenazo III was measured at varying total Ca 2+ concentrations with and without soluble proteins (Fig. 2). With 10 ~M arsenazo III at a soluble protein concentration of 14.7 mg/ml, the optical change caused by the addition of 12--446 pM Ca 2÷ is significantly reduced (Fig. 2B), compared with the absorbance change observed under similar conditions in the absence of soluble proteins (Fig. 2A). The effect of soluble proteins upon the Ca2+-induced absorbance change is less pronounced at 100 #M total arsenazo IIT concentration (Fig. 2C and D). Since at 5 mM CaC12 concentration a b o u t half of the total arsenazo III is still bound to soluble proteins,
A
B Ca
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450
500
550
600
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550
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Fig. 2. T h e e f f e c t o f soluble p r o t e i n s o n t h e o p t i c a l r e s p o n s e o f a r s e n a z o I I I t o Ca 2+. T h e axsenazo I I I s p e c t r a w e r e m e a s u r e d a t 4 ° C in a n A m i n c o DW-2 s p e c t r o p h o t o m e t e r in a m e d i u m o f 0.1 M K C I / I O m M i m i d a z o l e p H 7 . 0 / 2 m M MgC12 in t h e a b s e n c e ( A and C) o r p r e s e n c e (B a n d D ) o f 1 4 . 7 5 m g soluble m u s c l e p r o t e i n s p e r m l a t a z s e n a z o I I I c o n c e n t r a t i o n s o f 10 /~M (A a n d B) or 1 0 0 / ~ M (C a n d D), r e s p e c t i v e l y . Ca 2+ was a d d e d t o final c o n c e n t r a t i o n s i n d i c a t e d in the figure. T h e s p e c t r a w e r e m e a s u r e d a g a i n s t b u f f e r (A a n d C) or p r o t e i n s o l u t i o n s (B a n d D) c o n t a i n i n g t h e s a m e a m o u n t of Ca 2+ as t h e c o m p l e t e s a m p l e s . T h e s a m p l e s c o n t a i n i n g 10 /~M a r s e n a z o I I I (A a n d B) w e r e a s s a y e d in c u v e t t e s o f I e m p a t h l e n g t h while t h o s e c o n t a i n i n g 1 0 0 / J M a r s e n a z o I I I in c u v e t t e s of 1 m m p a t h l e n g t h .
322
some o f the protein b o u n d dye apparently responds to Ca 2÷ with a spectrum change similar to t h a t of the free dye. T h e diminished response of arsenazo III to Ca 2÷ in the presence of soluble proteins is n o t explained by Ca 2÷ binding to proteins, since t he high affinity Ca 2÷ binding sites of t he soluble fraction are saturated at a free Ca 2÷ concentration o f less than 10 ~M (Fig. 3) and no significant additional Ca 2÷ binding was observed between 11 and 213 /IM free Ca 2÷ concentrations. Even at 0.5 mM free Ca 2÷ less than 5% of the total Ca 2÷ was b o u n d to proteins. In experiments where soluble proteins were preequilibrated at free Ca 2÷ c o n c e n t r a t i o n s o f 25, 50, and 100 pM by dialysis against repeated changes of solutions adjusted t o these Ca 2÷ concentrations, additions of arsenazo III still p r o d u c e d a reduced Ca 2÷ response com pa r ed with protein-free buffer solutions o f the same free Ca 2÷ levels. Under these conditions the total Ca 2÷ c o n t e n t of protein solutions was greater than the total Ca 2÷ c o n t e n t of the protein-free c o n t ro l samples. Ca 2÷ binding to soluble proteins contributes to the reduced arsenazo III response only u n d e r conditions where the high affinity Ca 2÷ binding sites of t h e proteins are n o t saturated with Ca 2÷. For example, after com pl et e removal o f b o u n d Ca 2÷ f r o m soluble proteins by t r e a t m e n t with skeletal muscle microsomes, in the presence o f ATP and Mg, addition o f 15--20 pM Ca 2÷ caused little change in arsenazo III absorption (Fig. 4) because most of the added Ca 2÷ is b o u n d to proteins. F u r t h e r increase in Ca 2÷ c o n c e n t r a t i o n was reflected in an increase in the difference absorbance o f arsenazo III, but the change was m u c h less than th a t observed in a protein-free buffer solution or with soluble proteins saturated with a b o u t 40--60 #M Ca 2÷. This difference is due t o the binding o f Ca 2÷ and arsenazo III t o the soluble proteins.
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BOUND Ca, umoles/g PROTEIN F i g . 3. C a l c i u m b i n d i n g t o r a b b i t s k e l e t a l m u s c l e s u b f r a c t i o n s . M y o f i b r i l l a r , m i t o c h o n d r i a l , m i c r o s o m a l , a n d s o l u b l e p r o t e i n f r a c t i o n s w e r e t a k e n u p in 0.1 M K C L 1 0 m M i m i d a z o l e (pI-I 7.0), 2 m M MgC12 to f i n a l p r o t e i n c o n c e n t r a t i o n s o f 1 8 . 0 6 g/l, 4 . 5 g/l, 1 8 . 3 g/l a n d 1 4 . 7 5 g/l, r e s p e c t i v e l y . T o 2-ml a l i q u o t s o f t h e s e s o l u t i o n s w a s a d d e d 4 5 C a t o f i n a l c o n c e n t r a t i o n s o f 0 - - 5 0 0 ~M (0.1 ~ C i / m l ) . A f t e r e q u i l i b r a tion for 2 h at 4°C, the myofibrils, mitochondria, and microsomes were removed by centrifugation at 1 7 0 0 × g, 8 0 0 0 X g, a n d 7 8 0 0 0 X g r e s p e c t i v e l y . A l i q u o t s o f t h e s u p e r n a t a n t s w e r e c o u n t e d t o o b t a i n t h e c o n c e n t r a t i o n o f f r e e 4 5 C a 2 + . T h e a m o u n t o f Ca 2+ b o u n d t o t h e p a r t i c u l a t e e l e m e n t s w a s c a l c u l a t e d f r o m the differenee b e t w e e n total 45Ca2+ a n d free 45Ca2+ c o n c e n t r a t i o n s . Calcium binding to the soluble p r o t e i n f r a c t i o n s w a s m e a s u r e d b y e q u i l i b r i u m d i a l y s i s a t 4 ° C . T h e i n i t i a l Ca 2+ c o n c e n t r a t i o n o f t h e fract i o n s w a s : 7 0 . 9 ~M ( m y o f i b r i l s ) , 3 0 . 8 # M ( m i t o c h o n d x i a ) , 1 7 6 #M ( m i c r o s o m e s ) a n d 21 /JM ( s o l u b l e p r o t e i n f r a c t i o n ) . S y m b o l s : o m y o f i b r i l s ; &, m i t o c h o n d r i a ; D m i e r o s o m e s ; m s o l u b l e p r o t e i n s .
323
.7 .6
1
0
200
300
ADDEDCa, pM Fig, 4. C a l c i u m t i t r a t i o n of t h e soluble p r o t e i n f r a c t i o n in t h e p r e s e n c e o f a r s e n a z o I I I . T h e soluble p r o t e i n f r a c t i o n c o n t a i n e d 1 1 . 5 gfl P r o t e i n along w i t h 0.1 M K C I / I O m M i m i d a z o l e , p H 7 . 0 / 5 m M K 2 o x a l a t e / 5 m M MgC12, 80 stM a r s e n a z o I I I a n d 0 . 2 5 g/! o f r a b b i t m u s c l e m i c r o s o m e . A d d i t i o n o f 1 m M A T P a t r o o m t e m p e r a t u r e l o w e r e d t h e free Ca 2+ c o n c e n t r a t i o n b e l o w 1 ~tM. T h e m i c r o s o m e s w e r e separ a t e d f r o m t h e soluble p r o t e i n s a t 5°C b y c e n t r i f u g a t i o n f o r 30 m i n a t 9 0 0 0 0 X g. T h e d i f f e r e n c e a b s o r b a n c e ( 6 6 0 - - 6 8 5 rim) of t h e s u p e r n a t a n t was m e a s t t r e d at a d d e d CaCI 2 c o n c e n t r a t i o n s o f 0 . 1 - - 3 0 0 / I M a t 4 ° C ( e ) . I n o t h e r s a m p l e s , e i t h e r t h e s a m e p r o c e d u r e was r e p e a t e d b u t w i t h 1 DM A 2 3 1 8 7 a d d e d t o i n h i b i t t h e r e m o v a l of Ca (o), o r t h e soluble P r o t e i n s w e r e o m i t t e d ( s ) .
The binding of C a 2+ to subcellular fractions The m a x i m u m a m o u n t of Ca 2+ bound to the high affinity sites of the soluble protein fraction was 1.3 pmol/g protein (Fig. 3 and Table III), which corresponds to about 99 gmol bound Ca per kg wet muscle weight (Table III). These sites are expected to be saturated in activated muscle with free cytoplasmic Ca 2+ concentrations in the vicinity of 10 -s M. Similar Ca 2+ titrations on the myofibrillar, mitochondrial and microsomal fractions gave 3.2, 4.2 and 5.2 gmol Ca per g protein for the high affinity sites, which corresponds to 367.2, 8.8 and 63.4 pmol Ca per kg wet muscle weight for the three fractions respectively (Table III). All measurements were performed in media containing 0.1 M KC1 and 2 mM MgC12 to minimize contribution by nonspecific cation binding sites. These observations imply that the total a m o u n t of Ca 2+ bound to all constituents in activated muscle may be close to 0.5 mM, and of this approx. one-fifth is bound to the soluble proteins. Even if the mitochondrial and microsomal binding sites are not equilibrated with Ca 2÷ during single spikes of activity (due to internal localization) 0.46 mM bound Ca/kg muscle remains associated with the myofibrils and soluble proteins. Characterization of the Ca2+-binding component of the soluble pro rein fraction Polyacrylamide gel electrophoresis of the soluble protein fraction at p H 8 . 3 in the absence of sodium dodecyl sulfate followed by equilibration of the gel with 4SCa2÷ and autoradiography permitted the identification of the Ca 2÷binding components.
324
TABLE III BINDING OF CALCIUM TO RABBIT SKELETAL MUSCLE SUBFRACTIONS T h e b i n d i n g o f Ca 2+ to r a b b i t s k e l e t a l m u s c l e s u b f r a c t i o n s w a s m e a s u r e d as d e s c r i b e d in t h e l e g e n d o f Fig. 3. T h e n u m b e r o f h i g h a f f i n i t y s i t e s w a s d e t e r m i n e d in e a c h s u b f r a c t i o n b y e x t r a p o l a t i o n o f t h e s t e e p l i n e a r p o r t i o n o f t h e S c a t c h a r d p l o t s g i v e n in F i g . 3. T h e c o n c e n t r a t i o n o f h i g h a f f i n i t y Ca 2+ b i n d i n g s i t e s p e r k g m u s c l e w a s c a l c u l a t e d as t h e p r o d u c t o f t h e n u m b e r o f s i t e s p e r g p r o t e i n a n d t h e p r o t e i n c o n t e n t of each fraction per kg wet weight. g protein/kg wet weight
Myofibrils * Mitochondria Microsomes Soluble protein
108 2.1 12.2 76.8
Total
199.7
Max. number o f sites A (~umol/g) 3.4 4.2 ~ 5.2 1.3 2 . 7 0 **
Ca b o u n d t o sites A (pmol]kg muscle) 367.2 8.8 63.4 99.8 539.20
* Likely to include some mitochondria and microsomes. ** 5 3 9 . 2 / 1 9 9 . 7
The major Ca2+-binding protein of the soluble fraction of muscle homogenate migrated to the same position as parvalbumin (Fig. 5), and Ca2+ or EGTA influenced the mobilities of authentic parvalbumin and of the Ca2+binding component of the soluble protein fraction to a similar extent. The electrophoretic mobility of troponin C was much faster than that of parvalbumin due to its greater negative charge. Troponin C and the Ca2+-binding protein of sarcoplasmic reticulum had greater electrophoretic mobility in the presence of 0.1 mM Ca2+ than in 1 mM EGTA. In contrast, the mobilities of the parvalbumin and of the Ca2+-binding component of the supernatant fraction ('myogen') were reduced by Ca 2+. On the basis of these observations it is plausible to assume that the Ca2+binding protein detected in the soluble fraction of muscle homogenate is parvalbumin.
Fig. 5. Identification of the Ca 2+ binding proteins of rabbit skeletal muscle on polyacrylamide gels. Electrophoresis was performed on quadruplicate polyacrylamide gel slabs ( 1 0 % acrylamide/0.27% N , N pmethylene-bisacrylamide in 0.375 M Tris-HCl buffer, p H 8.9). T h e electrophoresis buffer contained 25 m M Tris and 1 9 2 m M glycine, p H 8.3, with additions as indicated. For t w o sets of gels (labeled 'Ca') the samples were applied in 10 m M Tris-HCl buffer p H 7.0, containing 0.1 m M CaCl 2 and 1 0 % glycerol. O n e set of 'Ca' samples was stained with Coomassie blue (Fig. 5A), the other was equilibrated with 45C'a to identify the C a 2+ binding proteins as described below (Fig. 5B). For t w o sets of gels (labeled 'EGTA') the samples were applied in 10 m M Tris-HCl, p H 7.0/1 m M E G T A / 1 0 % glycerol. During electrophoresis the upper electrophoresis c h a m b e r also contained 1 m M E G T A . O n e set of E G T A samples was stained with Coomassie blue (Fig. 5 A ) the other was equilibrated with 4 S C a as described below (Fig. 5B). Equilibration with 45Ca. After electrophoresis the gels were rinsed with electrophoresis buffer and incubated for 2 h with constant shaking at z o o m temperature in electrophoresis buffer containing 1 ~ M 45CAC12 (1.47 mCi//~mol) and 2 % glycerol. In the case of E G T A gels the equilibration solution was changed to fresh solution after 1 h to reduce E G T A content. After brief rinsing in electrophoresis buffer containing 2 % glycerol, followed by water, the wet gels were transferred to glass plates, covered with Saran w r a p and exposed to K o d a k X R - 5 film for 12--18 h. Samples: Troponin C (25/~g); Ca binding protein ('C protein') of rabbit sascoplasmic reticulum (400 ~g), rabbit parvalbumin (50 ~g); rabbit skeletal muscle 78 0 0 0 X g supernatant ('myogen', 720/~g).
325
326
Discussion The following main observations were made: (1) The particulate and soluble fractions of rabbit skeletal muscle bind arsenazo III in unexpectedly large amounts, with the result that in muscle cells even at millimolar arsenazo III concentrations an estimated 80--90% of the dye is bound to cell constituents. Most of the bound arsenazo III is associated with soluble proteins. (2) At 10--20 #M arsenazo III concentrations (Fig. 2B) the absorbance response of the dye to Ca is drastically reduced by the soluble fraction of rabbit skeletal muscle. This effect is attributable in part to a decrease in free arsenazo III concentration caused by the binding of the dye to proteins. The estimated free arsenazo III concentration under these conditions is about 1 pM, which is below the dissociation constant of arsenazo III for Ca 2÷ [9]. In these systems the arsenazo III bound to the high affinity binding sites of proteins apparently forms Ca 2÷ complexes with smaller affinity than the free dye. As the arsenazo III concentration is increased to 100 #M (Fig. 2D) the effect of soluble proteins on the Ca 2÷ response of the dye is diminished. Under these conditions the free dye concentration is about 27 pM, which is close to the dissociation constant of Ca-arsenazo III complex [9]. Since 73% of the dye is still bound to proteins it is likely t h a t some of the arsenazo III bound to low affinity binding sites responds with absorbance change to Ca 2÷. In living cells where the concentration of proteins is higher than in the in vitro test system a comparable Ca response would require total arsenazo III concentration of about 1 mM (Tables I and II). It is clear t h a t quantitative evaluation of free ionized Ca 2÷ concentration in living muscle, based on analysis of arsenazo III spectra, must take into account the actual concentration of free arsenazo III in the cell, and a simple relationship between spectral change and free Ca 2÷ concentration is expected only with arsenazo III concentrations of 1 mM or greater. Baylor et al. [10] observed in intact frog muscle a dichroic c o m p o n e n t in the arsenazo III signal during a twitch, indicating a change in alignment of some of the dye molecules with respect to the fiber axis. The contribution of the dichroic c o m p o n e n t to the total absorption diminished as the arsenazo III concentration within the fiber was increased. The dichroic c o m p o n e n t of absorption presumably reflects the binding of the dye to the myofibrils or to some other oriented structure. According to our observations this is only a fraction of the total a m o u n t of bound dye within the cell. As the dye concentration is increased the dichroic contribution is expected to be diluted by increasing free dye concentrations, and by increase in the binding of the dye to soluble proteins (Fig. 1). (3) Ca 2÷ binding to the soluble protein fraction of skeletal muscle was earlier observed by Briggs and Fleischman [11]. The estimated concentration of the corresponding Ca 2÷ binding sites in rabbit skeletal muscle was 2 × 10 -4 M with a dissociation constant of 6.67 pM. The a m o u n t of parvalbumin in rabbit skeletal muscle is 0.6--1.1 g/kg wet weight, capable of binding 100--182 pmol Ca 2÷ per kg muscle with a dissociation constant close to 1 ~M [12]. The Ca 2÷
327 bmding capacity of parvalbumin in rabbit skeletal muscle is comparable to the Ca :÷ binding observed in the soluble fraction. Electrophoretic studies also support the conclusion that the principal Ca 2÷ binding component of the soluble protein fraction is parvalbumin. The calmodulin content of rabbit skeletal muscle is less than 30 mg/kg wet weight or about 1.76 ~mol/kg [13,14] and contributes little to the observed Ca 2÷ binding. Troponin C and myosin light chains are associated with the myofibrils [15] and do not participate in the Ca 2÷ binding of the soluble fraction. The total Ca 2÷ binding capacity of the high affinity binding sites in the various cell fractions is about 0.54 mmol/kg muscle. The Ca 2÷ bound to F actm is apparently nonexchangeable [16] and the accessibility of mitochondrial and mlcrosomal sites to cytoplasmic Ca 2÷ is uncertain. Discounting these contributions the saturation of the remaining cytosolic and myofibnllar Ca 2÷ bindmg sites requires the release of about 0.35 mmol Ca/kg muscle during each contraction cycle. Acknowledgements This work was supported by research grants AM 18117 and AM 26832 from NIH, PCM 7705393 from the National Science Foundation and a grant in aid from the Muscular Dystrophy Association. T.J.B. was the recipient of a postdoctoral fellowship from the Muscular Dystrophy Association. The collaboration of Miss Debbie Hermann is gratefully acknowledged. References 1 Mfledl, R , P~trker, I a n d S c h a l o w , G. ( 1 9 7 7 ) P r o c R S o c L o n d o n B 1 9 8 , 2 0 1 - - 2 1 0 2 D1Polo, R , R e q u e n a , J., B n n l e y , F . J . , M u l h n s , L . S . , S c a r p a , A . a n d T l f f e r t , T ( 1 9 7 6 ) J . G e n . P h y s i o l . 67,433---467 3 S c a r p a , A., B r i n l e y , F . J . , T l f f e r t , T a n d D u b y a k , G . R . ( 1 9 7 8 ) A n n u N.Y A c a d Scl 3 0 7 , 8 6 - - 1 1 2 4 Thomas, M.V and Gorman, A.C.F. (1977) Science 196, 531--533 5 W e i s s m a n , G., Collins, T , Evers, A . a n d D u n h a m , P ( 1 9 7 6 ) P r o c . N a t l . A c a d . Sci U . S . A . 7 3 , 5 1 0 - 514 6 R u s s e l l , J . T , Beeler, T. a n d M a r t o n o s l , A . ( 1 9 7 9 ) J . Biol. C h e m . 2 5 4 , 2 0 4 0 - - 2 0 4 6 7 L o w r y , O H . , R o s e b r o u g h , N . J , F a r r , A . L . a n d R a n d a l l , R J ( 1 9 5 1 ) J Biol. C h e m 1 9 3 , 2 6 5 - - 2 7 5 8 Kendrlck, N.C. (1976) Anal. Bloehem. 76, 487--501 9 Russell, J.T. and Maxtonosi, A. (1978) Blochlm. Blophys. Aeta 544,418--429 1 0 B a y l o r , S.M., C h a n d l e r , W . K a n d M a r s h a l l , M.W. ( 1 9 7 9 ) B l o p h y s . J . 2 5 , 1 4 1 a I I B n g g s , F . N . a n d F l e l s h m a n , M. ( 1 9 6 5 ) J G e n . P h y s l o l 4 9 , 1 3 1 - - 1 4 9 1 2 B l u m , H E , L e h l a , P., K o h l e r , L., S t e i n , E . A a n d F i s c h e r , E ~ I ( 1 9 7 7 ) J . Blol C h e m 2 5 2 , 2 8 3 4 - 2838 1 3 Yagl, K . , Y a z a w a , M., K a k m c h l , S., O h s h i m a , M. a n d U e n l s h l , K . ( 1 9 7 8 ) J . B1ol C h e m . 2 5 3 , 1 3 3 8 - 1340 1 4 N a l r n , A C. a n d P e r r y , S . V ( 1 9 7 9 ) B l o c h e m . J . 1 7 9 , 8 9 - - 9 7 15 Head, J.F. and Perry, S.V. (1974) Biochem. J 137,145--154 1 6 S t r z e l e c k a - G o l a s z e w s k a 0 H , J a k u b l a k , M. a n d D r a b l k o w s k ~ , W. ( 1 9 7 5 ) E u r . J . B i o c h e m . 5 5 , 2 2 1 - 230