Phosphorylation of the l&000-Dalton Light Chain of Myosin during a Single Tetanus of Frog Muscle*

Changes in the 32P content of proteins due to muscle contraction were investigated, using muscles dissected from liver frogs injected with [32P]orthophosphate. The only significant change found was in the radioactivity of the 18,000-dalton light chain of myosin; during a single tetanus, an increase of 85 to 90% occurred as compared to the resting muscle. This increase corresponded to about 0.4 mol of 32P per mol of light chain. The same increase in radioactivity of this light chain was also found upon caffeine-induced contracture of the intact muscle. It is postulated that the increased Ca2+ concentration in the sarcoplasm resulting from electrical stimulus or caffeine treatment activates the myosin light chain kinase which phosphorylates the 18,000-dalton light chain.


SUMMARY
Changes in the '12P content of proteins due to muscle contraction were investigated, using muscles dissected from liver frogs injected with ["2Plorthophosphate.
The only significant change found was in the radioactivity of the l&000dalton light chain of myosin; during a single tetanus, an increase of 85 to 90% occurred as compared to the resting muscle. This increase corresponded to about 0.4 mol of azP per mol of light chain. The same increase in radioactivity of this light chain was also found upon caffeine-induced contracture of the intact muscle. It is postulated that the increased Ca"+ concentration in the sarcoplasm resulting from electrical stimulus or caffeine treatment activates the myosin light chain kinase which phosphorylates the l&000-dalton light chain. and were left at room temperature for periods of 2 to 3 days. The frogs were then pithed, and the paired sartorius and semitendinosus muscles were dissected.
The muscles were clamped at rest length in the electrode assembly which was adapted to allow freezing during isotonic contraction as previously described (11). One muscle, lifting a 5.0-g weight, was given tetanic stimulation at 25" for 30 s (frequency, 30 pulses/s), and then immersed into isopentane chilled by liquid nitrogen. The paired muscle was treated and frozen under identical conditions, without stimulation.
The pooled frozen muscles from three such runs were pulverized in a special all-stainless steel apparatus designed to allow all manipulations to be carried out under liquid nitrogen cooling. First, 6 ml of the appropriate homogenizing solution (see below) was frozen over a smooth surface in a col'd mortar.
The muscles were placed over the frozen solution and brought in contact with a chilled pestle. Pulverization was achieved by striking the pestle several times with a mallet. The muscle powder embedded in homogenizing solution was transferred from the mortar into a small Waring Blendor, where it was immediately homogenized in an additional 30 ml of the same solution, at 0" for 40 s. This procedure prevented a change in the 32P content of the muscle proteins since the muscle was thoroughly mixed with the homogenizing solution before it could be thawed. For the determination of 32P incorporation into total muscle proteins, homogenization was carried out in 5% trichloroacetic acid containing 5 rnM NaH*PO,. The homogenate was centrifuged at 40,000 x g for 5 min, and the residue was washed with this solution six more times. The final residue was dissolved in 3 ml of 2% SDS and 0.25 M sodium phosphate, pH 8.0, using a Polytron homogenizer (Brinkmann) at 0" and dialyzed against 2 liters of 0.1% SDS and 0.1 M sodium phosphate buffer, pH 7.0, 25" overnight. After clarification at 80,000 x g, 25", for 30 min, the samples from contracting and resting muscles were electrophoresed on 10% polyacrylamide gels as recently described (9). The distribution of radioactivity on the gels was determined by slicing the gels, followed by digestion with hydrogen peroxide, and subsequent counting (9). For the determination of arP incorporation into myofibrils, homog enization was carried out in a solution containing 20 rnM sodium phosphate, 1 rnM EDTA, and 1 mM iodoacetamide, pH 7.0, and the fibrils were sedimented at 600 x g. The pellet was washed with the same medium six times and prepared for gel electrophoresis as described before. Caffeine contracture was produced by incubating the dissected sartorius and semitendinosus muscles in Ringer's solution containing 10 rnM caffeine at 25" for 20 min; the paired muscles were kept in normal Ringer's solution.
The muscles were homogenized, without prior freezing, at 0" in one of the described solutions, and the '"Plabeled phosphoproteins were analyzed on SDS gels. The specific radioactivity of the phosphate groups of ATP was determined upon a 5% perchloric acid extract of the 'J2P-labeled frog leg muscles remaining after dissection of the sartorius and semitendinosus muscles.
Control experiments showed that the measured specific radioactivity of ATP was independent of the source of the muscles (mixed leg muscles compared with either sartorius or semitendinosus).
The specific radioactivity of the y-phosphate group of ATP was determined in two separate ways as previously described by us (11,12). The procedures of Ulbrecht and Ulbrecht (13) were used to determine the specific radioactivities of each one of the a-, /3-, and y-phosphates.

Phosphorylation of Myosin Light Chain during Muscle Contraction 4753
The molar content of y2P in the 19,000-dalton light chain of frog myosin was determined on the basis of counts eluted from the appropriate zone of the gel. The counts were standardized using the specific radioactivity of the terminal phosphate of ATP as a reference. Values were expressed in terms of mole of 32P per mole of myosin light chain from the known amount of protein applied to the gel and from the following conversions: 50% of the total frog muscle proteins or 70% of the myofibrils are myosin (141, and there are two 18,000-dalton light chains per 500,000-dalton myosin (4).

AND DISCUSSION
Live frogs injected with laPlorthophosphate and left at room temperature rapidly incorporate :12P into ATP. Within a day after the injection, the (Y-, /3-, and y-phosphate groups of ATP were found to be equally labeled; this finding indicates that the total s2P pool in the muscle had reached equilibrium. In our experiments, the frogs were kept alive for at least 1 day in order to ensure that any changes in the radioactivity of muscle proteins caused by the stimulus would represent differences in phosphoprotein concentration rather than in phosphoprotein turnover. A further requirement for the validity of these studies is the complete inhibition of protein kinase and phosphatase activities. We used trichloroacetic acid for denaturation of these enzymes. Recently, we reported that trichloroacetic acidinsoluble muscle proteins isolated from 32P-injected frogs contained considerable amounts of label (15). However, only a few of these phosphoproteins show changes in 32P content during a single tetanus. Fig. 1 compares the radioactivities of the total muscle proteins from the contracting muscle with those from the resting one. The only major change taking place, as a result of tetanus, was an 85% increase in radioactivity of the l&000-dalton protein. An increase amounting to 36% was also seen in the 34,000-dalton protein zone. The amount of protein isolated from contracting or resting muscle for these analyses Procedures" (see also Ref. 9). The radioactivity in the 200,000-dalton region derived from nucleic acids (11) and in the region below 10,000 derived from phospholipids, and proteolipids (15) is not shown. Staining intensities were measured with a Zeineh soft laser scanning densitometer and were the same for bands of contracting or resting muscles. was the same: on average 170 mg of total protein per g of muscle.
To identify the 18,000-dalton protein that became labeled during contraction, we compared the radioactivities of myofibrillar proteins from contracting and resting muscles. Fig. 2 shows a 90% increase in .12P label of the l&000-dalton protein in fibrils of contracting muscle under conditions when essentially no other change was found. A radioactivity profile similar to that shown in Fig. 2 was also obtained on actomyosins derived by further fractionation of these myofibrils. The mobility of the l&000-dalton protein zone was found to be identical with that of the DTNB light chain from rabbit skeletal myosin (16,17). The myofibrillar proteins were also analyzed by electrophoresis on SDS-urea gels (15) under conditions which separated the Ca2+-binding subunit of troponin (troponin C) from the DTNB light chain. This experiment showed clearly that the label is associated with the light chain.
The molar content of n2P in the l&000-dalton light chain of frog myosin was calculated using the specific radioactivity of the terminal phosphate of ATP as a reference, as further detailed under "Experimental Procedures." These values were 0.45 and 0.85 mol of 32P per mol of light chain before and after tetanus, respectively, when the frozen muscles had been treated with trichloroacetic acid, and 0.30 and 0.55 mol of R2P per mol of light chain in the myofibrillar fractions of resting and tetanized muscles, respectively. The fact the anP content of light chains in myofibrils was lower than that in the acidtreated muscles indicates that inhibition of the light chain phosphatase was not complete under our conditions. However, the data from trichloroacetic acid-treated muscles show that about 0.4 mol of phosphate is transferred from ATP to the 18,000-dalton light chain during a single tetanus.
Pires et al.
(2) established that rabbit myosin light chain kinase requires Ca2+ for its activity. Based on the assumption that the frog enzyme behaves similarly, we postulated that the phosphorylation of light chain in the stimulated muscle is caused by an elevated Ca2+ concentration demonstrated by Ashley and Ridgway (18). In order to test our hypothesis, we treated the frog muscles with caffeine, an agent known to release Ca2+ from the sarcoplasmic reticulum (19). Total proteins and myofibrils isolated from caffeine-treated muscle showed a specific increase in the radioactivity of myosin light