Structure of the actin-myosin complex produced by crosslinking in the presence of ATP

,I.

MoZ.Biol. (1986) 191, 107-116

Structure of the Actin-Myosin Complex Produced by Crosslinking in the Presence of ATP Toshiaki Arata Department of Biology, Faculty of Science Osaka University, Toyonaka, Osaka 560, Japan (Received 29 November

1985, and in revised form

1 May 1986)

The structure of the actin-myosin complex during ATP hydrolysis was studied by covalently crosslinking myosin subfragment 1 (Sl) to F-actin in the presence of nucleotides (especially ATP) using I-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The fluorescence energy transfer was measured between N-(iodoacetyl)-N’-( I-sulfo-5-naphthyl)ethylenediamine and 6-(iodoacetamide)fluorescein bound to the SHl thiol of Sl and the Cys374 thiol of actin. The covalent acto-Sl, produced by crosslinking in the absence of nucleotide or in the presence of ADP, showed transfer efficiency of 0.50 to 0.52 and intersite distance of 4.5 to 4.7 nm, which were equal to those obtained with non-crosslinked acto-Sl in the absence of nucleotide. However, the covalent acto-Sl, produced by crosslinking in the presence of either 5’-adenylyl imidodiphosphate (AMPPNP) at high ionic strength or ATP, showed a significant decrease in the efficiency to 0.26 to 0.34 and hence an increase in the distance to 5.2 to 5.5 nm. These results suggest that AM-ATP and/or AM-ADP-P (formed during ATP hydrolysis) and AM-AMPPNP have a very different conformation from AM and AM-ADP (in which A is actin and M is myosin).

1. Introduction Muscle contraction results from the sliding of the thick (myosin-containing) filaments past the thin (actin-containing) filaments (Huxley & Niedergerke, 1954; Huxley & Hanson, 1954). The sliding force is generated by cyclic interaction of myosin heads (Sit) with F-actin. This reaction is coupled to ATP hydrolysis. Structural change is thought to occur in the actomyosin complex to produce a force (Huxley, 1969; Huxley & Simmons, 1971; Morales et al., 1982). Recent time-resolved X-ray diffraction studies on contracting muscle provided some evidence for a change in myosin head orientation on the millisecond time-scale (Huxley et al., 1981, 1983). However, although myosin heads (Sl) in the absence of ATP have been observed to bind to actin filament at a uniform angle (Huxley, 1963; Reedy et al., 1965; Borejdo & Putnum, 1977; Thomas $ Cooke, 1980; Yanagida, 1981), direct evidence on the detailed structure of the attached Sl during active t Abbreviations used: Sl, myosin subfragment 1; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; AMPPNP, 5’-adenylyl imidodiphosphate; IAEDANS, N-(iodoecetyl)-N’-( 1-sulfo-5-naphthyl)ethylenediamine; 6AF, 6-(iodoacetamido)fluorescein; HEPES, N-2-(hydroxyethyl)-piperazine-N’-2-ethanesulfonic

acid;

SDS, sodium dodecyl sulfate. 0022%2836/86/170107-10

$03.00/O

107

contraction (at other chemical states during ATP hydrolysis) has been difficult to obtain. Measurement of fluorescence energy transfer between two points within an acto-Sl complex has been useful for detecting a large-scale structural change in vitro (Morales et al., 1982; Trayer & Trayer, 1983). Electron microscopy of a negatively stained actomyosin complex was first reported by Huxley (1963) and has been used to observe single Sl molecules bound to the actin filaments (Craig et al., 1980). Three-dimensional reconstitution of the electron microscopic image has been used for determining the detailed a&o-S1 structure (first analyzed by Moore et al., 1970). However, these methods have not proved successful in experiments with ATP, because acto-Sl dissociates in its presence

at low

concentrations

of actin,

and

Sl.

Recently, Yamamoto & Sekine (1979) and Mornet et al. (1981) developed a method for covalently crosslinking Sl to actin in the absence of ATP using dimethylsuberimidate or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). This crosslinked acto-Sl complex has been studied by electron microscopy in the presence of ATP (Craig et al., 1985). Arata (1984) developed a method for crosslinking Sl to actin in the presence of nucleotide using EDC. The covalent acto-Sl complexes, produced by 0 1986 Academic Press Inc. (London) Ltd.

T. Arata

crosslinking in the absence of nucleotide and in the presence of ADP, showed high MgATPase activity, whereas those produced by crosslinking in the presence of ATP and AMPPNP showed low activity 1984). Therefore, (Arata, covalent acto-Sl complexes produced by crosslinking inthe presence of ATP and AMPPNP may have very different conformations from the complexes produced in the absence of nucleotide and the presence of ADP. In this study, we report on the structures of these crosslinked acto-Sl complexes examined by the fluorescence energy transfer, and provide evidence for a large-scale structural difference between the covalent acto-Sl complexes produced in the absence and presence of ATP.

2. Materials and Methods (a) Proteins Myosin was purified from skeletal white muscle by the method of Perry (1955). Sl was prepared by chymotryptic digestion of myosin as described by Weeds & Taylor (1975). Actin was prepared from an acetone powder of rabbit skeletal white muscle by the method of Spudich k Watt (1971). Pyruvate kinase was prepared from rabbit skeletal muscle as described by Tietz & Ochoa (1958). Protein concentrations were determined by means of the Biuret reaction calibrated by nitrogen determination. The molecular weights of Sl and actin monomer were assumed to be 1.2 x 10’ (Lowey et al., 1969) and 4.2 x lo4 (Elzinga et al., 1973), respectively. (b) Chemicals and Jluorescent probes EDC was purchased from Nakarai Chemicals. Ltd, Kyoto. ATP and ADP were purchased from Kohjin Co. Ltd, Tokyo. AMPPNP, phosphoenolpyruvate and diadenosine pentaphosphate were purchased from Sigma Chemical Co., St Louis, MO. IAEDANS and 6IAF were purchased from Molecular Probes, Plano, TX. The concentrations of IAEDANS and 6IAF were calculated using the molar extinction coefficients at 337 and 496 nm of 6.8 x lo3 and 7.1 X 104 M-l cm-’ (at pH 82), respectively. (c) Fluorescence labeling of Sl and

The reaction was terminated with 10 miw-dithiothreitol. F-actin was then precipitated by centrifugation at 120,OOOg for 1 h. The pellet was gently homogenized using a Teflon-glass homogenizer and dissolved in 50 mMKCl, 1 miu-MgCl,, 10 mi+%-Tris* HCl (pH 8.2). The F-actin solution was centrifuged again at 120,OOOg for 1 h and the resulting pellet was homogenized and dissolved in the same buffer. The denatured protein was removed by centrifugation at 10,000 g for 15 min. (ii) Determination of the amount of bound Jluorophore The concentration of AEDANS bound to Sl or actin was determined by comparison of the fluorescence intensity of labeled protein with that of standard solutions of AEDANS-N-acetylcysteine adduct,, both denatured in 6 iw-guanidine ’ HCl. In the second method, from these data we made a calibration curve relating the concentration of bound AEDANS to the absorbance at 337 nm under non-denaturing conditions. Using this calibration curve, the concentration of AEDANS bound to Sl or actin was determined from the absorbance at 337 nm. The concentration of 6AF bound to Sl or actin was determined from the absorbance at 496 nm in the presence of 5 M-urea using an extinction coefficient of 7.7 x lo4 M-l cm-’ (Takashi, 1979; Ando, 1984). The concentration of fluorophore bound to protein ranged from 0.7 to 0.9 mol of fluorophore per Sl and from 0.9 to 1.2 mol per actin monomer. (iii) Acto-Sl ATPase activity of labeled protein The actin-activated ATPase activities of unlabeled and labeled Sl were examined in 0.5 m&r-ATP, 6 mM-KCl, 2 m&i-MgCl,, 10 mM-HEPES (pH 7.0) at 2O”C, as a function of increasing concentration of unlabeled and labeled F-actin. The maximum activities (V,,,) and the concentrations of actin required for half-maximum activity (K,) of various acto-Sl systems were as follows: actin + Sl, 11 s-l, 51 PM; actin + AEDANS-Sl, 8 s-i, 14 PM; actin + 6AF-Sl. 8 s-l, 16 PM; AEDANS-act,in + sl. 9S-‘. 15jiM; 6AF-actin + Sl, 10~~‘. 15~~; AEDANS-actin + BAF-Sl, 7 s- ‘. 11 PM; 6AF-actin + AEDANS-Sl, 6 s- ‘, 9 PM. In general, K, was less in a doubly or singly labeled acto-Sl system than in the unlabeled acto-Sl system, but V,,, was always lower. This agrees with the reports of Takashi (1979) and Trayer & Trayer (1983), and shows that the labeling of either Sl or actin does not seriously abolish the cyclic interaction of Sl with actin and ATP.

actin

(i) Fluorescence labeling Sl was labeled at the SHl thiol with IAEDANS or 61AF using the method of Arata (1984), which is essentially the same as those of Botts et al. (1979) and Marsh & Lowey (1980). Sl (10 to 50 mg/ml) was incubated with a l.l-fold molar excess of IAEDANS or 6IAF in 50 m&r-KC1 and 10 mmimidazole at pH 7.0 and 0°C for 24 h in the dark. The reaction was terminated with 10 miw-dithiothreitol, and unreacted probe was removed by exhaustive dialysis against 10 miw-imidazole, 6.1 mM-EDTA at pH 7.0 and 4°C. The denatured protein was removed by centrifugation at 120,OOOg for 1 h. Actin was labeled at the Cys374 thiol with IAEDANS or 6IAF using essentially the same method as Lin & Dowben (1983) and Yasui et al. (1984). F-actin (x 2 mg/ml) was incubated with a Z-fold molar excess of probe over actin monomer in 0.15 M-KCl, 1 mrvr-MgCl,, 10 miv-Tris. HCl (pH 8.2) at 0°C for 18 h in the dark.

(d) Crosslinking

of acto-Sl

complex

SI was crosslinked to actin using EDC. under 4 different conditions: A, -nucleotide: B. +ATP: C. +AMPPNP; D, +ADP (Arata, 1984). For the energytransfer measurements, fluorescently labeled or unlabeled Sl (6 to 10 PM) was mixed with fluorescently labeled or unlabeled F-actin (3 PM-monomer) in 2 m&r-MgCl,, 20 mM-HEPES (pH 7.0) at 20°C. The crosslinking reaction was started by adding 2 to 4 miw-EDC, which had been dissolved immediately prior to use in 0.1 MHEPES at pH 7.0, to 40 mm In an alternative method for condition A, F-actin was incubated with 2 to 4 mmEDC for 2 min and then mixed with Sl. This procedure resulted in a large extent of crosslinking. For condition B, MgATP (~2 InM) was added to the reaction medium at 15-min intervals. For condition C, MgAMPPNP (5 mm) was added initially to the medium at KC1 concentrations of 0, 0.5 or 1.0 M. For condition D, MgADP (5 InM) and 0.1 mM-diadenosine pentaphosphate (adenylate kinase

Structure of Act&31 inhibitor) were added initially. The reaction was allowed to proceed for 90 min. At high ionic strength (condition C) where the reactivity of EDC was low (Arata, 1984), the crosslinking reaction was allowed to proceed for 3 to 4 h. In all the cases, the crosslinking was terminated by adding 50 mw-mercaptoethanol, 5 mM-ATP, 1.0 M-KCl, 6 mM-MgCl,. The mixture was centrifuged at 120,OOOg for 30 min at 4°C to remove the non-crosslinked Sl. Control experiments with a mixture of the noncrosslinked Sl (10 FM) and actin (3 PM) showed that less than 5% of the Sl was sedimented with actin under these conditions as determined by fluorimetry (section (f)). The pellet was washed with a buffer solution (20 mM-HEPES at pH 7.0) at 0°C and then gently homogenized in 20 mMHEPES (pH 7.0) at 0°C and finally dissolved in 0.1 MKCl, 5 mM-MgCl,, 20 mM-Tris . HCl (pH 8.2). For condition B, the crosslinking was sometimes repeated 2 or 3 times; the crosslinked preparations (in 20 PM-HEPES) isolated by removing the non-crosslinked Sl were mixed again with 61 (6 to 10~~) in the presence of EDC and ATP, and the crosslinking reaction in each cycle was allowed to proceed for 50 min. The amounts of free Sl that remained in all the crosslinked preparations were determined from SDS/polyacrylamide gel electrophoresis to be less than 1.5% of the amount of Sl added to the reaction mixture. Immediately after crosslinking, the preparation was used for fluorescence measurements.

Crosslinked in ATP

109

because there was no AEDANS fluorescence and no energy transfer from AEDANS. Close agreement in the ratio of crosslinked Sl to the actin was found between the direct and the indirect methods. The fluorimetric quantification of the concentration of AEDANS-labeled protein requires that no energy transfer between the probes occurs under the denaturing conditions. The singly and doubly labeled preparations were set to contain the same concentration of 6AF using the above determination. The fluorescence intensities of 6AF in the singly and doubly labeled preparations were measured under the denaturing conditions, at 520 nm emission upon excitation at 340 nm, and were corrected for the contribution of AEDANS fluorescence. In this wavelength setting, the intensity of doubly labeled preparation would include sensitized emission and would be higher than that of singly labeled preparation if transfer occurs. However, the measured intensities were found to be the same in the singly and doubly labeled preparations. In the second experiment, the singly and doubly labeled acto-Sl mixtures were set to contain the same concentration of AEDANS. The intensity of AEDANS at 480 nm emission upon excitation at 340 nm was measured under the denaturing conditions in the singly and doubly labeled acto-Sl mixtures which were just crosslinked but not separated from non-crosslinked Sl. Again, the measured intensities were found to be the same in the single and double labeled mixtures.

(e) Fluorescence measurements Steady-state fluorescence measurements were made using cells of 1 cm path-length (sample volume of 2.5 ml) with a Hitachi Perkin-Elmer MPF-4 fluorescence spect’rophotometer equipped with a spectrum-correcting capability. a temperature-controlled cell holder, and a built-in magnetic stirrer. Concentrations of labeled which proteins were in the range 0.1 to 1 PM, corresponded to absorbances of less than 0.02 at the exciting wavelength. For an experiment with a noncarosslinked arto-81 preparation at high concentrat,ions (2 PM-AEDAX-Sl and 4 PM-6AF-a&in). a cell of 2 mm path-lrnpth was used. (f) Concentration of Sl and actin in the crosslinked preparation The concentrations of labeled Sl and actin in the crosslinked preparation were determined indirectly by subtracting from the concentrations of total Sl and actin added, those of Sl and actin remaining in the supernatant after centrifugation with ATP (see section (d), above). The concentrations were also determined directly with the crosslinked preparation isolated by resuspending the pellet after centrifugation with ATP. The concentrations of fractions or preparations were quantified fluorimetrically by comparison with EDCtreated, singly labeled proteins of known concentration. In both indirect and direct determinations, fluorescence emission spectra were recorded under denaturing conditions: 6 M-guanidine . HCI, 0.1 M-KCl, 5 mM-MgCl,, 20 mM-Tris. HCl (pH 8.2) and 2O”C, and were corrected for the small contribution of light-scattering with unlabeled samples. For the concentration of AEDANSlabeled proteins, direct comparison was made at 480 nm emission upon excitation at 340 nm because there was no contribution of the fluorescence of the second fluorophore 6AF. For the concent’ration of BAF-labeled proteins in the doubly labeled samples, direct comparison was also made at 520nm emission upon excitation at 460nm

(g) Measurement of energy transfer Transfer of resonance energy from the donor (AED48S) to the acceptor (6AF) both attached to Sl and actin was measured by the steady-state method. Typically, 4 preparations were used for each set of experiments: (1) AEDANS-Sl crosslinked to actin; (2) Sl crosslinked to GAB-actin; (3) AEDANS-51 crosslinked to BAF-actin; (4) AEDANS-Sl alone treated with EDC. Alternatively, the labeled proteins were reversed such that AEDANS-actin was crosslinked to GAF-Sl. Preparations (1). (3) and (4) were set to contain t)hc same concentration of AEDANS-Sl, while preparations (2) and (3) were set to contain the same caoncentration of HAF-actin. Transfer efficiency was determined at an excitation wavelength of 340 nm and an emission wavelength of 475 nm, which contains no acceptor emission. The transfer efficiency. E, was given by the equation: E = 1- FDAjFD, (1) where F, is the molar fluorescence intensity of donorlabeled Sl (actin) crosslinked to unlabeled actin (Sl) and FDA is the molar intensity of donor-labeled 81 (actin) crosslinked to acceptor-labeled acein (Sl). This equation is only applicable to the situation where each protein js labeled with 1 mol of donor or 1 mol of acceptor und complete crosslinking of donor-labeled protein is achieved. Therefore, it was necessary to correct for nonstoichiometric protein labeling and for the presence of any non-crosslinked donor-labeled protein as given by Takashi (1979): FD = @W’Cfo-fdl-4) FDA = (daz)-l(fD,-d(l-a)rFD-f,(l-~)).

(2)

(:I) where fn. fDA and fd are the total fluorescence intensities of preparations (l), (3) and (4), respectively, d and a are the fractions of incorporation of donor and acceptor into proteins, respectively, and z is the crosslinking extent of

110

T. Arata

the donor-labeled protein. Efficiency E was calculated using equations (1) to (3). The experiments were done mainly with the crosslinked complex of AEDANS(donor)-Sl and 6AF(acceptor)-actin, which was separated from the non-crosslinked Sl. Absence of non-crosslinked donor-labeled Sl provides the best condition, i.e. x = 1, which simplifies the calculation to give an accurate value of E. The measured transfer efficiency was used to calculate the distance, R, between the donor and acceptor by the equation: E = R;/(R:+ R6). (4) R, is the critical transfer distance at which the efficiency of transfer is 0.5 and is given by the equation: Rg = (8.79 x 10-11)n-4Qk2J

and 6AF (acceptor) on Cys374 of actin in the crosslinked acto-Sl preparation. The crosslinked acto-Sl was produced in the absence of nucleotide by using EDC and then isolated by dissociating the non-crosslinked Sl in the presence of ATP. The ratio of crosslinked AEDANS-SI to 6AF-actin was determined fluorimetrically (see Materials and Methods), and was 0.42 in this preparation. The

presence of energy transfer can be detected as quenched donor emission at 475 nm. The donor emission intensity was significantly less (46%) in the doubly labeled, crosslinked acto-Sl complex (curve 3) than in the singly donor-labeled complex (curve

(nm6).

(5) In this equation, n is the refractive index (taken as 1.4), Q is the quantum yield of the donor, k2 is the orientation factor, which takes on values from 0 to 4, and J is the spectral overlap integral between the emission spectrum of the donor and the absorption spectrum of the acceptor. Q was determined using a standard quinine sulfate solution (in 0.02 M-H,SO,) with a quantum yield assumed to be 0.70 (Scott et al., 1970). J was approximated by the following summation:

(6)

J = (Cfd(n)e.(n,n4A~XCfd(1)A~)-l,

where f,(n) is the fluorescence intensity of the singly donor-labeled protein and E,(L) is the molar extinction coefficient, of the acceptor at wavelength 1. The summation was taken over 5-nm intervals. k2 was initially given the value 2/3. For AEDANS-Sl and BAF-actin, Q = 0.59, J = 1.14 x 1015 M-~ cm-’ (nm)4 and R,(2/3) = 4.6 nm. For AEDANS-actin and 6IAF-Sl. cm-’ and J = 1.31 X 1o15 M-l Q = 0.62, (W4 R,(2/3) = 4.7 nm. These values were almost the same before and after the treatment of proteins with EDC.

1). Energy

transfer

was

also

detected

as

sensitized acceptor emission at 520 nm by comparing the emission of doubly labeled complex (curve 3) with the spectrum obtained by direct addition of the spectra of the singly labeled complexes (curve 1 + curve 2). Addition of MgATP to the crosslinked preparation caused a small change from 46 to 42% in the quenching of donor emission (broken curves). The transfer efficiency, E, per mol of crosslinked acto-Sl complex (molar transfer efficiency) was calculated from the quenched donor emission using equations (1) to (3) (Materials and Methods). Since calculation of the efficiency from the sensitized acceptor emission is complicated by the presence of

1001

I

I

I

(h) ATPase measurements All the ATPase assay systems contained 0.2 mg and 2 mM-phosphoenolpyruvate. pyruvate kinase/ml The ATPase activity was determined from pyruvate which was measured by the methods of liberation, Reynard et al. (1961). Acto-Sl MgATPase activity of the labeled proteins was measured in 1 PM-Sl, 0.05 to 2 mg F-actin/ml, 0.5 mM-ATP, 6 mM-KC& 2 miw-MgCl,, 10 mM-HEPES (pH 7.0), at 20°C. The maximum ATPase activity and the actin concentration for half-maximal activity were determined from double reciprocal plots of concentration. The the activity versus the actin MgATPase activity of the crosslinked acto-Sl preparation was measured in 0.5 mM-ATP, 50 mM-KCl, 2 m&i-Mgcl,, 10 mM-HEPES (pH 7.0), at 20°C. (i) SDSlpolyacrylamide

gel electrophoresis

SDS/polyacrylamide gel electrophoresis (8 or 12.5% (W/V) acrylamide) was carried out according to Laemmli (1970) with staining by Coomassie blue. Gels were scanned with a Fuji-Riken densitometer FD-A4.

3. Results (a) Fluorescence energy transfer in the covalent complex formed by crosslinking Sl to actin in the absence of nucleotide

Figure transfer

1 shows a typical between

AEDANS

example (donor)

of energy

on SHl

of Sl

Wavelength

(nm)

Figure 1. Fluorescence energy transfer in the covalent acto-Sl complex produced by crosslinking in the absence of nucleotide. Emission spectra were recorded in O-1 MKCl, 5 mM-MgCl,, 20 mmTris . HCl (pH 82), at 20°C. Excitation wavelength was 340 nm. Curve 1, AEDANSSl crosslinked to actin; curve 2, Sl crosslinked to 6AFactin; curve 3, AEDANS-Sl crosslinked to 6AF-actin. The concentratiohs of AEDANS-Sl and BAF-actin in the crosslinked preparations for curves 1 to 3 were 0.21 and 0.50 P(M, respectively. The concentrations of unlabeled actin and Sl in the preparation for curves 1 and 2 were not, determined. Labeling stoichiometry: 0.88 mol. AEDANS/mol Sl; 1.08 mol GAF/mol actin. The preparations contain virtually no non-crosslinked Sl and hence all the Sl is crosslinked to actin (crosslinking extent z = 1). Broken curves represent the spectra obtained after the addition of 2 mM-MgATP to the crosslinked preparation.

Structure of Acto-Sl

111

Crosslinked in ATP

Table 1 Eflciency

of fluorescence energy transfer in crosslinked acto-Sl complexes

Location of AEDANS

Nucleotide in crosslinking medium

BAF

Crosslink

Sl

Actin

-

Sl

Sl

Actin Actin Actin Actin Actin

+ + + + +

None ADP AMPPNP AMPPNP. ATP

Actin Actin

Sl Sl

+ +

None ATP

Sl Sl Sl

Sl/actin (mol/mol)

1.0 ~-Kc1

R(213)

Et (mean+S.D.(n))

(nm)

0.50

0.48f0.02(2)

4.7

0.4-0.6 0.67 0.40 0.2-0.4 0.2-0.7

0.52+0.05(3) 0.50 0.52 0.3350.04(3) 0.26+0.04(T)

4.5 4.6 4.5 5.2 5.5

0.5-0.7

0.52+0.04(3) 0.34&0.03(3)

4.7 5.3

0.2-0.9

t E was calculated by assuming that all of the Sl present binds to actin.

considerable donor fluorescence at 520 nm, it was not attempted. In this preparation, non-crosslinked AEDANS-Sl was removed by sedimentation with MgATP (i.e. the crosslinking extent of AEDANSSl (z) = l), to simplify the calculation. From the data of Figure 1, the efficiency E was calculated to be 0.46. For ratios of crosslinked Sl to actin from O-4 to 0.6, the value of E remained constant at 0.46 to 0.56 (Table 1). The energy transfer was also measured using the crosslinked acto-Sl mixture in the presence of MgATP, just before centrifugal isolation (data not shown). Although this mixture still contained much non-crosslinked Sl (the crosslinking extent of Sl (x) = 0.29), the calculated value of E was found to remain unchanged (~0.45). When the probes were reversed (i.e. 6AF-SI and AEDANS-actin were crosslinked), the transfer efficiency E was also unchanged (0.48 to 0.56; Table 1). The energy transfer was also measured in the non-crosslinked complex of AEDANS-Sl and BAF-actin in the absence of ATP. The value of E was estimated to be 0.46 to 0.50 (Table 1). that fluorophores are in randomizing Assumin motion (k F-- 2/3), the distance R(2/3) between donor and acceptor was calculated from R,(2/3) as described in Materials and Methods. The results are shown in Table 1; the distance between AEDANS and 6AF in the crosslinked complex was 4.5 to 4.7 nm.

(b) Energy transfer in the complex formed by crosslinking Sl to actin in the presence of ATP Figure 2 shows a typical example of energy transfer between AEDANS-Sl and 6AF-actin in the crosslinked preparation that was produced in the presence of MgATP and then isolated by dissociating the non-crosslinked Sl. In this preparation, crosslinking was repeated twice and the ratio of crosslinked AEDANS-Sl to 6AF-actin was 0.40. The donor fluorescence at 475 nm was quenched by only 24% (compare curve 3 with curve 1). The molar efficiency E was calculated to

be 0.26. The value of E remained constant at 0.22 to O-34, when the crosslinking conditions were changed; the length of crosslinking (50 to 120 min) and the number of crosslinking cycles (1 to 3) varied the ratio of crosslinked Sl to actin from 0.2 to 0.7 (Table 2). This E value was much less than that obtained for the complex produced in the absence of nucleotide (0.52). The addition of MgATP to the crosslinked preparation caused no significant change in the energy transfer (broken curves in Fig. 2). The addition of unlabeled Sl

100,

I

400

450

3

500 Wavelength

I

550

600

(nm)

Figure 2. Energy transfer in the covalent acto-Sl complex produced by crosslinking in the presence of MgATP. Emission spectra were recorded under the same conditions as for Fig. 1. Curve 1, AEDANS-Sl crosslinked to actin; curve 2. Sl crosslinked to 6AF-actin: curve 3, AEDANS-Sl crosslinked to 6AF-actin. The crosslinking was repeated twice. The concentrations of AEDANS-Sl and GAF-actin in the crosslinked preparations for curves 1 to 3 were 0.16 and 0.40~~, respectively. The concentrations of unlabeled actin and 81 in the preparations for curves 1 and 2 were not determined. Labeling stoichiometry: 0438 mol AEDANS/mol Sl; 0.91 mol 6AF/mol actin. The preparations contain virtually no non-crosslinked Sl and hence all the Sl is crosslinked to actin (z = 1). Broken curves represent the spectra obtained after the addition of 2 mM-MgATP.

112

T. Arata

Table 2 Effects of crosslinking cycles and time on the energy-transfer efficiency of the covalent a&o-S1 complex produced by crosslinking in the presence of ATP

Cycles

Crosslinking time/cycle (min)

AEDANS-Sl/GAF-actin (mol/mol)

E

R(2/3) (nm)

3 3

50 50

0.61 0.68

0.34 0.26

5.1 5.5

2 2

50 50

0.40 0.28

0.26 0.25

5.5 5.5

1 1

50 50

0.26 0.18

0.24 0.22

5.6 5.7

1

120

0.54

0.26

5.5

(5 PM) or actin (10 PM) caused no significant change in the energy transfer (data not shown). The energy transfer was also measured using the acto-Sl mixture in the presence of MgATP before centrifugal isolation (data not shown). The calculated E value was x0.15, although this value may not be accurate because this mixture contained non-crosslinked AEDANS-Sl (x = 0.26). When the probes were reversed, the transfer efficiency was a little higher; E = 0.30 to 0.38 (Table 1). Assuming k2 = 213, the distance R(2/3) between donor and acceptor in the crosslinked complex, produced in the presence of ATP, was calculated to be 5.3 to 5.5 nm (Table 1). (c) Energy transfer in the complexes formed by crosslinking Sl to actin in the presence of AMPPNP and ADP Figure 3(a) and (b) shows typical examples of energy transfer in the covalent acto-Sl complexes

Wavelength

produced by crosslinking in the presence of MgAMPPNP at low and high ionic strengths, respectively. The transfer efficiency, E, in the covalent acto-Sl complex produced by crosslinking in the presence of 5 mM-MgAMPPNP at low ionic strength (in 0 M-KCl) was 0.52, which provides 4.5 nm as the distance R(2/3). However, when the covalent acto-Sl complex was produced by crosslinking in the presence of MgAMPPNP at high ionic strength (in 1-OM-KCI), the E value decreased to 0.33. It should be noted that the affinity of Sl for actin in the presence of MgAMPPNP is still high at high ionic strength when the SHl on Sl has been modified

(Furukawa

&

Tonomura,

1982).

The

distance R(2/3) obtained at high ionic strength increased to 5.2 nm and became closer to that of the crosslinked complex produced in the presence of ATP (5.5 nm). The transfer efficiency, E, in the covalent complexes obtained by crosslinking AEDANS-Sl to 6AF-actin in the presence of 5 mM-MgADP was

(nm)

Figure 3. Energy transfer in the covalent acto-Sl complexes produced by crosslinking in the presence of MgAMPPNP at (a) low (OM-KCI) and (b) high (~.OM-KCI) ionic strengths. Emission spectra were recorded under the same conditions as for Fig. 1. Curve 1, AEDANS-Sl crosslinked to actin: curve 2, Sl crosslinked to GAF-actin; curve 3. AEDANS-Sl crosslinked to 6AF-actin. The concentrations of AEDANS-Sl and GAF-actin in the crosslinked preparations for curves 1 to 3 of (a) were 0.18 and 0.45 /AM, respectively. The concentrations of AEDANS-Sl and 6AF-actin in the preparations for curves 1 to 3 of (b) were 0.09 and 0.48 PM. respectively. The concentrations of unlabeled actin and Sl in the preparations for curves 1 and 2 of (a) or (b) were not determined. Labeling stoichiometry: 0.88 mol AtiDANS/mol Sl: I.08 mol 6AF/mol actin. The preparations contain virtually no non-crosslinked Sl and hence all the Sl is crosslinked to actin (z = 1).

Structure of Acto-Sl 0.50 and indistinguishable from that obtained by crosslinking in the absence of nucleotide. The distance R(2/3) was calculated to be 4.6 nm (Table 1).

4. Discussion By covalently crosslinking Sl to actin in the presence of various nucleotides with the zero-length crosslinker EDC, we have been able to use fluorescence energy transfer to study the structure of the acto-Sl complexes formed during ATP hydrolysis. Fluorescence energy transfer is useful for detecting large-scale structural changes in the acto-Sl complex in vitro (Morales et al., 1982). However, this method has not been used for noncrosslinked acto-Sl in the presence of ATP, because Sl binds too weakly to actin at low concentrations of Sl and actin. The present study shows that the structure of the covalent acto-Sl complex formed by crosslinking in the presence of ATP is very different’ from the covalent’ complex formed in its absence, suggesting that a non-crosslinked acto-Sl complex also has very different structures in the presence and absence of ATP. The structure of the acto-Sl complex in the absence of nucleotide, the AM (A is actin and M is myosin) or rigor state, has been extensively studied with various techniques including fluorescence energy transfer. Takashi (1979) and Trayer & Trayer (1983) measured the energy transfer between AEDANS and 5-(ac$amido)fluorescein attached to the Cys374 on actin and the SHl on Sl. They found that 30 to 50% of the energy absorbed to the dansyl was transferred to fluorescein and that the same efficiency could be obtained upon reversal of the donor and acceptor attachment sites. It was therefore concluded that, assuming two fluorophores were in a randomizing motion (L? = 2/3), the distance R(2/3) between SHl and Cys374 could be 4.5 to 6 nm. These results were confirmed in the present study for both the noncrosslinked and the crosslinked a&o-S1 complexes. The energy transfer between AEDANS and 6AF attached to the SHl on Sl and the Cys374 on actin was measured, and the efficiency was found to be 0.52 in the acto-Sl complex that was crosslinked in the absence of nucleotide (Fig. 1 and Table 1). The distance R(2j3) was calculated to be 4.5 nm. This distance was unaffected by reversal of the donor and acceptor sites (4.7 nm) and was very close to that in the non-crosslinked acto-Sl complex (4.7 nm). Thus, crosslinking does not seem essentially to alter the structure of the rigor complex. In the presence of ATP, Sl bound to F-actin exists as the species, AM-ATP and AM-ADP-P (Inoue et al., 1981; Stein et al., 1984; Rosenfeld & Taylor, 1984). The cross-linked acto-Sl produced in the presence of MgATP showed a small energy transfer; the efficiency was only 0.26 to 0.34 (Fig. 2, and Tables 1 and 2). The distance between the SHl on Sl and the Cys374 on actin was 5.3 to 5.5 nm,

Crosslinked in ATP

113

which was significantly greater than that in the absence of nucleotide (4-5 to 4-7 nm). Thus, the crosslinked acto-Sl complex produced in the presence of ATP has a different structure from that produced in its absence, strongly suggesting that AM-ATP and/or AM-ADP-P have very different structures from AM. This is consistent with the previous finding that the crosslinked acto-Sl produced in the presence of ATP showed lower ATPase activity than that produced in it,s absence (Arata, 1984). There can be lit’tle doubt that, all of the Sl is crosslinked specifically to act’in in the efficiency is presence of ATP. The transfer to crosslinking cycle and t,ime, insensitive indicating that EDC treatment does not provide artifactual species (Table 2). Moreover, EDC treatment of actin or Sl does not significantly affect the acto-Sl ATPase characteristics (Arata, 1984; King & Greene, 1985). The finding that the ATPase activity of crosslinked complex was not enhanced further by exogeneous F-actin showed that the act,in binding site of Sl in the crosslinked complex does not interact with exogenous F-actin and is completely occupied by actin (Arata. 1984), although steric constraints of non-specific crosslinking may prevent this interaction. Moreover, the crosslinking rate was proportional to the population of acto-Sl complex present prior to the start of the crosslinking reaction (Arata, 1984). It is also important to examine the structure of the crosslinked acto-Sl complex produced from the other biochemically defined states by the use of ATP analogs. The intermolecular distance R(2/3) estimated from energy-transfer measurement was rigor-like (4.6 nm) in the crosslinked acto-Sl complex that was produced in the presence of MgADP (Table 1). However, the distance R(2/3) in the crosslinked acto-Sl that was produced in the presence of MgAMPPNP depended on the ionic strength of the crosslinking medium. The R(2/3) value was rigor-like (4.5 nm) in the crosslinked acto-Sl produced at, low ionic strength. On the other hand, the crosslinked acto-Sl produced at high ionic strength showed a significant decrease in efficiency and hence an increase in R(2/3) to 5.2 nm (Fig. 3 and Table 1). These data suggest that the AM-ADP and AM-AMPPNP at low ionic strength have structures similar to AM, and that .4MAMPPNP at high ionic strength has a structure similar to AM-ATP and/or AM-ADP-P. However, the AM-AMPPNP structure at high ionic strength seems still to be an intermediate between rigor and ATP-induced structures. Therefore. it is possible that AM-AMPPNP exists in (at, least) two fundamental configurations with their proportion being varied by nucleotide species and ionic strength, or that there are intermediate configuration(s) that can be made by adding various nucleotides and by varying ionic strength. This may be related to the AMPPNPor ATP-induced structure undergoing a serial motion as it changes from one configuration to another.

114

T. Arata

AM-AT-AWAtP-P FlUOEhSCUlC~ energy transfer

10

0.26-0.34 5.2-5.5

0.50-0.52 “m

4.5-4.7

“In

Figure 4. Diagram relating the actomyosin ATPase mechanism to the structural change in the acto-Sl complex. The acto-Sl structures were drawn on the basis of the data of the energy-transfer measurements of the covalent acto-Sl complexes produced by crosslinking in the presence of various nucleotides. See the text for details.

A potential problem arises from the fact that the relative orientation of the donor and acceptor pair is not known. The orientation factor, k2, was taken as 213, which assumes that both the donor and acceptor are in random motion. The possible range of dipole motion is restricted when a fluorophore is bound to a protein. Then this approximation becomes questionable. Upper and lower limits can be placed on k2 and hence on an interprobe distance R, by using fluorescence anisotropy to estimate the degree of rapid probe randomization (Dale et al., 1979). If we take the limits estimated by Trayer & Trayer (1983) and Torgerson & Morales (1984), for IAEDANS and 61AF located on the SHl of Sl and the Cys374 of actin, an uncertainty of 24 to 30% needs to be applied to R in the present measurements. The crosslinked complex produced in the absence of nucleotide or in the presence of ADP shows an interprobe distance R ranging from 3.2 to 6.1 nm (R(2/3) = 4.5 to 4.7 nm). The crosslinked complex produced in the presence of ATP or AMPPNP shows the R ranging from 3.6 to 7.2 nm (R(2/3) = 5.2 to 55 nm). These ranges in R overlap considerably. A change in k2 (probably due to a change in the relative orientation of actin and Sl) would explain the apparent difference of 0.6 to 1.0 nm in the distance R(2/3) that is observed between two groups of crosslinked complexes. Calculations using equation (5) (Materials and Methods) indicate that k2 would have to change by a factor of 2 to 3. However. the multiplicity of emission transition dipole absorption or orientations would promote k2 + 213 and reduce uncertainty further. IAEDANS is known to have more than one transition dipole orientations (Hudson & Weber, 1973). Since R is insensitive to a reversal of the attachment sites of donor and acceptor, which would induce a change in their relative dipole orientations, the choice of 213 for k2 may be a valid approximation. Crosslinked acto-Sl complex showed high which is similar to the MgATPase activity, maximum activity of the non-crosslinked acto-Sl

system (Mornet et al., 1981). However, addition of MgATP to the crosslinked complex induced no, or a small (0.02 to O-04), decrease in the energy-transfer efficiency (Figs 1 and 2). Moreover, both ATPase activity (Arata, 1984) and energy-transfer efficiency (Table 1) of the crosslinked complex depend on the presence and species of nucleotide during the crosslinking reaction. Therefore, crosslinking seems to stabilize the structure of the acto-Sl complex and no, or a limited, large-scale structural change seems to occur in the crosslinked acto-Sl complex during high ATPase activity. (Since the ATPinduced change in the transfer efficiency is of the right direction but of low magnitude, the structures imposed on the complexes by the crosslinking process may retain less flexibility.) However, the addition of MgATP caused a drastic change in the structure of crosslinked acto-Sl observed by electron microscopy; the Sl became shorter and fatter (Arata, unpublished results; Craig et al., 1985). It is important to identify primary sequences involved in the crosslinking of Sl to actin in the presence of either ATP or AMPPMP. In a rigor acto-Sl complex, the crosslinking reaction occurs between the amino-terminal acidic se ment of aetin and either the 20 x lo3 M, or 50 x 105 M, fragment of Sl to produce a doublet with a molecular weight of 180 x lo3 on SDS/polyacrylamide gels (Mornet et al., 1981; Sutoh, 1982, 1983). Preliminary experiments showed that either the 20x lo3 M, or 50 x lo3 M, fragment of Xl was crosslinked to actin even in the presence of ATP or AMPPNP and a similar doublet was produced (Arata, unpublished results; cf. Chen et al., 1985). Although a more detailed analysis is required, this result suggests that similar crosslinking stabilizes the a&o-S1 structure in different ways. Mornet & Ue (1985) raised the possibility that Sl can move by alternating 20 x lo3 M, and 50 x lo3 M, actinbinding sites, since they never found a covalent complex of both 20x lo3 M, and 50 x lo3 M, fragments with actin. The evidence from energy-transfer measurements is closely related to the mechanism by which the energy of ATP hydrolysis is converted to mechanical motion (Fig. 4). SHl is located in the center of the Sl molecule (Sutoh et al., 1984) and Cys374 at the outer radius of the actin filament (Safer et al., 1985). The distance R(2/3) between SHI and Cys374 is 4-5 to 4.7 nm in the AM and AM-ADP states, On the other hand, the R(2/3) is 5.2 to 5.5 nm in the AM-ATP and/or AM-ADP-P states. Recently, Bhandari et al. (1985) reported that the distance R(2/3) between the single cysteine of the light chain 1 on Sl and the Cys374 of actin was 5 to 6 nm in the AM and AM-ADP states, whereas it decreased to less than 3 nm in the AMATP and/or AM-ADP-P states. Numerous studies on the actomyosin ATPase mechanism in solution have led to the conclusion that ATP is hydrolyzed via a dissociating pathway (AM -+ AM-ATP + A+M-ATP + A+M-ADP-P

Struhure of A&-S1 + AM-ADP-P -+ AM-ADP + AM +ADP + Pi) and also via a non-dissociating pathway (AM + + AM-ADP-P + AM-ADP + AM-ATP AM+ADP+Pi; Inoue et al., 1973, 1981; Stein et al., 1979; Rosenfeld & Taylor, 1984). Recent studies have focused on the question of which chemical step is coupled to force generation (Arata & Shimizu, 1981; Yanagida et al., 1982; Nagano & Yanagida, 1984: Hibberd et al., 1985). One attractive possibility raised by the present study is that force generation occurs as a result of transition between long and short-R(2/3) structures, which is driven by a chemical reaction AM-ADP-P + AM-ADP (see Fig. 4). This mechanism is essentially the same as those originally proposed by Huxley (1969) and Huxley & Simmons (1971). Moreover, the reaction AM-ADP-P + AM-ADP is a step accompanying most of the decreases in basic free-energy (Yasui, Arata & Inoue, unpublished results). The long&(2/3) structure itself may reflect a wide range of fundamental configurations, as suggested above. The two different (long and short-R(2/3)) structures of the acto-Sl complex should be detected in a contracting muscle, although it remains unclear which is the predominant intermediate during steady-state actomyosin ATPase activity in muscle. Data from electron paramagnetic resonance and fluorescence polarization using probes suggested that a portion of the probes bound to Sl show an ordered orientation like that in the rigor state but the rest show a disordered orientat’ion like that in the relaxed state (Cooke et al., 1982: Yanagida, 1981). It seems difficult to accommodate this with the present results. Although these authors emphasized that the disordered probes are due only to the dissociated, randomly oriented Sl, the disordered probes are likely to indicate complete or limited disorder of the Sl still bound to the thin filament (see Mendelson & Wilson (1982) for the limited disorder). However, two recent reports agree much better with the results presented here. Burghardt et al. (1983) reported that during isometric contraction a large portion of fluorescent probes bound to Sl showed an ordered orientation very distinct from rigor. Tsukita & Yano (1985) reported some difference in the tilting angle of crossbridges that is observed by electron microscopy on muscles rapidly frozen in rigor and during isometric contraction. It will be important to use chemical crosslinking to study the acto-Sl structure in contracting muscle. I am grateful to Dr A. Inoue of my laboratory for his valuable comments and discussions. This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and the Muscular Dystrophy Association, Inc.

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by H. E. Huxley