Vol. 254, No. 9, Issue of May 10, pp. 36174623, 1979 Printed in U.S.A.

Phosphorylation-Dephosphorylation of the l&000-dalton Light Chain of Myosin during the Contraction-Relaxation Cycle of Frog Muscle*

(Received for publication, September 25, 1978)

Kate B&r&y and Michael B&r&y From the Departments of Physiology and Biophysics, and Biological Chemistry, University of Illinois at the Medical Center, Chicago, Illinois 60612

Jean M. Gillis From the Department of Physiology, Catholic University of Louvain School of Medicine, B-1200 Brunelles, Belgium

Martin J. KushmerickS From the Department of Physiology, Harvard Medical School, Boston, Massachusetts 02115

The l&000-dalton light chain of myosin was phospho- rylated and dephosphorylated during the contraction and relaxation cycle of frog muscle.

The extent of light chain phosphorylation was a func- tion of stimulus duration in the early phase of contrac- tion. At O”C, 0.14 and 0.22 mol of [S2P]phosphate was transferred/m01 of light chain in muscles which were stimulated for 200 and 500 ms, respectively, and pro- duced 40 and 80% of the maximal tetanic tension, re- spectively. During a few seconds of tetanization at var- ious temperatures, 0.2 to 0.3 mol of [3eP]phosphate was transferred, whereas electrical stimulation for 20 to 30 s or caffeine treatment for 20 min resulted in esterifi- cation of 0.35 to 0.4 mol of [3”P]phosphate. This increase of phosphorylation seems to be related to the continued presence of sarcoplasmic Ca2+ at activating levels, that is, >lO-8 M.

Light chain phosphorylation in stimulated semiten- dinosus muscles stretched beyond the point of overlap of the thick and thin filaments was identical to that in muscles stimulated at standard rest length when the time of stimulation was at least 3 s. The data indicate that the stimulation of the muscle per se and not the mechanical event initiates the phosphorylation.

Light chain dephosphorylation took place in the re- laxing phase of muscle activity. After short tetani, de- phosphorylation approximately followed relaxation. After longer tetani, relaxation occurred before dephos- phorylation. In vitro experiments demonstrated that the absence of CaZ+ was required for light chain de- phosphorylation and that phosphoryl groups did not turn over during active myofibrillar ATPase.

Light chain phosphorylation may be involved in ac- tivation of muscle contraction by providing a driving force to place the cross-bridges in the vicinity of actin filaments.

Phosphorylation of a specific light chain of myosin, called the P-light chain by Frearson and Perry (I), has been studied

* This work was supported by Grants NS-12172 and AM 14485 from the United States National Institutes of Health and the Mus- cular Dystrophy Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

*Recipient of Research Career Development Award K04 AM 00178.

extensively both in vitro and in Go. Light chain phosphoryl- ation enhances the actin-activated ATPase activity of skeletal muscle myosin (2), of smooth muscle myosin (3-5), and of myosin from primitive motile systems (6). Light chain phos- phorylation was shown to take place in the canine heart in uiuo (7), as well as in perfused rabbit heart (8), and in the skeletal muscle of live frog (9) or rabbit (10). Recently, we detected light chain phosphorylation during contraction of frog muscle; about 0.4 mol of [“*PIphosphate was incorpo- rated/m01 of light chain during a 30-s tetanus (9). Stull and High have found the same extent of phosphorylation in rabbit muscle tetanized for 15 s (11).

A further clarification of the role of light chain phosphoryl- ation in muscle contraction requires study of this reaction during the entire contraction-relaxation cycle. Such experi- ments are reported in this paper. Furthermore, we have meas- ured light chain phosphorylation in stimulated semitendino- sus muscle stretched beyond the point of overlap of the myofiiaments in order to assess the role of stimulus per se in the phosphorylation. A preliminary report of this work has appeared (12).

EXPERIMENTAL PROCEDURES

Frogs (Rana pipiens, temporaria, or esculenta) were injected intraperitoneally or in the lymph sacs with about 1 mCi of carrier- free [32P]orthophosphate in 0.1 M Tris-HCl buffer, pH 7.0. The animals were kept at room temperature for 2 to 3 days before use. The frogs were chilled and pithed, and the paired muscles, sartorius and semitendinosus (dorsal heads), were dissected. The muscles were allowed to recover either in oxygenated frog Ringer’s solution which contained 2 mM Tris-HCl buffer, pH 7.0, instead of the usual phos- phate buffer, or in a bicarbonate-Ringer solution, gased with 95% 02, 5% COZ, or in a closed chamber filled with moistened oxygen.

The specific radioactivities of the proteins (in counts per min/mg) from the paired sartorius or semitendinosus muscles were determined eight times in the course of these experiments. No difference was found in the specific activities when the muscles from the left and right leg were compared.

Stimulation-The muscles were mounted on electrode assembly (13-15). One of the paired muscles was tetanically and maximally stimulated (100/s at 25°C or 25/s at O-2’%) while the other muscle was resting. For isotonic contraction, the muscles were loaded with 5 g so that considerable shortening of the muscles took place. For isometric contraction, the muscles were held near their in vivo length by applying a 1 g force to the relaxed muscle. About 50 g of passive tension was applied to the semitendinosus muscles which were thereby stretched to 1.4 times the rest length; no detectable active tension was developed upon stimulation. The sarcomere length of the stretched semitendinosus muscles was monitored occasionally by measuring the first order of the diffraction pattern obtained with a

3617

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3618 Light Chain Phosphorylation during Contraction-Relaxation

laser beam (16). Both muscles of a pair were kept at the same length. Freezing-Stimulated and resting muscles were frozen simultane-

ously while the tensions were recorded. For automatic freezing the muscles mounted on a multielectrode assembly (13) were immersed into isopentane cooled by liquid nitrogen near its freezing point (-16O’C) using the machine of Mommaerts and Shilling (17). The time necessary for a complete immersion of the muscles ranged from 40 to 60 ms, and about 150 ms additional time was needed to cool the muscles below -10°C. Alternatively, the muscles were frozen within 100 ms in an apparatus of the type described by Kretzschmar and Wilkie (18). Muscles which were stimulated for several seconds were frozen manually (14).

For each experiment described in this paper, three to four frozen muscles, stimulated or resting, were pooled.

Caffeine Contra&we-This was produced by incubating four sar- torius or semitendinosus muscles in Tris/Ringer solution, pH 7, containing 10 mru caffeine, at 25’C for 20 min, while the paired muscles were kept in Tris/Ringer solution. Subsequently, within 5 min, the caffeine-treated and untreated muscles were washed with large volumes of cold Tris/Ringer solution, then homogenized, with- out prior freezing, at 0°C.

Preparation of Muscle Proteins-In most experiments, myofibrils were used. The frozen muscles were pulverized (9) and then homog- enized in ice-jacketed Waring Blendors in a solution of 0.1 M NaF, 0.01 M KPi, 1 mM EDTA, 1 IIIM iodoacetamide, pH 7, at 0°C for 40 s and the tibrils were sedimented at 600 x g, 0°C for 40 min. Unfrozen muscles were homogenized in the same solution and centrifuged the same way. The pellet was washed with the same medium five times, concentrated by centrifugation at 11,000 x g for 90 min, and dissolved in 2% SDS,’ 0.1 M sodium phosphate buffer, pH 7.0, using a Polytron homogenizer.

In a few cases, myosin or actomyosin were isolated from the muscles (19). The frozen or unfrozen muscles were homogenized in a solution containing 0.5 M KCl, 0.1 M NaF, 0.01 M KPi, 1 mru EDTA, pH 7.0, for 5 s and extracted by stirring at 4°C for 20 min or 24 h, respectively.

For the determination of 32P incorporation into total muscle pro- teins the frozen muscles were pulverized according to Serayderian et al. (20) or Barany and Barany (9) and subsequently homogenized in 5% trichloroacetic acid at 0°C. Unfrozen muscles were homogenized identically. The suspension was centrifuged at 40,060 X g for 5 min, and the residue was washed with 2% trichloroacetic acid containing 5 mM NaH2P04 six more times. The final residue was dissolved in 2% SDS, 0.25 M NaPi, pH 8.0, with aid of the Polytron.

Trichloroacetic acid homogenization was used in all experiments carried out in Belgium. The residues obtained after the first centrif- ugation were shipped to Chicago at room temperature; the duration of shipping varied from 3 to 7 days. After arrival, the pellets were washed with the trichloroacetic acid/NaHzPOd solution and dissolved. Control experiments showed that 5% trichloroacetic acid-treated mus- cle residues which were left at 25°C over 1 week did not lose any protein-bound radioactivity as compared to another part of the same muscle residues which were analyzed immediately.

Isolation of the 18,ooO-da&on Light Chain from Caffeine-treated and Untreated 32P-labeled Frog Leg Muscles-For the experiment shown in Fig. 2, myofibrils were prepared from 12 g each of caffeine- treated and untreated muscles. The final residues, containing about 650 mg of myofibrils from each type of muscle, were suspended in 40 ml of the homogenizing solution at 25°C and 480 mg of urea were added/ml to solubilize the proteins. Subsequently, dithiothreitol was added to 5 mM final concentration and the solutions were gently stirred at 25’C for 1 h. After chillina to 0“C. 50 ml of ethanol (-20°C) were added and the mixtures were stirred at 4’C for 30 min. The precipitates were removed at 12,000 X g, 0°C for 30 min, and super- natants were dialyzed against 150 volumes of 50 mu Tris-HCl buffer, pH 8.0, in the cold room overnight. The small amount of precipitate formed during dialysis were removed at 40,000 x g, and the total supernatants (156 ml for caffeine-treated and 170 ml for untreated sample) were applied to DE52 cellulose columns (20 x 1.5 cm, each) equilibrated with 50 mM Tris-HCl, pH 8.0, at 25’C. The columns were washed with 150 ml of the equilibration buffer. Elutions of the 18,000- dalton light chains were initiated with a solution of 100 mu KC1 and 50 mru Tris-HCl, pH 8.0, 3-ml fractions were collected at 25’C, and

’ The abbreviations used are: SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(&uninoethyl ether)N,N-tetraacetic acid; TN-T, the tropomyosin-binding subunit of troponin.

the optical densities were measured at 280 mn. Fractions 8 to 13 were pooled for both types of samples and concentrated by Amicon ultra- filtration to about 2 ml. After dialysis against 0.1% SDS, 0.1 M phosphate buffer, pH 7.0, overnight, the protein content of the isolated fractions were determined. About 4 mg of protein were obtained from 12 g of caffeine-treated or untreated muscles.

Gel Electrophoresis-Disc gel (5 x 131 mm) electrophoresis was performed on 10% polyacrylamide gels containing 0.1% SDS, 0.1 M sodium phosphate buffer, pH 7.0, and 8 M urea (21). The reservoir buffer contained 0.1% SDS and 0.1 M sodium phosphate, pH 7.0. Prior to electrophoresis, samples were dialyzed overnight at 25°C against 500 volumes of 0.1% SDS and 0.1 M sodium phosphate. The samples were clarified in a Spinco preparative ultracentrifuge at 100,000 x g for 30 min. The protein concentrations of experimental and control solutions were equalized. The solutions were reduced with 50 mM dithiothreitol in boiling water for 2 min, then solid urea was added to a final concentration of 8 M.

About 150 ag of proteins were placed on each gel tube and electro- phoresis was carried out for 20 h at room temperature at a current of 2 mA/tube. Gels were fixed in a solution containing 10% trichloroa- cetic acid and 50% methanol for 3 h, stained in a solution of 0.25% Coomassie blue, 25% isopropyl alcohol, and 10% acetic acid for 6 h. They were destained by diffusion in a solution containing 40% meth- anol and 10% acetic acid.

Staining intensities were measured with a Zeineh soft laser scan- ning densitometer. No difference was observed between proteins of stimulated and resting muscles.

The gels were cut according to protein bands and the same bands of six gels were pooled in a glass vial; 0.5 ml of 30% H202 was added and the gels were digested at 90°C until complete dissolution was obtained. For counting the xylene-based liquid scintillation fluid (22) was used.

The counts given in Figs. 1 to 3 and in Table IV are counts above the background value, 20 cpm. After determining the radioactivity in all slices of the gels, the slices from the 18,000-d&on light chain and tropomyosin zones were recounted for 80 to 100 min, so that the accuracy of the counting was within a statistical error of 1 to 2%.

RESULTS

Evidence for Phosphorylation of the 18,000-dalton Light Chain during Muscle Contraction-Fig. 1 compares the dis- tribution of radioactivity in proteins of myofibrils from isoton- ically tetanixed and resting muscles. The SDS-urea gels sep- arate three major radioactive peaks; these correspond to the (Y component of tropomyosin (52,000 daltons), the two forms of TN-T (32,000 to 34,000 daltons), and the 18,000-dalton light chain. Only the light chain shows a change in 32P incorporation as a result of muscle activity; this amounts to a 2-fold increase for the tetanixed muscle in this experiment.

Results similar to those shown in Fig. 1 were obtained when

1 I

50,caJ 4Qooo 3qom 2Qm3

Molecular Weqht

FIG. 1. SDS-urea gel comparison of the distribution of radioactiv- ity in myofibrils of frog semitendinosus muscle frozen 20 s after isotonic tetanic stimulus at 25°C (W, upper gel) and resting (O--O, lower ge0. Samples containing 160 ag of protein were electrophoresed.

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Light Chain Phosphorylation during Contraction-Relaxation

myosin or actomyosin were prepared from contracting and resting muscles. However, the yield of these proteins was much less than that of myofibrils, 50 to 80 mg/g of original muscle. Furthermore, the simplicity of myofibril preparation was a great advantage for rapid comparison of various muscles.

The radioactivity observed in the 18,000-d&on zone of myotibrils is associated with the light chain itself. 32P-labeled light chains were isolated from caffeine-treated and untreated muscles (see “Experimental Procedures”) and electrophoresed on SDS-urea gels. Fig. 2 shows that these proteins were essentially homogeneous and that all of the radioactivity was associated with the 18,000-dalton bands. A comparison of the radioactivity in the peak areas of the light chains indicates a 2.1-fold increase for the caffeine-treated muscle uersus the untreated one. This increase was the same as was found in the original myotibrils which were used for the preparation of the light chains of Fig. 2. These data suggest that the differ- ence in the 32P content of these fibrils is derived from the light chains and not from any other component.

In the majority of the experiments reported in this paper, myofibrils were used for final analysis, although in some cases the trichloroacetic acid-washed residue was used for electro- phoresis. Fig. 3 illustrates an example of the latter; three major radioactive peaks may be seen, at 52,000, 33,000, and 18,000 daltons. These had mobilities identical with tropomyo- sin, TN-T, and the P-light chain, respectively. Fig. 3 shows that, upon a short stimulation of the muscle, the radioactivity of the light chain was increased about 1.5fold, but no change was found either in the tropomyosin or TN-T regions.

Tropomyosin as an Internal Standard for Light Chain Phosphorylation-It was noted in the early part of this work that tropomyosin may be used as a reference point for light chain phosphorylation since its [32P]phosphate content is the same in resting and stimulated muscles (Figs. 1 and 3) and it can be well separated, on SDS-urea gels, from the other labeled proteins either in myofibrils or in trichloroacetic acid- washed muscle residues. The [32P]phosphate content of tro- pomyosin in frog muscles has been determined recently, under the conditions of the current experiments (23). Thus, the [“PIphosphate of the light chains may be calculated from the ratio of radioactivity in the light chain zone to the radio- activity in the tropomyosin zone. Table I shows these ratios in trichloroacetic acid-washed residues and myotibrils of rest- ing muscles and of muscles contracted by caffeine or by tetanic stimulation. The ratio is higher in resting muscles which were rapidly frozen than in the resting muscles which were homog-

I I I I

54000 40,000 5Ko3J 20,000

Molecular Weight

FIG. 2. SDS-urea gel comparison of the distribution of radioactiv- ity in the 18,000-dalton light chain of caffeine-treated (W, upper gel) and untreated (M, lower gel) frog leg muscle. Samples containing 15 pg of protein were electrophoresed.

50,000 30,000 zqooo Molecular Weight

lO,CXXl

FIG. 3. SDS-urea gel comparison of the distribution of radioactiv- ity in trichloroacetic acid-insoluble residue of frog sartorius muscle, frozen 200 ms after isometric tetanic stimulus at 25“C (W, upper gen and resting (M, lower gel). Samples containing 220 pg of protein were electrophoresed.

TABLE I Ratio of radioactivities of the 18,000-dalton light chain to

tropomyosin in resting and stimulated frog muscles Ratios are given -I standard error for all the resting muscles and

for caffeine-contracted muscles. For tetanically stimulated contracted muscles, the ratio is not constant but it varies with the time of stimulation; the values given correspond to the range of ratios ob- served.

Type of Ratio stimulation Type of sample analyzed n

Resting Contracted

Caffeine Trichloroacetic acid- 0.73 + 0.11 1.75 + 0.13 8 washed residue from unfrozen muscle

Caffeine Myofibrils from un- 0.80 f 0.08 2.00 + 0.09 9 frozen muscle

Tetanic Trichloroacetic acid- 1.12 + 0.20 1.43-2.04 21 washed residue from rapidly frozen muscle

Tetanic Myofibrils from rapidly 1.14 -I 0.16 1.46-2.11 35 frozen muscle

enized without freezing. This implies that the freezing causes an increase in the [U2P]phosphate content of the light chain. A large number of observations demonstrate this effect in muscles which were frozen in our different laboratories. In contracted muscles, the ratio is furthtr increased in proportion to the extent of phosphorylation of the light chain.

In the following we shall use the ratios of 0.80 and 2.00 in myofibrils of resting and caffeine-trested muscles to illustrate the calculation2 of the [32P]phosphate content of their light

’ The formula used is:

Mol [3ZP]phosphate Mol 18,000-dalton LC =

mol [32P]phosphate

> mol TM subunit

radioactivity in LC zone mol TM subunit radioactivity in TM

Mol 18,000-dalton LC

where LC is light chain and TM is tropomyosin.

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3620 Light Chain Phosphorylation during Contraction-Relaxation

chains. According to Ebashi and Nomura (24) myofibrils contain 57.5% myosin and 4.5% tropomyosin, amounting to 2.30 mol of the l&000-dalton light chain and 1.36 mol of the 33,000-dalton subunit of tropomyosin in 100,000 g of myofi- brils. Since frog tropomyosin contains 0.5 mol of [“‘Plphos- phate/mol of subunit (23), 1.36 mol of tropomyosin subunit correspond to 0.68 mol of [“‘PIphosphate in either resting or caffeine-treated muscle, and 2.30 mol of light chain correspond to 0.54 and 1.36 mol of [“2P]phosphate in the light chain of resting and caffeine-treated muscle, respectively. Hence, 0.24 and 0.59 mol of [“2P]phosphate were esterified/mol of light chain in resting and caffeine-treated muscle. For an independ- ent check on the validity of this calculation, we determined the [32P]phosphate content of the light chain in these muscles using the specific radioactivity of the terminal phosphate of ATP isolated from the same muscles as a reference (9, 14). By this method, we obtained 0.26 and 0.67 mol of [32P]phosphate/ mol of light chain in resting and caffeine-treated muscle. These values are similar to those calculated from the ratio of light chain radioactivity to tropomyosin radioactivity.

Phosphorylation of the 18,000-dalton Light Chain in Stim- ulated Muscles-Table II summarizes our results on the phosphorylation of the l&000-dalton light chain in stimulated frog muscles as compared to resting muscles. The extent of phosphorylation was determined on SDS-urea gels from the radioactivity in the separated zones using either myofibrils or trichloroacetic acid-washed residues. The phosphorylation is expressed both as a percentage increase (sixth column) and as moles of [“‘PIphosphate transferred/m01 of light chain (sev- enth column). Furthermore, since these experiments were carried out in three different laboratories, they are organized in a way that the experiments from a particular laboratory are grouped together. When the muscle was stimulated at 0°C and frozen 200 ms after the stimulus, the isometric tension produced by the muscle was about 40% of the maximal, and light chain phosphorylation reached 135% of the resting value (Table II, 1A). Phosphorylation increased to 156% in muscles

stimulated for 500 ms where the isometric force was about 80% of maximal (Table II, 1C). However, stimulation for 5 s did not increase the phosphorylation very much more (Table II, 1E). In Experimental Series 2, carried out at 2”C, about 170% phosphorylation was found already after 300 ms (Table II, 2A) which reached 180% after 3 s (Table II, 2C). The absolute level of phosphorylation in this series was relatively high, but consistent. At 25”C, the maximum increase in phos- phorylation was already obtained at the earliest time of freez- ing, nominally 40 ms, and remained essentially unchanged for tetani up to 3 s duration (Table II, 3A and 3B). Longer tetanization at room temperature increased the phosphoryl- ation to 185% (Table II, 4A). In all these cases, frozen stimu- lated muscles were compared with frozen unstimulated mus- cles. In the case of caffeine-treatment which does not require freezing, 246% phosphorylation was found (Table II, 5), which is due to the low level of light chain phosphorylation in resting muscle, similarly unfrozen. In the other cases when the resting muscle was frozen, the value of reference phosphorylation was greater and thereby the relative increase of the percentage phosphorylation was less. This is evidenced by the fact that the absolute values for moles of [32P]phosphate transferred/ mol of light chain in caffeine-treated unfrozen (Table II, 5) and 20- to 30-s tetanized frozen muscles (Table II, 4A) were virtually the same, 0.36 and 0.35, respectively.

Table II also shows light chain phosphorylation in stimu- lated semitendinosus muscles stretched to a sarcomere length of 3.9 to 4.0 pm so that no active tension was observed upon stimulation. The extent of phosphorylation in these muscles at various temperatures was identical with that in muscles stimulated at 2.2 to 2.4 pm when the time of stimulation was at least 3 s (cf Table II, 1E and lF, 3B and 3C, 4A and 4B). Shorter stimulations at 0°C 200 and 500 ms, revealed a somewhat slower rate of phosphorylation in the stretched muscles than that in muscles of standard rest length (cf Table II, IA and lB, 1C and 1D).

Table II lists the moles of [32P]phosphate transferred/m01

TABLE II Phosphorylation of the 18,000-dalton light chain of myosin in stimulated muscles as compared with resting muscles

Results are given f standard error in cases of four or more experiments. Tempera- Percentage phos- [‘“PIPhosphate

Number Activation of muscle Time of activation ture of acti- Type of contraction phorylation” transferredh/light n vation chain

“C m01/l?l01

1A Tetanic stimulation 206 ms 0 Isometric’ 135 0.14 3 1B Tetanic stimulation of 200 ms 0 Noned 128 0.11 3

stretched muscle 1c Tetanic stimulation 500 ms 0 Isometric’ 156 f 9 0.22 f 0.03 5 1D Tetanic stimulation of 500 ms 0 Noned 142 0.17 2

stretched muscle 1E Tetanic stimulation 5s 0 Isometric 159 0.24 1 1F Tetanic stimulation of 5s 0 None” 160 0.24 1

stretched muscle 2A Tetanic stimulation 300 ms 2 Isometric 168 0.27 1 2B Tetanic stimulation 600 ms 2 Isometric 169 0.28 1 2c Tetanic stimulation 3s 2 Isometric 182 0.33 2 3A Tetanic stimulation 40-200 ms 25 Isometric 145 f 7 0.18 + 0.02 5 3B Tetanic stimulation l-3 s 25 Isometric 147 f 7 0.19 f 0.02 5 3c Tetanic stimulation of 3s 25 Isometric 150 + 5 0.20 * 0.02 4

stretched muscle 4A Tetanic stimulation 20-30 s 25 Isotonic 185 + 14 0.35 + 0.06 12 4B Tetanic stimulation of 20-30 s 25 Noned 183 f 12 0.34 f 0.05 11

stretched muscle 5 Caffeine 20 min 25 Isotonic 246 f 15 0.36 f 0.06 17

” The phosphorylation in the resting muscle was taken as 100%. b Calculated from the ratio of radioactivity of light chain to tropomyosin, and using 0.5 mol of [32P]phosphate/33,000-dalton tropomyosin. ’ About 40% of the maximal isometric tension was produced. ’ No detectable isometric tension was produced. ’ About 80% of the maximal isometric tension was produced.

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Light Chain Phosphorylation during Contraction-Relaxation 3621

of light chain, calculated from the ratio of light chain radio- activity to tropomyosin radioactivity (seventh column), as described before. In all experiments, with the exception of caffeine, the 100% phosphorylation values in the resting mus- cle ranged from 0.36 to 0.40 mol of [“‘P]phosphate/mol of light chain. Thus, the 150 to 170% phosphorylation upon short tetanic stimulation corresponds to an increase of 0.20 to 0.28 mol of [32P]phosphate transfer/m01 of light chain and the 185% phosphorylation with the 20- to 30-s tetani corresponds to an increased incorporation of 0.35 mol of [32P]phosphate. The total phosphate content of the light chain in muscles which reached the peak tension varied from 0.54 to 0.75 mol of [32P]phosphate/mol of protein; that is, stimulation causes a quantitatively significant phosphorylation of the light chain.

stimulation (5 min in 4B and 4D of Table III), light chain

Dephosphorylation of the 18,000-da&on Light Chain dur- ing Muscle Relaxation-The phosphorylation of the light chain associated with tetanic stimulation is reversible; this is illustrated in Table III. At O”C, when the muscle was frozen 3 s after the last stimulus of a 5-s isometric tetanus (when it is near complete relaxation), light chain phosphorylation was 125% of the resting value (Table III, 1); this is to be compared with the value of 159% for a muscle frozen after 5 s of sustained tetanic tension (Table II, 1E). In another series of experiments at 2”C, relaxation of the muscle from a 3-s isometric tetanus required about 3 s, which coincided with complete dephos- phorylation of the light chain (Table III, 2); contracted mus- cles analyzed under these conditions possessed as much as 182% phosphorylation (Table II, 2C). At 25’C, the rate of relaxation is much faster than at 0 or 2”C, therefore, the time interval between the last stimulus of the tetanus and the time of freezing was reduced accordingly: 0.3 s was just sufficient to allow full relaxation of the muscles after a 2.5-s tetanus. In this case, 114% phosphorylation was observed (Table III, 3); this is to be compared with the l- to 3-s stimulated muscles in this series which showed 147% phosphorylation (Table II, 3B). Thus, after brief tetani most of the phosphate incorporated into the light chain during stimulation is dephosphorylated accompanying muscle relaxation. However, after longer tetani the rate of relaxation is faster than the rate of light chain dephosphorylation. Thus, at 25°C for a 10-s or 30-s isotonic contraction, the muscles relaxed within 1 s, but even at 30 s after the end of stimulation, light chain phosphorylation re- mamined elevated, 117 or 148%, respectively (Table III, 4A and 4C). These values may be compared with 185% phospho- rylation found for the 20- to 30-s stimulated muscles (Table II, 4A). Nevertheless, after a sufficiently long interval after

TABLE III Dephosphorylation of the 18,000-dalton light chain of myosin in

muscles relaxing from tetanus One of the paired muscles was tetanized and frozen at various times

after the end of stimulation as indicated in the table; the resting muscle was frozen at the same time. Values represent percentage phosphorylation of stimulated muscle as compared to a 100% value of resting muscle. For recoveries lasting 5 min, the muscles were oxy- genated in Ringer’s solution before freezing.

Time of freez-

Num- Type of tetanus Duration Tempera- ing of tetanized Percentage

ber ture muscle after phospho- the end of stim- rvlation

ulation

1 Isometric 5 0 3s 125 2 Isometric 3 2 3s 99 3 Isometric 2.5 25 0.3 s 114 4A Isotonic 10 25 30s 117 4B Isotonic 10 25 5 min 94 4c Isotonic 30 25 30s 148 4D Isotonic 30 25 5 min 100

dephosphorylation has returned to the resting values even in those muscles tetanized for 10 or 30 s.

Dephosphorylation of the l&000-dalton Light Chain in Myofibrils in Vitro-We have found a partial dephosphoryl- ation of the light chain in a system reconstituted from myofi- brils and the sarcoplasmic protein fraction. This is shown in Table IV. The radioactivity of the light chain decreased in fibrils treated with EGTA and Mg2+ (Table IV, 2A to 2D) as compared to the fibrils treated with CaZc and Mg”+ (Table IV, 3A to 3D) or untreated (Table IV, 1). This decrease amounted to about 50% in the presence of sarcoplasmic proteins (cfi Table IV, 2C and 3C, 2D and 3D) and to about 20% in their absence (I$ Table IV, 2A and 3A, 2B and 38). Under the same conditions, a reduction in the “‘P label of TN-T was also observed in an extent of 30 and 12%, respectively. However, the counts in tropomyosin did not change. The constancy of

TABLE IV

Changes in the radioactivity of the 18,000-dalton light chain, troponin-T, and tropomyosin in “‘P-labeled frog myofibrils

32P-labeled leg muscle from freshly killed frogs was minced at 4°C and homogenized in 10 volumes of a solution containing 0.1 M NaF, 1 mM EDITA, and 1 mM Tris-HCl, pH 7, at O’C for-l min. The suspension was centrifuged at 0°C with 600 x g for 30 min. and the residue was washed twice with a solution of 0.04 M NaCl, 1 mM Tris- HCl, pH 7. An aliquot of these myotibrils was treated with 10% trichloroacetic acid at O°C immediately for determination of the ‘*P content of the light chain, TN-T, and tropomyosin in the fib&. This aliquot corresponds to “None” addition in the table. The sarcoplasmic proteins were prepared simultaneously from muscles of freshly killed nonradioactive frogs. The minced muscle was homogenized in 10 volumes of a solution of 0.1 M KC1 and 2 mM Tris-HCl, pH 7, at 0°C for 1 min and centrifuged at 600 x g for 30 min, and the supernatant was saved as the sarcoplasmic protein fraction. When the nonradio- active sarcoplasmic proteins were added to the ““P-labeled myofibrils, the weight ratio of s&coplasmic proteins to myofibrils was 1:2. In the exueriment shown in the table. “‘P-labeled mvofibrils (1 m&ml) were incubated at 25°C in a medium containing 12 mM MgCl2,50 mM Tris- HCI, pH 7.4, and in the presence of the indicated additions. The final ionic strength was 0.15, adjusted by the addition of KCl. The final volume was 25 ml and the suspensions were stirred magnetically. After 30 min, the reaction was stopped by the addition of trichloroa- cetic acid to 108, and the precipitated proteins were spun down at 40,000 x g; bot,h the supernatant and residue were saved. The supernatant was assayed for the P, liberated from ATP by the myofibrils. The residue was washed with 2% trichloroacetic acid, 5 mM NaH?PO, solution, dissolved in 2% SDS, 0.25 M NaP, solution at pH 8, and dialyzed against 0.1% SDS, 0.10 M NaP, solution at pH 7. Equal amounts of proteins were subjected to SDS-urea gel electro- phoresis, and the radioactivity of the various protein bands was determined after digestion. The dilution of the radioactive myofibrils by the nonradioactive sarcoplasmic proteins was taken into account when the radioactivity of the proteins was calculated.

Num- ber Additions to labeled myofibrils

1 None

2A 2 mM EGTA 2B 2 mM EGTA + 5 mM ATP 2c 2 mM EGTA + sarcoplasmic

2D proteins

2 mu EGTA + 5 mM ATP + sarcoplasmic proteins

3A 3B 3c

3D

0.1 mM CaCl2 0.1 mM CaCL + 5 mM ATP 0.1 mra CaC& + sarco-

plasmic proteins 0.1 mM CaCl2 + 5 mM ATP

+ sarcoplasmic proteins

Counts/20 min in the zone of:

Light Troponin- Tropo- chain T myosin

2404 4293 2503

1897 3733 2468 1974 3569 2512 1215 3084 2429

1152 2926 2575

2576 4098 2646 2483 4155 2441 2681 4237 2632

2239 4430 2387

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3622 Light Chain Phosphorylation during Contraction-Relaxation

light chain radioactivity in the presence of Cazt and its reduc- tion in the absence of Ca”+ mimic the in vivo situations prevailing in the stimulated and resting muscle, respectively.

The data in Table IV show that the presence of ATP virtually does not influence the “‘P content of the light chain (cf Table IV, 3A and 3B, 2A and 2B, 3C and 3D, 2C and 2D). Not shown in the table are the data which revealed that in the presence of 12 mM Mg’+ and 0.1 mM Ca2+ the fibrils hydrolyzed all the added ATP, but as much as 80% of the ATP remained in the presence of 12 mM MgZc and 2 mM EGTA. It appears that the phosphoryl group attached to the light chain does not turn over in the physiologically active ATPase.

DISCUSSION

We have shown a reversible phosphorylation-dephospho- rylation of the 18,000-dalton light chain during contraction and subsequent relaxation of frog muscle. Maximal phospho- rylation was obtained during a few seconds of tetanization at various temperatures; it amounted to 0.2 to 0.3 mol of ]“P]phosphat t e ransferred/mol of light chain. This value represents a difference in the ,laP content between light chains of tetanically stimulated and resting muscles, when both of the paired muscles were frozen (Table II). The increase of 0.2 to 0.3 mol of phosphate esterified must be considered as minimal since, as shown in this paper, the freezing itself induces a phosphorylation of the light chain. The mechanism of the freezing-mediated phosphorylation is unknown. Ten- sion records of resting muscles show no sign of contraction; hence, one may assume a subtle change in the internal milieu of muscle, e.g. ice formation in the sarcoplasmic reticulum. Another explanation might be that freezing inactivates the light chain phosphatase which normally keeps the phospho- rylation at a low level. We find 0.20 to 0.25 mol of phosphate/ mol of light chain in unfrozen resting muscles compared to 0.35 to 0.40 mol in frozen resting muscles; thus, the effect of freezing is manifested by 0.15 mol of [“‘PIphosphate incorpo- ration. Considering all the data, it appears that upon Full activation of a muscle 0.35 to 0.45 mol of phosphate may be transferred from the terminal phosphate group of ATP to a serine residue’ of the light chain. In this case, the light chain may contain as much [“2P]phosphate as 0.55 to 0.70 mol/mol of protein. Prolongation of the stimulus to 20 to 30 s results only in a slight additional increase of incorporation of the labeled phosphate (Table II).

The extent of light chain phosphorylation is a function of stimulus duration in the early phase of contraction. This is best evidenced by the experiments carried out at 0°C with muscles stimulated for 200 and 500 ms, before freezing, so that tension produced by these muscles was about 40 and 80% of maximal tetanic tension, respectively. Under these conditions, the increment in phosphorylation was 0.14 and 0.22 mol, respectively (Table II). It seems likely that the extent of the phosphorylation reflects the Ca”’ activation of the light chain kinase (27,28) by the progressive increase of the sarcoplasmic Ca” concentration (29). This idea is supported by experiments with caffeine, an agent known to release Ca2+ from the retic- ulum (30), which demonstrate phosphorylation of light chain without an electrical stimulus (Ref. 9 and Table II).

The results with stretched muscles (Table II) indicate clearly that the events of activation per se and not the me- chanical response initiates the light chain phosphorylation. From experiments specially designed to compare light chain phosphorylation between muscles left at standard length or

,‘In rabbit muscle this serine residue is located near the NH2 terminus of the light chain (25,26).

stretched to the point of no overlap (Table II), it appears that the rate of phosphorylation may be somewhat less at the 140% rest length. This could be due to the reduced rate of Ca2+ release in stretched and stimulated semitendinosus fibers (29).

Localization of light chain phosphorylation to the activation of muscle raises the question of what is the role of this phosphorylation in the contraction cycle. Maximal phospho- rylation of a serine residue in the light chain adds 1.8 negative charges to one myosin head at the physiological pH, 7.4. According to current concepts (31, 32), there are probably 3 myosin molecules in a cross-bridge. Thus, phosphorylation of the light chain may add about 11 negative charges/cross- bridge. This may be the cause for the swinging of the cross- bridges (33) from the negatively charged backbone of the thick filaments toward the thin filaments. Recent results of Sutoh et al. (34) suggest that the heads of the myosin molecule move away from the thick filament surface at alkaline pH, that is, with increasing negative charge of the proteins. This may be the biochemical equivalent for the early change of the equatorial reflections (11,~/11,1) observed with fast x-ray dif- fraction techniques on live muscle first by Huxley (35) and subsequently by Matsubara and Yagi (36). Moreover, light chain phosphorylation in stretched and stimulated muscles may be correlated with the x-ray data of Haselgrove (37) which show changes in the x-ray diffraction pattern of these muscles, indicating movement of the cross-bridges. In addition to its possible role in the activation of contraction, light chain phosphorylation may also facilitate the attachment between myosin and actin filaments. The phosphoryl group attached to the myosin head could provide an ionic attractive force with actin-bound calcium (38, 39) and thus tend to increase the affinity of cross-bridge attachment to the thin filament.

Light chain phosphorylation is reversible. Although the precise temporal relationship between dephosphorylation and relaxation has not been determined, our data indicate that after short tetani dephosphorylation approximately followed relaxation and after longer tetani dephosphorylation lagged behind relaxation. Light chain dephosphorylation after short tetani is reminiscent of the behavior of the equatorial reflec- tions of the x-ray pattern which returned to the resting value only after the tension had fallen to zero (36). The delay in dephosphorylation after longer tetani may be related to a delay in the reduction of Ca2+ levels (40,41) or to the phenom- enon of post-tetanic potentiation (42).

In vitro experiments demonstrate that the absence of Ca2+ is required for light chain dephosphorylation (Table IV). However, according to Morgan et al. (43), Ca”+ is not an inhibitor of purified rabbit myosin light chain phosphatase. Assuming that the frog enzyme behaves similarly to the rabbit enzyme, it is reasonable to postulate a Ca”‘-dependent regu- lator of frog light chain phosphatase. Several reports have appeared describing protein inhibitors of protein phosphatases (44-46). Such a protein inhibitor may be bound through Ca’+ ions to the light chain phosphatase, similarly to the binding of troponin-C to troponin-I (47). Accordingly, until Ca2+ is pres- ent, light chain phosphatase would be inhibited, and only after the dissociation of the phosphatase . inhibitor complex by EGTA could the phosphatase act on the phosphorylated myosin light chain.

A phosphorylation-dephosphorylation of light chain linked to contractile activity of frog muscles raises the question of its relationship to the well established troponin-tropomyosin con- trol of contraction. Both processes are Ca”+-dependent at micromolar concentration, i.e. the physiological range which triggers contraction. The binding of Ca*’ to troponin-C initi- ates a molecular rearrangement of the thin filament structure which is reversible through a dissociation of the troponin-C .

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Light Chain Phosphorylation during Contraction-Relaxation 3623

Ca2’ complex. Although this process is believed to be fast, its kinetics in live muscle has not yet been studied. The light chain phosphorylation is a covalent modification of the cross- bridge through a specific enzyme and is reversed by another specific enzyme. Application of classical physiological tech- niques allowed us to demonstrate light chain phosphorylation during the early phase of contraction. It would appear that light chain phosphorylation is another factor for the activation of contraction in frog muscle in addition to the Ca”+ switch mechanism on the thin filament.

Acknowledgments-We wish to thank Mr. Scott T. Sayers, John D. Glowicki, and Mrs. Claire Vercruysse-Megank for their expert and enthusiastic assistance. We are grateful to Dr. R. J. Solar0 for giving us his unpublished procedure for the preparation of the 18,000-dalton light chain, to Mr. J. G. Sarmiento for his help in the isolation of the “‘P-labeled light chain, and to Dr. J. D. Potter for a sample of troponin-C.

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K Bárány, M Bárány, J M Gillis and M J Kushmerickduring the contraction-relaxation cycle of frog muscle.

Phosphorylation-dephosphorylation of the 18,000-dalton light chain of myosin

1979, 254:3617-3623.J. Biol. Chem. 

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