k,ur J Biochcm 117, 201 -206 (1981) ( FLBS 1981

Studies on the Actomyosin ATPase and the Role of the Alkali Light Chains

Brian POPE, Paul D. WAGNER, and Alan G. WEEDS

Medical Research Council Laboratory of Molecular Biology, Cambridge

(Received November 7. 1980/March 3, 1981)

Myosin isoenzymes, highly enriched in either alkali 1 or alkali 2 light chains have becn preparcd by light chain exchange in 4.7 M ammonium chloride, under conditions where there is minimal loss of ATPase activity. While the actin-activated ATPase measurements were complicated by a biphasic dependence on actin concentration, the two myosin isoenzymes behaved in a similar manner; at a variety of ionic strength conditions their maximum rates of ATP hydrolysis wcre nearly identical. Furthermore, under conditions where their K, values could be reliably determined, their apparent affinities for actin in the presence of ATP did not differ greatly. These results suggest that the presence of a particular alkali light chain does not influence the maximum rate of ATP turnover by actomyosin under ionic strength conditions approximating physiological.

Rabbit fast-twitch muscle myosin contains two closely related light chains of M , 21 000 (alkali 1 = A l ) and 17000 (alkali 2 = A2) [I ,2] . Subfragment-1 isoenzymes, S-l(A1) and S-I(A2), separated on the basis of their alkali light chains showed no differences in ATPase activities in the absence of actin, but in the presencc of actin the maximum velocity, Vfor S-l(A1) was about one half the value for S-I (A2), while the K, for actin (the apparent dissociation constant in the presence of ATP) was much lower for S-l(A1) [3]. Light chain exchange shows that these kinetic differences could be attri- buted solely to the light chains [4]. Separation of heavy mero- myosin isoenzymes by affinity chromatography showed the same kinetic differences in the two-headed proteolytic frag- ments [5]. These kinetic differences are maintained with regu- lated actin (i.e. actin containing tropomyosin and tropo- nin) [6].

Reliable values for the extrapolated Vrequire assays to be carried out at actin concentrations above K,. Since the K , for actin increases with increasing ionic strength, the initial ATPasc activities were measured at very low ionic strength ( I = 0.021 M) [4]. Reccnt experiments have shown that the kinetic differences betwecn S-l(A1) and S-l(A2) are eliminated as the salt concentration is increased, though because of the high K, values, the extrapolations used to obtain V become less and less reliable [6]. The experiments reported here extend these studies by using myosin isoenzymes under a variety of ionic strength conditions. The filamentous structure of myosin at physiological ionic strength and the lower K, thereby produced, allow the actin-activated ATPase to be measured under conditions which would not be feasible with the pro- teolytic subfragments.

While separation of subfragment 1 and heavy mero- myosin isoenzymes can readily be achieved [3,5,7], the only method for separating parent myosin isoenzymes is by im- munoadsorbance using antibody columns specific for the A1 and A2 light chains [8,9]. This method is severely limited in the amounts of purified myosins that can be obtained. In this report, light chain exchange has been used to prepare myosin

Ahhreviafions. Myosin light chains: alkali 1, A l ; alkali 2, A2; light chain removed with 5,5'-dithiobis(2-nitrobenzoic acid), Nbsz light chain.

Ennzyme. Myosin ATPase (EC 3.6.1.3).

isoenzymes which are highly enriched in either A1 or A2, under conditions where the ATPase activity of the myosin is preserved. Actin-activated ATPase data have been analysed by both Lineweaver-Burk and Eadie-Hofstee plots. Unlike similar plots for subfragment 1 or heavy meromyosin, which gave single straight lines, it is consistently observed that those for myosin are biphasic, with two values for V , one corre- sponding to a low K, for actin and the other to a much higher K,. Control experiments have been carried out to explore the nature of this biphasic response.

MATERIALS AND METHODS

Protein Preparations

Myosin, actin and the tropomyosin-troponin complex were prepared as described previously [6]. Protein concen- trations were determined by absorbance at 280 nm using values for the absorption coefficient, A:& as follows: 5.60 cm for myosin, 2.2 cm-' for alkali light chains, 11 .0 cm-' for actin, 6.0 cm-l for tropomyosin-troponin and 8.75 cm-' for actin-tropomyosin-troponin. The purity of the proteins was checked by polyacrylamide gel electrophoresis as described [lo] and the relative proportions of the alkali light chains determined by gel densitometry using a Camag flat bed densitometer. Further evidence for the purity of the myosin homodimers formed by light chain exchange was obtained by gel electrophoresis under nondissociating con- ditions in pyrophosphate buffers [I I]. This method was also used to detect the presence of free light chains, whose mobil- ities are much greater than native myosins.

Myosin light chains were dissociated in 6 M guanidine . HCI, 40 mM Tris/HCl pH 8.0, 2 mM EDTA and 2 mM dithiothreitol and heavy chains precipitated in 66 (vlv) ethanol, after the method of Perrie and Perry [12]. After rcinoving the ethanol and guanidine . IICl and concen- trating the light chains, the bulk of thc Nbsz light chains were precipitated by ethanol [13]. The alkali light chains were recovcrcd from the supernatant solution containing 26 ?<) ethanol and fractionated on a column (60 x 2.5 cm) of DEAE- cellulose in 2 M urea, 25 mM TrislHC1 pH 8.0, 35 mM NaCI, 0.5 mM EDTA and 0.1 mM dithiothreitol at 20'C. This

method is similar to that published by Jakes et al. 1141. The A1 and residual Nbs2 light chains were eluted consecutively with a linear gradient of 1600 ml to 0.15 M NaCl in the same urea buffer. The A2 light chain was eluted after application of a step to 1.0 M NaCI. Except where otherwise indicated, all operations were carried out at below 4 "C.

Light Chain Exchange

Light chain exchange was carried out as previously described with minor modifications 141. Solid ammonium chloride was added to the light chain solution (to give a final concentration of 4.7 M) in 100 mM Tris/HCI, pH 8.0, 2 mM EDTA and 2 mM dithiothreitol. Myosin was then added to a final concentration of 10-15 pM and the solution mixed carefully on ice for 75 min. The final volume was routinely between 1 ml and 3 ml and the light chain concentration 10-20-fold in molar excess of the myosin. Ammonium chlo- ride was removed by dialysing the solution overnight against 0.4 M KCI, 10 mM Tris/HCI pH 8.0, 1 mM sodium azide, 0.05 mM phenylmethanesulphonyl fluoride and 0.1 mM dithiothreitol. The myosin was then precipitated by dilution with 20 vol. of ice-cold water and collected by centrifugation for ,I5 min at 30000 xg. It was redissolved in 0.6 M KCI, 10 mM sodium phosphate pH 7.0 and reprecipitated once again by dilution with 20vol. of water to ensure complete removal of excess light chains. After redissolving the myosin in 0.6 M KCI, 10 mM Tris/HCl pH 8.0, the solution was dialysed overnight against 0.4 M KCI, 10 inM Tris/HCI pH 8.0 containing 0.1 mM dithiothreitol and clarified by centrifugation as 40000 x g for 1 h.

ATPuse Activities

ATPase activities were measured in a Radiometer pH-stat at pH 8.0 25°C in tubes constantly flushed with nitrogen. EDTA ATPase assays were carried out in 0.6 M KCI, 1 mM EDTA and 5 mM ATP; Ca2+ ATPase in 0.4 M KCI, 10 mM CaC12, 5 mM ATP, with myosin concentrations of 100 pg/ml or greater. Actin-activated ATPase activities were measured in 2.5 mM ATP, 3.75 mM MgC12 and KC1 between 10 mM and 125 mM. Actin concentrations were varied between 1.5 pM and 100 pM. The data were corrected for the ATPase of myosin alone, then analysed using either Lineweaver-Burk or Eadie-Hofstee plots to obtain the maximum velocity of ATP hydrolysis, V , and Km. Mg2+ ATPase activities in the absence of actin were measured using the linked assay system 1151 and occasionally assays were carried out by measuring the hydrolysis of [y-32P]ATP, using a method based on that of Seals et al. 1161, where the volumes were reduced tenfold so that extraction with organic solvents could be carried out in Eppendorf tubes in a microfuge. All activities are expressed as turnover rates in terms of mol ATP hydrolysedx(mo1 myosin site)-' x s-', using a molecular weight for myosin of 470000 and assuming two independent hydrolytic sites. Because light-chain-exchanged myosins were less stable than controls, all ATPase activity ineasurements were completed within two days of carrying out the exchange reaction.

RESULTS

Conditions .for Light Chain Exchange

In earlier experiments using subfragment 1, conditions were established to achieve a high level of light chain exchange

without significant loss of ATPase activity [4]. Experiments were carried out using NH4CI [4,17] or KSCN [I 81 to optimise exchange conditions. While incubation at 21 "C in NHdCI concentrations above 2 M gave rapid loss of ATPase activity, similar incubation at 0 "C did not. Activity was lost in KSCN at concentrations above 0.6 M at both temperatures; thus 4.7 M NH4C1 was chosen as the most suitable solvent for light chain exchange and the ATPase activity was most stable following incubation at pH values between 7.5 and 8.5. The extent of light chain exchange, monitored by densitometry of polyacrylamide gels, showed that levels of exchange were low for incubations of 30 min, while those of 2-4 h gave maximal exchange but the ATPase activity declined. For example, exchange for 4 h regularly yielded a myosin with greater than 90 % A1 or 80 % A2 light chain, but the EDTA ATPase was only 75 control. (In rabbit fast-twitch muscle myosin there is about 50% more A1 than A2 [19,20], which makes it easier lo achieve higher enrichments of Al . ) The conditions chosen to optimise light chain exchange with minimal loss of ATPase activity were a 75-min incubation at 0 "C using a 10 - 20-fold molar excess of added light chains over myosin 'heads'. In 12 separate experiments, exchange with A1 yielded a myosin whose A1 content had increased from an average of 1.16 ( & 0.06) to 1.72 (k 0.07) mol/mol myosin. The same 12 preparations using A2 light chains for the exchange gave an increase from 0.84 to 1.42 (k 0.08) mol/mol myosin. While the A1 honiodimer was nearly 90 :< pure, the A2 form was less highly enriched, but sufficient enrichment was achieved to compare the actin-activated ATPase activities of these two isoenzymes.

While gel electrophoresis in the presence of sodium dodecyl sulphate provided a means to determine the relative amounts of A1 and A2 light chains, it was important to establish that these light chains were bound to the myosin and not present as contaminants. Electrophoresis of myosin in pyrophosphate buffer under non-dissociating conditions was used to deter- mine the amount of contaminating light chains. Although trace amounts of free light chain could be detected by this method in myosin following a single precipitation, none was present following the second precipitation. These dilution and precipitation steps involved inevitable losses of myosin and usually only 50 o/, of the myosin was recovered following this procedure.

These experiments show that it is possible to obtain myosins with marked enrichment in A1 or A2 light chains. While the method does not give the levels of purity obtained by affinity chromatography on antibody columns [8,9], im- munoadsorbance methods are limited both by the concen- tration and the amount of myosin that can be purified. Further- more, the specific antibodies are not readily available. The method described here provides a means of obtaining rela- tively large amounts of highly enriched myosin isoenzymes.

Table 1 summarises the ATPase activities of a number of preparations together with the extent of light chain enrichment achieved by exchange. Whilst there is some variation in the absolute activities between the different preparations, com- parison within each preparation shows that it is possible to obtain almost pure myosin isoenzymes without significant loss of ATPase activity.

Measurement of Actin-Activated ATPase

The principal advantage of the pH-stat for measuring ATPase rates is that it gives a continuous trace of proton liberation. In order to make reliable comparisons of the

203

Table 1. A TPme uctivitic~s of'myo.sins .following light chain inkwhunge ATPase assays were carried out as described in Materials and Methods. Values in parenthesis are the ratios of A1 to A2 light chains obtained by gel dcnsitometry. Mg2+-ATPase assays for these samples gave a mean value of 0.032 (k 0.004) s- '

Preparation ATPase activity of ~ . ~

control myosin A1 myosin A2

K + Ca" K + Ca2+ K + Ca2'

mol ATP (mol myosin site)-' . s C 1

~~~~~ . - ~ ~ C - C C ~-

~C~~ ~ ~~~ ~~

18 7.72 - 7.52 - x.10 -

(58 : 42) (91 : 9) (23 : 77) 20 8.8 2.98 8.8 2.94 9.12 3.00

(59: 41) (83 : 17) (25: 75) 21 7.9 2.23 7.8 2.27 7.44 1.88

(62 : 38) (90: 10) (23 : 77) 23 6.74 2.15 6.38 2.00 6.46 2.12

(54 : 46) (83:17) (30:70)

ATPase activities of different myosins under a variety of con- ditions of actin concentration and ionic strength, it was im- portant to obtain reproducible and linear rates. As others have observed with actomyosin [21,22], hydrolysis of ATP is not linear with time. During the first minute or two either there were bursts of proton liberation, particularly at the Idwest KCl concentrations used (10 mM), or more frequently slower initial rates. Following this early lag, the traces of proton liberation were linear for 10-15 min and thereafter the rate declined. Experiments to test the order of mixing the proteins and ATP showed little or no difference in observed rates except at very low KCI concentrations. In many of the earlier experiments reported here, actin and myosin were premixed and incubated for about 2 min before adding the ATP to initiate reaction, but in later experiments reaction was started by adding myosin to the solution containing actin and ATP. No differences were observed whether myosin was added in 0.6 M salt and precipitated in the reaction vessel, or was dialysed into 0.14 M salt to form filaments [23] and then mixed with actin prior to adding ATP. Thus experi- ments were routinely carried out by adding myosin in solution in 0.4 M KCI to the reaction vessel. This results in rapid reduc- tion in ionic strength of the added myosin and the formation of synthetic filaments.

The rate of ATP hydrolysis was found to increase with increasing myosin concentration, but the specific activity became constant only above 50-70 pg/ml. For this reason a standard myosin concentration of 100 pg/ml was chosen, though much higher concentrations were used when necessary. In experiments with heavy ineroinyosin or subfragment I it was possible to increase the actin concentration in individual assays and obtain increased rates of ATP hydrolysis. Similar experiments with actomyosin did not result in reproducibly increased ATPase activities and in some cases there was none. Thus new assays were set up for each actin concentration. However, assays could be carried out at 45 mM, 85 mM and 125 mM KCI using the same samples, but the longer times of these assays required an increase in ATP concentration from 1.0 mM [6] to 2.5 mM.

Fig. 1 shows the effects of increasing actin concentration on the rate of ATP hydrolysis by myosin. With actin concen- trations in the range 1-20 pM, a linear double-reciprocal

081 A Y

071 B

0.6 1 ; @ ,

l / [Ac t i n ] (~M 1) - I-- I , - 1 - 5 L

-002 0 @02 004 0 6 6

l/[Actin] (LM-')

2 00

1.75

1.50

1.25 h - (0 s 1.00

0.75

0.50

0.25

L

-0.1

4.0

3.5

3.0

h '

Y

1 5 2.0

1.5

1.0

0.5

- 0.1

l/[Actin] (LM-') ~~C L

-0.01 0 0.01 0.03 0 0 5

0 2 04 0 6 08 l/[Actin] (KM I )

, -e C 0 /'

0,,'

1 I ,

l/[Actin] (pM~ ' ) I ,

, /' 1 , , L ' -0.01 0 0.01 0.03 0.05

-. 1 _.L~_LP

) 0~1 0.3 0.5 0 7 l/[Actin] (pM-1)

Fig. 1. Liriewenvev-Burk plol.7 of' uc,ion7,r.ositi ATPusr. (A) 46 mM KCI; (B) 86 mM KCI; (C) 126 mM KCI. Other conditions as in Materials and Methods. In each case {he inset shows an expanded plot of the points at high actin concentrations. Values for V (with K,,, for actin in parenthesis) are as follows: (A) 3.19 s - ' (2.63 pM) and 7.73 s - ' (38.3 pM); (B) 0.96s-' (1.56pM) and 4.60s-I (83pM); (C) 0 .39s - ' (1.17pM) and 1.84 s - l (74 pM)

plot was obtained, extrapolating to a maximum velocity, V, with a low K,,, for actin. Experiments at higher actin concen- trations extrapolated to a significantly higher V with a corre- spondingly higher K,,, for actin. Results similar to these have been obtained for numerous myosin preparations, not only from rabbit skeletal muscle but also cat and rat skeletal muscles (unpublished work) and rat cardiac myosins [24].

o.80 0.70 i 060

0.50

.& 040 - -

0 3 0 ;

-\ -

\ \

-

0.201 j I

O d 0.00: ' 0.012 ' 0,020 ' 0 . 0 2 8 ' 0.036 v/[Act in] (pM- 's '1

Fig. 2. Eadie-Hofstee plot of uctnmjosin ATPusc. Actomyosin ATPase measured in 0.25 M KC1. Values for V and K , (in parenthesis) are: 0.074s-' (1.1 pM actin) and 0.71 sC1 (117 pM actin). The change in slope occurs at 16 pM actin. Myosin concentration = 0.7 - 1.8 mg/ml

l 6 (i 1.4 1

0.6 o.8 t :: '0 0.05 ' 0:15 ' 0.25 ' 0.35 ' 0.45

v / [Ac t i n ] (KM-' s-I)

Fig. 3. Eudie-Hqfstec plot qf actomjo.rin A TPu.sc. Myosin concentration was 1.6 mgiml (6.8 pM sites) in the asbay, compared to 0.53 pM in Fig. 1, The KCl concentration was 66 mM. Extrapolated values for V = 1 . 1 s - l and 1.8 s-', with corresponding K,, values of 0.1 pM and 10 pM actin. The change in slope is at 16.5 pM actin

Experiments carried out at ionic strengths between 0.034 M and 0.15 M showed biphasic plots: over 36 separate experi- ments the transition point between the two phases occurred at 20 _+ 6 pM actin. Even in 0.25 M KC1, where the myosin filaments should be substantially dispersed, the plots remained biphasic (Fig. 2). Changing the order of mixing did not affect the results and direct measurment of 32Pi release from [y-32P]- ATP gave similar profiles.

Since the transition between the two phases of actin activa- tion occurred at an actin concentration which was about 50 times the myosin 'heads' concentration, it was possible that a minor contaminant in the actin, not detectable by gel electrophoresis, might be responsible for this second phase of activation. Actomyosin ATPase assays were carried out at myosin concentrations up to 1.6 mg/ml (6.5 yM 'heads'). All these experiments gave biphasic actin activation with similar transition points to those found under other conditions (Fig. 3) . It seems unlikely that actin contaminants are respon- sible for these observations.

Table 2 summarises extrapolated values for V and K,,, obtained under a variety of ionic conditions. The general trend is towards a lower value for V with increasing salt con- centration in both the low and high actin concentration ranges.

Table 2. Values ,for V and K, at different salt concentrations V and K , values are averaged for the number of experiments given in parentheses and standard deviations are calculated where appropriate. The ionic strength may be determined from the KCl concentration used plus 0.024 M for the substrate and other salts present

KCI Low actin High actin .. ~~ ~~~ ~ ~~.

V k=, V K,"

P M S - I PM mM s-l

- 10 7.5 2.5 (2) -

26 3.8 1 (2) -

46 4.2 f 1.5 1.5 t 0.4 (3) 6.5 2 38 12 (5) 66 2.9 1.4 (1) 8.9 38 (1) 86 1.6 k 1.1 1.8 k 0.4 (3) 5.4 2.4 62 30 (8)

126 0.6 i- 0.2 1.9 k 1.1 (3) 2.5 k 0.9 66 f 23 (7) 250 0.07 1.1 (1) 0.71 117 (1)

-

Table 3. Valuesfor V und K, f o r myo.rin.y eiiriched by light chain mchunge The control myosin had an average A1 light chain content of 1.18 0.05 mol/mol myosin; myosin A1 had an average A1 light chain content of 1.76 & 0.08 mol/mol myosin and myosin A2 an average A2 light chain content of 1.48 0.07 mol/mol myosin. The asterisk indicates values for V at low actin concentrations, with low K,, for actin. Other values obtained for actin concentrations between 2 0 p M a n d 100pM

Sample KCI Control Myosin A1 Myosin A2

V K , V K , V K , - ~ . .

pM s- l PM mM s-' pM s-'

14* 26 3.3 0.6 2.9 0.4 3.1 0.8 14 46 3.1 18 3.0 22 2.5 9 12* 70 2.9 1.4 2.9 1.2 2.9 0.6 14* 86 0.8 2.2 1.3 2.1 0.8 1.1 14 86 1.7 28 1.5 28 1.3 18 18 86 3.5 42 3.4 39 3.3 55 20 126 2.1 46 2.1 46 2.1 46 21 126 1.7 42 1.8 34 1.8 39

K, values tend to increase with salt concentration in the high actin range, but show a fairly constant value in the low actin range. It should be emphasized however, that as the salt concentration increases, the observed values become much slower and measurements therefore less reliable because of the low rate of proton release relative to background drift.

Comparutive Studies

Fig.4 shows a set of experiments at 46 mM, 86 mM and 126 mM KCl, using actin concentrations corresponding to the second phase of actin activation (i.e. above 15 yM). At both 46 mM KCl and 86 mM KCl there is apparent inhibi- tion of the ATPase at the highest actin concentrations; these lower rates probably reflect the difficulty of obtaining adequate mixing of the viscous actomyosin suspensions under these conditions. (These data points were omitted from the regres- sion analysis.) Extrapolated values of Vindicate no significant differences between the two myosin isoenzymes. At low actin concentrations the two myosins also showed similar ATPase activities. Table 3 suminarises data from a number of experi- ments showing that myosins enriched in either A1 or A2 light chains do not differ significantly in their maximum rates of

- . 0.151

O ’ I 07

5 0.51

0.02 0.04 0.06 l / [ A c t i n ] (LM-’)

I , 0 002 0 04 006

l / [Ac t i n ] (pM-I)

A 5 i 0 04 006

/ L# 0 0 02

l / [ A c t i n ] (pM ’)

Fig. 4. Linenea,.rr-Rrtrli plots of’actomyosin A TPase orn7?.o.sin i .wetzzpzcs. (A) 461nM KCI. (€3) 8 6 m M KCI; (C) 126mM KCI. Myosin A1 (*--~--@); myorin A2 ( O - - - ~ ~ 0). Table 3 summarises results from a number of similar experimenis

ATP turnover over a wide range of salt concentrations up to physiological. The K , values show greater variation partic- ularly at lower ionic strength values, but under conditions approaching physiological these differences were minimal.

DISCUSSION

In comparing these results with earlier experiments using soluble myosin subfragments, two important differences

emerge. The first is that the values obtained for V are much lower than those observed with heavy meromyosin or sub- fragment 1, while the second relates to the biphasic nature of actin activation. Table 2 shows values for V in the range 6-8 S - ’ for myosin, under conditions where subfragmcnt 1 or heavy meromyosin gave turnover ratcs of 20- 35 s ~ ’ [6]. These values are in good agreement with those reported else- where [21,26]. While lack of accessibility of individual myosin ‘heads’ in these synthetic filaments may account in part for the differences in turnover rates, it is also possible that myosin ‘heads’ are constrained in the filaments so that even when they are accessible to actin they hydrolyse ATP at a slower rate than isolated subfragment 1.

The biphasic response does not appear to be an artefact of the assay system used. Nor does it depend on ionic strength or order of mixing the components. The two phases of actin activation must be related to the interaction of the two fila- mentous proteins since subfragments of myosin do not behave in this way. At present it is unclear whether the biphasic response relates to particular properties of myosin alone or actin alone, but probably both contribute. Synthetic myosin filaments are in rapid equilibrium with myosin monomer and both monomer and polymer will interact with actin and contri- bute towards the overall ATPase activity [23,25]. Increasing the ionic strength will increase the proportion of monomer in the mixture. While synthetic filaments will have a low K, for actin, monomeric myosin would be expected to behave more like heavy meromyosin. Thus if this equilibrium were the source of the biphasic response, the ratio of V at high actin to that obtained at low actin concentration should increase with increasing ionic strength, while increasing myosin con- centration will reduce this ratio. Both these predictcd effects have been observed (Table 2 and Fig.3). However, the ob- served K, at high actin concentration is much lower than that expected for heavy meromyosin at the same ionic strength [6] and biphasic activation has been observed in 0.25 M KCI, where filament formation should be minimal. Thus other factors must also be involved to explain the biphasic response. ‘Sleeve-like’ aggregates of actomyosin have been reported, whose formation depends critically on actin concentration [27,28]. Such organised structures might permit w higher proportion of myosin ‘heads’ to interact with actin a n d hmce increase the ATPase activity.

Although the biphasic actin activation complicated thcse ATPase assays it was nevertheless possible to compare the synthetic myosin isoenzymes under a variety of conditions (Table 3). Under all conditions tested the differences in V between myosin Al , myosin A2 and controls were very small. In general the values for K, obtained for the two isoenzymes at high actin concentration agreed reasonable well (prepara- tion 14 being an exception to this, see Table 3). The K, v‘ ‘1 I ues obtained at low actin concentrations are subject to rclatively large error since it was not possible to obtain accura~e mea- surements at low enough actin concentrations. Fortunately the condition of greatest interest is that where the ionic strength approximates physiological and here the K,, values are in closest agreement (Fig.4C and Table 3). While these enriched myosins are not pure, the results indicate that the actin-activated ATPase activities of the individual isoenzymes are unlikely to differ significantly under conditions closest to physiological. Myosins purified by affinity chromatography give similar results [29]. While it is not possible in vifro to mimic the organised structure of the filament lattice found in vivo, there is no evidence to believe that the homodimers of myosin are kinetically different in muscle. The observation

that heterodimers of myosin occur naturally [9,30,31] and that both light chains can be detected in the same myofibril [32], indicate that there is no specific localisation of fast myosin isoenzymes within the myofibril. Other experimental approaches with muscle fibres are needed to test this, but at present there is no obvious explanation for the existence of polymorphic forms of myosin light chains in either fast- twitch or slow-twitch fibres.

We would like to thank Dr H. E. Huxley for criticism of this manu- script and helpful suggestions.

REFERENCES

1. Weeds, A. G. & Lowey, S. (1971) J . Mol. B i d . 61, 701 -725. 2. Frank, G. & Weeds, A. G. (1974) Eur. J . Biochem. 44, 317-334. 3. Weeds, A. G. &Taylor, R. S. (1975) Nature jL(1nd.j 257. 54-56. 4. Wagner, P. D. & Weeds, A. G. (1977) J . Mol. Bid. 109, 455-473. 5. Wagner, P. D . (1977) FEBS Lef t . 81, 81 -85. 6. Wagner, P. D., Slater, C. S., Pope, B. & Weeds, A. G. (1979) Eur.

7. Trayer, H. R., Winstanley, M. A. & Trayer, I . P. (1977) FEBS Lett.

8. Holt, J . C. & Lowey, S. (1977) Biochemistry, 16, 4398-4402. 9. Lowey, S., Benfield, P. A,, Silberstein, L. & Lang, L. M. (1979)

J . Biochem. YY, 385-394.

83, I41 - 144.

Nature (Lond.) 282, 522 - 524. 10. Wedds, A. G . & Pope, B. (1977) J . Mol. Biol. I l l , 129-157. 11. Hoh, J. F. Y., McGrath, P. A. & White, R. I. (1976) Biochem. J .

157, 87-95.

12. Perrie, W. T. RC Perry, S. V. (1970) Biochem. J . IN, 31 -38. 13.

14.

15. 16.

17.

18. 19. 20.

21. 22.

23. 24.

25. 26. 27.

28.

29. 30 31.

32.

Perrie, W. T., Smillie, L. B. & Perry, S. V. (1973) Biochern. J . 135,

Jakes, R., Northrop, F. & Kendrick-Jones, J . (1976) FEBS Lett. 70,

Taylor, R. S . & Weeds, A. G. (1976) Biochem. J. 15Y, 301 -315. Seals, J . R., McDonald, J . M., Burns, D. & Jarett, L. (1978) Anal.

Dreizen, P. & Richards, D. H. (1972) Cold Spring Harbor Synip.

Okomoto, Y . & Yagi, K . (1977) J . Biochem. (Tokyoj 82, 17-23. Sarkar, S . (1972) Cold Spring Harbor Symp. Quant. B id . 37, 14-17. Weeds, A. G., Hall, R. RC Spurway, N. C. (1975) FEBS Lett. 49,

320-324. Mulhern, S. A. & Eisenberg, E. (1978) Biochemistry, 17, 4419-4425. Strzelecka-Golaszewska, H., Klimaszewska, U. & Dydynska, M.

Josephs, R. Rc Harrington, W. F. (1966) Biochemistry, 5. 3474- 3487. Pope, B., Hoh, J . F . Y . Rc Weed>. A. G. (19x0) FEBS Lrlt. 118,

Harringlon, W. F. & Josephs, R. (1968) Dev. Biol. Suppl. 2, 21 -62. Schliselfeld, L. H. (3975) Biochim. Biophys. Acta, 445, 234-245. Ikernoto, N., Kitagawa, S. RC Gergely, J. (1966) Biochem. Z . 345,

Nonomura, Y . Rc Ebashi, S. (1974) J . Mechanochem. Cell Motility, 3,

Pastra-Landis, S . C. & Huiatt, T. W. (1980) Fed. Proc. 39, 2169.

d’Albis, A, , Pantaloni, C. & Bechet, J.-J. (1979) Eur. J . Biochenz. 9Y,

Silberstein, L. & Lowey, S. (1978) Biophys. J . 21, 45a.

151 -164.

229 - 234.

Biochem. YO, 785 - 795.

Quant. Bid. 37, 29-45.

(1979) Eur. J . Biochem. 101, 523-530.

205 - 208.

410-426.

1-8.

Hoh, J . F. Y. (1978) FEBS Lett. YO, 297-300.

261 -272.

B. Pope and A. G. Weeds, Laboratory of Molecular Biology of the Medical Research Council, Postgraduate Medical School, University of Cambridge, Hills Road, Cambridge, Great Britain, CB2 2QH

P. D. Wagner, Cardiovascular Research Institute, School of Medicine, University of California San Francisco, San Francisco, California, USA 94143