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The thin filament of vertebrate skeletal muscle co-operatively activates as a unit
J. Mol. Biol. (1984) 180, 379-384
The T h i n F i l a m e n t o f Vertebrate Skeletal M u s c l e Co-operatively Activates as a U n i t
We find that extraction of as little as one troponin C molecule per troponintropomyosin strand on a thin filament reduces the slope of the pCa/tension relation. We interpret this to mean that the regulatory units along a thin filament of rabbit psoas fibers are linked co-operatively so that a thin filament activates as a unit. The presence of extended co-operativity explains why the pCa/tension relation in skinned fibers has a slope much higher than predicted by binding of Ca 2+ to one regulatory unit. Replacement of the extracted troponin C with purified troponin C fully reverses the effect of extraction and shows it to be the essential Ca 2+ binding protein responsible for the steep slope of the pCa/tension relation.
I n t a c t muscle is activated when depolarization of the plasma m e m b r a n e leads to a rise in intracellular Ca 2+. Binding of Ca 2+ to troponin C, a subunit of the t r o p o n i n - t r o p o m y o s i n regulatory complex, allows the myosin cross-bridges of the thick filament to a t t a c h to aetin. In skinned muscle fibers the plasma m e m b r a n e is removed or permeabilized so t h a t activation can be studied with direct control over the concentration of Ca 2+, substrate and other solutes bathing the myofibrils. The increase in tension in a skinned fiber (Fig. l(a)) is plotted against the Ca 2+ (shown here as the pCa or - l o g [Ca:+]). To compare curves we empirically fit the data points to a form of the Hill equation (as explained in the legend to Table 1), and extract two parameters, one describing the midpoint (pK) and one proportional to the slope (n~). The latter might be expected to be between 1 (no co-operativity in calcium binding) and 2 ( m a x i m u m co-operativity) because it is known t h a t only two of the four Ca 2+ binding sites on TnC$ regulate contraction (Potter & Gergely, 1975). Instead it is greater t h a n 5 (Brandt et al., 1980,1982). TnC is rapidly extracted from skinned rabbit psoas fibers by exposing t h e m to 5 mM-EDTA and 10 m ~ - M O P S (pH 7.2) at 30°C, a modification of the procedures of Cox et al. (1981). Densitometric analysis of sodium dodeeyl sulfate/ polyacrylamide gels (Fig. 2) of extracted fibers indicates t h a t TnC loss is a p p r o x i m a t e l y first order with a half-time of a b o u t eight minutes (Table 1) and t h a t TnC is the only myofibrillar protein extracted. Comparison of the loss of tension with the extent of TnC extraction reveals that, at least for extraction of 30 to 40% of the TnC, these values are a p p r o x i m a t e l y proportional (Kerrick et al., 1983). The effect of a five-minute extraction on the pCa/tension curve for a single fiber can be seen in Figure l(b). Comparison of this curve with t h a t for an $ Abbreviations used: TnC, troponin C; S1, myosin subfragment 1. 379 0022-2836/84/340379-06 $03.00/0 © I984 Academic Press Inc. (London) Ltd.
380
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Fro. l. (a) The pOa/tension relation of a control rabbit, psoas skinned fiber, n H = 8.3, p K = 5.96, Pc = 57 mg, (Fiber Clf.5, diameter 62.#m.) (b) The fiber was first exposed for 5 min to a solution t h a t extracts TnC (see the text), then a pCa/tension curve (O) was determined, n H = 1-9, p K = 5.43, Pc = 27 mg. Subsequent to this the fiber was incubated in TnC solution (1.5 m g / m l in a pCa 8 relaxing solution) for 20 rain and the second pCa/tension curve (D) was determined, n H = 4.6, p K = 5.83, Pc = 46 mg. (Fiber C2i.10, diam. 51 pro.) (e) The fiber was first exposed to extracting solution for I min before the pCa/tension relation (O) was determined, n H = 3.6, p K = 5.66, Pc = 5 3 r a g . Subsequently the fiber was incubated in TnC solution for 29 rain t h e n the pCa/tension relation ([]) was measured again, n H = 6.0, p K = 5.79, Pc = 62 rag. (Fiber C2i.23, diam. 56 pro.) The d a t a of (a) to (c) were normalized, t h e n fitted by a least-squares computer program to a form of the Hill equation (Table 1 legend). The 2 parameters n H and p K are used in the equation in the legend to Table 1 to construct the smooth lines fitted to the d a t a points. The methods for preparing the fibers, collecting and treating the d a t a have been described previously (Brandt et aI., 1980,1982). The sarcomere lengths were all set to between 2.4 ttm and 2.6 #m. The s t a n d a r d pCa 8 "relaxing" solution contains (Na salts): 9-8 mM-EGTA, 0.2 mM-CaEGTA (ethylene glycol bis(fl-aminoethylether)N,N,N'N'-tetraaeetic acid), 5 mM-MgATP, 5 mM-Na,ATP, 7-5 raM-NaP i, 40.1 mM-sodium propionate, 17.2 mM-Na2SO 4, 10 m ~ MOPS (morpholinopropane sulfonie acid) at p H 7"00. The "activating" solutions contain 10 ms~ total EGTA (the ratio of CaEGTA : EGTA according to the pCa and the other components as above). Ratios of pCa 8 and pCa 4-865 solution are automatically mixed in the chamber during an experiment to produce the intermediate pCa values.
LETTERS
TO T H E A
EDITOR
B
381
c
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-
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.
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~
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.
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Fro. 2. Sodium dodeeyl sulfate/polyacry[amide gel analysis of psoas single fibers as described by Laemmli (1970) and stained by the method of Oaklay et ol. (1980). An unextraeted fiber is shown in lane A. Fibers extracted for 10 mira 15 rain and 50 min are shown in lanes B. C and D, respectively. The positions of the major proteins are labeled. Tm, tropomyosin; role, myosin light chain. Note that the lanes were not exactly equally loaded (see also Table 1 legend).
unextraeted fiber (Fig. l(a)) shows that the n H of the curve is reduced from the control range (5 to 8) to approximately 2 and that the tension is reduced to about half. These effects are typical for extractions of 40 to 500/o of the TnC (Table 1). With less TnC extraction less reduction in n H and tension is observed. To show that the only significant correlate of the change in n H is loss of TnC, extracted fibers were incubated at 20°C in a relaxing solution of pCa 8 containing !'5 mg TnC/ml purified by the procedure of Eisenberg & Kielley (1974). Sodium dodecyl sulfate/polyaerylamide gels heavily loaded with the purified TnC show a single band. When the extracted fiber of Figure l(b) was incubated in TnC solution for 20 minutes, and the pCa/tension curve again determined, both
P. W. BRANDT, M. S. DIAMOND AND F. H. SCHACHAT
382
TABLE 1
Changes in physical and chemical parameters for TnC with time of extraction Time in extraction media (min) Parameter TnC (%) Tension (%) nH pK No. of fibers
0
2
100_+4 100 + 8 6"3 +0"5 5.97+0.02 11
5
77_+3 69_+8 76 _+8 40 __6 3'3 _+0"2 2'1 _+O'l 5.65___0.03 5.38+0'05 13 12
10
20
30
40_+6 28 _+6 2-0 _+0"3 5.21+0-08 8
31_+2
18_+2
To determine the TnC content of fibers after various times in the extraction media, 1O to 20 single fibers were extracted and frozen until electrophoresis was done. The gels were stained by the Coomassie method, then photographed and the densities of the bands were determined either with a Zeinhe Soft Laser densitometer or a Joyee Loebl microdensitometer. TnC was normalized by division by the sum of TnT+tropomyosin+light chains. Each time point is the mean of 6 different determinations. To ensure that the sum used to normalize the TnC does not include a component that is also extracted, we normalized each component separately against the sum of the others and found no systematic loss of any component. Our extraction procedure, in contrast to that of Moss et al. (1982,1983), does not extract myosin light chain 2fl Similar, but qualitative data were obtained from silver-stained gels of single fibers (Fig. 2). pK is the midpoint of the pCa/tension curve and nH is the Hill coefficientcalculated by fitting the data to the equation:
P/Po = {[Ca2+] • K}'/(1 + {[Ca2+] • K}"). pK = log (K) (Brandt et al., 1980,1982): Po is the maximum tension. The number of fibers refers to all the rows except the TnC (%) row. The S.E.M. values are listed after each value in the Table.
a b s o l u t e t e n s i o n a n d n H increased d r a m a t i c a l l y . T h e effect of p a r t i a l e x t r a c t i o n of TnC on the slope of the p C a / t e n s i o n r e l a t i o n is m a r k e d y e t reversed (Greaser & Moss, 1984) b y the a d d i t i o n of purified TnC. We conclude from this t h a t gaps in the i n t e g r i t y of the c h a i n of r e g u l a t o r y u n i t s on the t h i n filament, i n t r o d u c e d b y T n C e x t r a c t i o n , b r e a k t h e co-operative spread of a c t i v a t i o n along the t h i n filament. The t h i n f i l a m e n t consists of two helically i n t e r t w i n e d s t r a n d s of a e t i n a n d two s t r a n d s of t r o p o m y o s i n t h a t lie in the grooves of the a c t i n d o u b l e helix. T r o p o n i n is b o u n d to t r o p o m y o s i n e v e r y 38"5 n m along t h e t h i n filament. This m e a n s t h a t there are some 26 r e g u l a t o r y u n i t s (1000/38"5) per s t r a n d of t r o p o m y o s i n on a 1 p m t h i n f i l a m e n t of r a b b i t psoas. To d e t e r m i n e how m a n y u n i t s in a n u n b r o k e n sequence are r e q u i r e d to g e n e r a t e the n o r m a l large n H value, we h a v e e x t r a c t e d fibers for one m i n u t e or less, t h e n i n c u b a t e d t h e m with T n C a n d characterized the p C a / t e n s i o n r e l a t i o n after e x t r a c t i o n a n d a g a i n after i n c u b a t i o n . W e c a n n o t a c c u r a t e l y d e t e r m i n e small fractions of T n C loss b y gel electrophoresis of single fibers so we rely on the difference in t e n s i o n b e t w e e n e x t r a c t e d a n d i n c u b a t e d fiber to e s t i m a t e the e x t r a c t i o n of TnC. Gels of briefly e x t r a c t e d single fibers, however, do show less e x t r a c t i o n of T n C t h a n do gels of fibers e x t r a c t e d for two m i n u t e s or longer. The slope a n d t e n s i o n are higher for the p C a / t e n s i o n c u r v e t h a t follows i n c u b a t i o n w i t h T n C (Fig. l(e)) t h a n for the curve t a k e n after one m i n u t e of e x t r a c t i o n . F r o m such e x p e r i m e n t s we find t h a t e x t r a c t i o n of a n e s t i m a t e d 10% of the T n C s u b s t a n t i a l l y reduces the c o - o p e r a t i v i t y ; 10~/o of the 26 T n C s u b u n i t s
LETTERS TO THE EDITOR
383
of an intact regulatory strand consists of three subunits and three breaks generate four pieces. Thus, average sequences of intact regulatory units as long as six, or about one-quarter of the normal size, are less co-operative than the intact regulatory strand of 26 units. F r o m experiments similar to that shown in Figure l(c), but with extraction for 30 seconds, we found t h a t the n H of the pCa/tension relation was diminished, even if so little of the TnC was removed t h a t there was a 5% or smaller difference in tension between the extracted and incubated conditions. In 11 experiments, the mean n H after extraction was 3.5_+ 0.1 (S.E.M.), and following incubation in TnC for 25 minutes it was 5-0___0"19. The increase in n~ with TnC addition is significant at the 99~o level and into the control range (Brandt et al., 1982). Because co-operativity decreases with a loss of TnC estimated at 5~o or approximately one break per regulatory strand, we suggest that the intact thin filament co-operatively activates as a unit. Co-operativity in the thin filament control mechanism was originally proposed by Bremel & Weber (1972), who suggested that binding of myosin subfragment l to the thin filaments turns on the regulatory unit and enhances its affinity for calcium. Activation of the filament by S1 binding is supported by observations t h a t N-ethylmaleimide-treated S1, bound irreversibly to actin strands, enhances hydrolysis (Pemrick & Weber, 1976; Nagashima & Asakura, 1982). Greene & Eisenberg (1980) found t h a t S1 binds co-operatively to regulated actin in the absence of Ca :+ and substrate, but in their presence the high co-operativity of S1 binding almost disappears (Greene, 1982). These observations have been incorporated into models of thin filament regulation with assumptions of near neighbor tropomyosin-tropomyosin co-operativity (Hill et al., 1980,1983; Hill, 1984; Trueblood et al., 1982). Co-operative activation of hydrolysis by S1 binding or co-operative S1 binding in soluble protein systems differ from that reported here in several important ways. In our skinned fibers the contractile proteins are intact and the in vivo geometric constraints of the myofilament lattice are preserved. We studied activation of tension by Ca :+ in high concentrations of MgATP, conditions in which soluble systems show little co-operativity, and we found a high degree of co-operativity with m a n y regulatory units acting in concert. Although the mechanism for the co-ordination of regulatory unit activation along the thin filament is unknown it apparently depends on the presence of TnC in the regulatory unit. This work was supported by grants NIH NS-18228 (to F.H.S.) and the Muscular Dystrophy Association of America and NIH NS-117660 (to P.W.B.). Department of Anatomy and Cell Biology Columbia University New York, NY 10032, U.S.A.
P. W. BRANDT M. S. DIAMOND
Department of Anatomy Duke University Medical Center Durham, NC 27710, U.S.A.
F. H. SCHACHAT
Received 29 March 1984
384
P . W . BI%ANDT, M. S. DIAMOND AND F. H. SCHA(~HAT
REFERENCES Brandt, P. W., Cox, R. N. & Kawai, M. (1980). Proc. Nat. Aead. Sci., U.S.A. 77, 4717 4720. Brandt, P. W., Cox, R. N , Kawai, M. & Robinson, T. (1982). J. (Ion. Physiol. 79, 9971016. Bremel, R. D. & Weber, A. (1972). Nature New Biol. 238, 97-101. Cox, J. A, Comte, M. & Stein, E. A. (1981). Biochem. J. 195,205-211. Eisenberg, E. & Kielley, W. W. (1974). J. Biol. Chem. 249 (15), 4742-4748. Greaser, M. L. & Moss, R. L. (1984). Biophys. J. 45,344a. Greene, L. (1982). J. Biol. Chem. 257, 13993-13999. Greene, L. E. & Eisenberg, E. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 2616-2620. Hill, T. L. (1984). Biophys. J. 44, 383 396. Hill, T. L., Eisenberg, E. & Greene, L. (1980). Pro< Nat. Acad. Sci., U.S.A. 77, 3186-3190. Hill, T. L., Eisenberg, E. & Greene, L. (1983). Prec. Ned. Acad. Sci., U.S.A. 80, 60-64. Moss, R. L., Giuliani, G. G. & Greaser, M. L. (1982). J. Biol. Chem. 257, 8588-8591. Moss, 1%. L., Giuliani, G. G. & Greaser, M. L. (1983). J. Cell Biol. 96, 970 978. Kerrick, W. G. L., Zot, H., Hoar, P. E. & Potter, J. D. (1983). Biopl~ys. J. 41~ 148a. Laemmli, U. K. (1970). Nature (London), 227 (259), 680-685. Nagashima, H. & Asakura, S. (1982). J. Mot. Biol. 155,409-428. Pemrick, S. & Weber, A. (1976). Biochemistry, 15, 5193-5198. Potter, J. D. & Gergely, J. (1975). J. Blot. Chem. 250, 4628 4633. Trueblood, C. E., Watsh, T. P. & Weber, A. (1982). In Basic Biology of Muscle: A Comparative Approach (Twarog, B. 1}I., Levine, R. J. C. & Dewey, M., eds), pp. 223243, Raven Press, New York.
Edited by H. E. ttuxley
The T h i n F i l a m e n t o f Vertebrate Skeletal M u s c l e Co-operatively Activates as a U n i t
We find that extraction of as little as one troponin C molecule per troponintropomyosin strand on a thin filament reduces the slope of the pCa/tension relation. We interpret this to mean that the regulatory units along a thin filament of rabbit psoas fibers are linked co-operatively so that a thin filament activates as a unit. The presence of extended co-operativity explains why the pCa/tension relation in skinned fibers has a slope much higher than predicted by binding of Ca 2+ to one regulatory unit. Replacement of the extracted troponin C with purified troponin C fully reverses the effect of extraction and shows it to be the essential Ca 2+ binding protein responsible for the steep slope of the pCa/tension relation.
I n t a c t muscle is activated when depolarization of the plasma m e m b r a n e leads to a rise in intracellular Ca 2+. Binding of Ca 2+ to troponin C, a subunit of the t r o p o n i n - t r o p o m y o s i n regulatory complex, allows the myosin cross-bridges of the thick filament to a t t a c h to aetin. In skinned muscle fibers the plasma m e m b r a n e is removed or permeabilized so t h a t activation can be studied with direct control over the concentration of Ca 2+, substrate and other solutes bathing the myofibrils. The increase in tension in a skinned fiber (Fig. l(a)) is plotted against the Ca 2+ (shown here as the pCa or - l o g [Ca:+]). To compare curves we empirically fit the data points to a form of the Hill equation (as explained in the legend to Table 1), and extract two parameters, one describing the midpoint (pK) and one proportional to the slope (n~). The latter might be expected to be between 1 (no co-operativity in calcium binding) and 2 ( m a x i m u m co-operativity) because it is known t h a t only two of the four Ca 2+ binding sites on TnC$ regulate contraction (Potter & Gergely, 1975). Instead it is greater t h a n 5 (Brandt et al., 1980,1982). TnC is rapidly extracted from skinned rabbit psoas fibers by exposing t h e m to 5 mM-EDTA and 10 m ~ - M O P S (pH 7.2) at 30°C, a modification of the procedures of Cox et al. (1981). Densitometric analysis of sodium dodeeyl sulfate/ polyacrylamide gels (Fig. 2) of extracted fibers indicates t h a t TnC loss is a p p r o x i m a t e l y first order with a half-time of a b o u t eight minutes (Table 1) and t h a t TnC is the only myofibrillar protein extracted. Comparison of the loss of tension with the extent of TnC extraction reveals that, at least for extraction of 30 to 40% of the TnC, these values are a p p r o x i m a t e l y proportional (Kerrick et al., 1983). The effect of a five-minute extraction on the pCa/tension curve for a single fiber can be seen in Figure l(b). Comparison of this curve with t h a t for an $ Abbreviations used: TnC, troponin C; S1, myosin subfragment 1. 379 0022-2836/84/340379-06 $03.00/0 © I984 Academic Press Inc. (London) Ltd.
380
P. W . B t ~ A N D T ,
~
[.0
M. S. D I A M O N D
0 DClO
A N D F . H. S C H A C H A T
l.O
OD
D
o
0.5
0-5
15
2
6' 0
o j _ _ _ _
6'0
'5' pCa (a)
4'.5
pCa (b)
g o
,5 g, 0.5 c
6.0
5.5
5.0
pCo (c)
Fro. l. (a) The pOa/tension relation of a control rabbit, psoas skinned fiber, n H = 8.3, p K = 5.96, Pc = 57 mg, (Fiber Clf.5, diameter 62.#m.) (b) The fiber was first exposed for 5 min to a solution t h a t extracts TnC (see the text), then a pCa/tension curve (O) was determined, n H = 1-9, p K = 5.43, Pc = 27 mg. Subsequent to this the fiber was incubated in TnC solution (1.5 m g / m l in a pCa 8 relaxing solution) for 20 rain and the second pCa/tension curve (D) was determined, n H = 4.6, p K = 5.83, Pc = 46 mg. (Fiber C2i.10, diam. 51 pro.) (e) The fiber was first exposed to extracting solution for I min before the pCa/tension relation (O) was determined, n H = 3.6, p K = 5.66, Pc = 5 3 r a g . Subsequently the fiber was incubated in TnC solution for 29 rain t h e n the pCa/tension relation ([]) was measured again, n H = 6.0, p K = 5.79, Pc = 62 rag. (Fiber C2i.23, diam. 56 pro.) The d a t a of (a) to (c) were normalized, t h e n fitted by a least-squares computer program to a form of the Hill equation (Table 1 legend). The 2 parameters n H and p K are used in the equation in the legend to Table 1 to construct the smooth lines fitted to the d a t a points. The methods for preparing the fibers, collecting and treating the d a t a have been described previously (Brandt et aI., 1980,1982). The sarcomere lengths were all set to between 2.4 ttm and 2.6 #m. The s t a n d a r d pCa 8 "relaxing" solution contains (Na salts): 9-8 mM-EGTA, 0.2 mM-CaEGTA (ethylene glycol bis(fl-aminoethylether)N,N,N'N'-tetraaeetic acid), 5 mM-MgATP, 5 mM-Na,ATP, 7-5 raM-NaP i, 40.1 mM-sodium propionate, 17.2 mM-Na2SO 4, 10 m ~ MOPS (morpholinopropane sulfonie acid) at p H 7"00. The "activating" solutions contain 10 ms~ total EGTA (the ratio of CaEGTA : EGTA according to the pCa and the other components as above). Ratios of pCa 8 and pCa 4-865 solution are automatically mixed in the chamber during an experiment to produce the intermediate pCa values.
LETTERS
TO T H E A
EDITOR
B
381
c
Myosin
Actin -
-
T n T ~
.
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~
~
~
.
~
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Fro. 2. Sodium dodeeyl sulfate/polyacry[amide gel analysis of psoas single fibers as described by Laemmli (1970) and stained by the method of Oaklay et ol. (1980). An unextraeted fiber is shown in lane A. Fibers extracted for 10 mira 15 rain and 50 min are shown in lanes B. C and D, respectively. The positions of the major proteins are labeled. Tm, tropomyosin; role, myosin light chain. Note that the lanes were not exactly equally loaded (see also Table 1 legend).
unextraeted fiber (Fig. l(a)) shows that the n H of the curve is reduced from the control range (5 to 8) to approximately 2 and that the tension is reduced to about half. These effects are typical for extractions of 40 to 500/o of the TnC (Table 1). With less TnC extraction less reduction in n H and tension is observed. To show that the only significant correlate of the change in n H is loss of TnC, extracted fibers were incubated at 20°C in a relaxing solution of pCa 8 containing !'5 mg TnC/ml purified by the procedure of Eisenberg & Kielley (1974). Sodium dodecyl sulfate/polyaerylamide gels heavily loaded with the purified TnC show a single band. When the extracted fiber of Figure l(b) was incubated in TnC solution for 20 minutes, and the pCa/tension curve again determined, both
P. W. BRANDT, M. S. DIAMOND AND F. H. SCHACHAT
382
TABLE 1
Changes in physical and chemical parameters for TnC with time of extraction Time in extraction media (min) Parameter TnC (%) Tension (%) nH pK No. of fibers
0
2
100_+4 100 + 8 6"3 +0"5 5.97+0.02 11
5
77_+3 69_+8 76 _+8 40 __6 3'3 _+0"2 2'1 _+O'l 5.65___0.03 5.38+0'05 13 12
10
20
30
40_+6 28 _+6 2-0 _+0"3 5.21+0-08 8
31_+2
18_+2
To determine the TnC content of fibers after various times in the extraction media, 1O to 20 single fibers were extracted and frozen until electrophoresis was done. The gels were stained by the Coomassie method, then photographed and the densities of the bands were determined either with a Zeinhe Soft Laser densitometer or a Joyee Loebl microdensitometer. TnC was normalized by division by the sum of TnT+tropomyosin+light chains. Each time point is the mean of 6 different determinations. To ensure that the sum used to normalize the TnC does not include a component that is also extracted, we normalized each component separately against the sum of the others and found no systematic loss of any component. Our extraction procedure, in contrast to that of Moss et al. (1982,1983), does not extract myosin light chain 2fl Similar, but qualitative data were obtained from silver-stained gels of single fibers (Fig. 2). pK is the midpoint of the pCa/tension curve and nH is the Hill coefficientcalculated by fitting the data to the equation:
P/Po = {[Ca2+] • K}'/(1 + {[Ca2+] • K}"). pK = log (K) (Brandt et al., 1980,1982): Po is the maximum tension. The number of fibers refers to all the rows except the TnC (%) row. The S.E.M. values are listed after each value in the Table.
a b s o l u t e t e n s i o n a n d n H increased d r a m a t i c a l l y . T h e effect of p a r t i a l e x t r a c t i o n of TnC on the slope of the p C a / t e n s i o n r e l a t i o n is m a r k e d y e t reversed (Greaser & Moss, 1984) b y the a d d i t i o n of purified TnC. We conclude from this t h a t gaps in the i n t e g r i t y of the c h a i n of r e g u l a t o r y u n i t s on the t h i n filament, i n t r o d u c e d b y T n C e x t r a c t i o n , b r e a k t h e co-operative spread of a c t i v a t i o n along the t h i n filament. The t h i n f i l a m e n t consists of two helically i n t e r t w i n e d s t r a n d s of a e t i n a n d two s t r a n d s of t r o p o m y o s i n t h a t lie in the grooves of the a c t i n d o u b l e helix. T r o p o n i n is b o u n d to t r o p o m y o s i n e v e r y 38"5 n m along t h e t h i n filament. This m e a n s t h a t there are some 26 r e g u l a t o r y u n i t s (1000/38"5) per s t r a n d of t r o p o m y o s i n on a 1 p m t h i n f i l a m e n t of r a b b i t psoas. To d e t e r m i n e how m a n y u n i t s in a n u n b r o k e n sequence are r e q u i r e d to g e n e r a t e the n o r m a l large n H value, we h a v e e x t r a c t e d fibers for one m i n u t e or less, t h e n i n c u b a t e d t h e m with T n C a n d characterized the p C a / t e n s i o n r e l a t i o n after e x t r a c t i o n a n d a g a i n after i n c u b a t i o n . W e c a n n o t a c c u r a t e l y d e t e r m i n e small fractions of T n C loss b y gel electrophoresis of single fibers so we rely on the difference in t e n s i o n b e t w e e n e x t r a c t e d a n d i n c u b a t e d fiber to e s t i m a t e the e x t r a c t i o n of TnC. Gels of briefly e x t r a c t e d single fibers, however, do show less e x t r a c t i o n of T n C t h a n do gels of fibers e x t r a c t e d for two m i n u t e s or longer. The slope a n d t e n s i o n are higher for the p C a / t e n s i o n c u r v e t h a t follows i n c u b a t i o n w i t h T n C (Fig. l(e)) t h a n for the curve t a k e n after one m i n u t e of e x t r a c t i o n . F r o m such e x p e r i m e n t s we find t h a t e x t r a c t i o n of a n e s t i m a t e d 10% of the T n C s u b s t a n t i a l l y reduces the c o - o p e r a t i v i t y ; 10~/o of the 26 T n C s u b u n i t s
LETTERS TO THE EDITOR
383
of an intact regulatory strand consists of three subunits and three breaks generate four pieces. Thus, average sequences of intact regulatory units as long as six, or about one-quarter of the normal size, are less co-operative than the intact regulatory strand of 26 units. F r o m experiments similar to that shown in Figure l(c), but with extraction for 30 seconds, we found t h a t the n H of the pCa/tension relation was diminished, even if so little of the TnC was removed t h a t there was a 5% or smaller difference in tension between the extracted and incubated conditions. In 11 experiments, the mean n H after extraction was 3.5_+ 0.1 (S.E.M.), and following incubation in TnC for 25 minutes it was 5-0___0"19. The increase in n~ with TnC addition is significant at the 99~o level and into the control range (Brandt et al., 1982). Because co-operativity decreases with a loss of TnC estimated at 5~o or approximately one break per regulatory strand, we suggest that the intact thin filament co-operatively activates as a unit. Co-operativity in the thin filament control mechanism was originally proposed by Bremel & Weber (1972), who suggested that binding of myosin subfragment l to the thin filaments turns on the regulatory unit and enhances its affinity for calcium. Activation of the filament by S1 binding is supported by observations t h a t N-ethylmaleimide-treated S1, bound irreversibly to actin strands, enhances hydrolysis (Pemrick & Weber, 1976; Nagashima & Asakura, 1982). Greene & Eisenberg (1980) found t h a t S1 binds co-operatively to regulated actin in the absence of Ca :+ and substrate, but in their presence the high co-operativity of S1 binding almost disappears (Greene, 1982). These observations have been incorporated into models of thin filament regulation with assumptions of near neighbor tropomyosin-tropomyosin co-operativity (Hill et al., 1980,1983; Hill, 1984; Trueblood et al., 1982). Co-operative activation of hydrolysis by S1 binding or co-operative S1 binding in soluble protein systems differ from that reported here in several important ways. In our skinned fibers the contractile proteins are intact and the in vivo geometric constraints of the myofilament lattice are preserved. We studied activation of tension by Ca :+ in high concentrations of MgATP, conditions in which soluble systems show little co-operativity, and we found a high degree of co-operativity with m a n y regulatory units acting in concert. Although the mechanism for the co-ordination of regulatory unit activation along the thin filament is unknown it apparently depends on the presence of TnC in the regulatory unit. This work was supported by grants NIH NS-18228 (to F.H.S.) and the Muscular Dystrophy Association of America and NIH NS-117660 (to P.W.B.). Department of Anatomy and Cell Biology Columbia University New York, NY 10032, U.S.A.
P. W. BRANDT M. S. DIAMOND
Department of Anatomy Duke University Medical Center Durham, NC 27710, U.S.A.
F. H. SCHACHAT
Received 29 March 1984
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P . W . BI%ANDT, M. S. DIAMOND AND F. H. SCHA(~HAT
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Edited by H. E. ttuxley