A Comparison of Rat Myosin from Fast and Slow Skeletal Muscle the Effect of Disuse *

Certain enzymatic and structural features of myosin, purified from rat skeletal muscles representative of the fast twitch glycolytic (type IIb), the fast twitch oxidative (type IIa), and the slow twitch oxidative (type I) fiber, were determined and the results were compared with the measured contractile properties. Good correlation was found between the shortening velocities and Ca(2+)-activated ATPase activity for each fiber type. Short term hind limb immobilization caused prolongation of contraction time and one-half relaxation time in the fast twitch muscles and a reduction of these contractile properties in slow twitch soleus. Furthermore, the increased maximum shortening velocity in the immobilized soleus could be correlated with increased Ca(2+)-ATPase, but no change was observed in the enzymatic activity of the fast twitch muscles. No alteration in light chain distribution with disuse was observed in any of the fiber types. The myosin from slow twitch soleus could be distinguished from fast twitch myosins on the basis of the pattern of peptides generated by proteolysis of the heavy chains. Six weeks of hind limb immobilization resulted in both an increased ATPase activity and an altered heavy chain primary structure in the slow twitch soleus muscle.

I Certain enzymatic and structural features of myosin, purified from rat skeletal muscles representative of the fast twitc.i glycolytic (type IIb), the fast twitch oxidative (type IIa), and the slow twitch oxidative (type I) fiber, were determined and the results wer -compared with the measured contractile properties. hoot. correlation was found between the shortening velocities and Ca"-activated ATPase activity for each fiber type. Fast twitch white (type IIb) and mixed fast twitch red (type IIa/IIb) muscles could not be distinguished physiologically and showed three identical isomyosins (FM1, FM2, and FM3) by nondenaturing electrophoresis. The relative abundance of fast twitch light chains (LC) in rat white muscle ( type IIb) is comparable with other mammalian fast twitch muscles, except for the reduced amount of LC,f in the rat. The low level of LC, f in the rat is corroborated by rorrelation between light chain distribution and the ratio of fast myosin isomyosins. Fast twitch red type IIa and fast twitch white type Ilb musa les have similar myosin ATPase activities and maximal sht,rtening velocity, but differ in terms of isomyosin profile and in the percentage of light chains and light chain ^-toichiometry.
Short term hind limb immobilization caused prolongation of contraction time and one-half relaxation time in the fast twitch muscles and a reduction of these contractile properties in slow twitch soleus. Furthermore, the increased maximum shortening velocity in the immobilized soleus could be correlated with increased Ca'*-ATPase, but no change was observed in the enzymatic activity of the fast twitch muscles. No alteration in light chain distribution with disuse was observed in any of the fiber types. The myosin from slow twitch soleus could be distinguished from fast twitch myosins on the basis of the pattern of peptides generated by proteol j vis of the heavy chains. Six weeks of hind limb immobilization resulted in both an increased ATPase activity and an altered heavy chain primary structure in the slow twitch soleus muscle.
Skeletal muscle function is reflected in the physiological and biochemical properties of the contributing fiber types (1)(2)(3)(4). Fast twit: h white fibers (type IIb) have relatively short isometric twitch contraction times, high maximal shortening velocities W...), and high myosin ATPase activities and fatigue rapidly. Fast twita a red fibers (type IIa) also demonstrate relatively short isometric twitch contraction times and high V,,,.. and myosin ATPase activity, but are more resistant to fatigue. Slow twitch oxidative fibers (type I) show a prolonged isometric contraction time, a low V,,,.. and low specific activity of myosin ATI'ase and a high resistance to fatigue.
The myosin molecule of vertebrate striated muscle has been well characterized and contains two heav y chain: and two pairs of light chains . The catalytic site for myosin ATPase and the actin-binding site appear to reside solely on the myosin heavy chains (10)(11)(12), but the role that the light chains play in modulating the actomyosin system is not well understood. The abilit y of chicken fast m yosin S-1 to retain both high ATPase activity and actin binding, when stripped of the alkali light chains (12), has been interpreted in tefms of the subunits stabilizing the conformation of the mvositf heavy chains. Although vertebrate skeletal fast tv: , " t ch piuscle m y osin heavy chain exhibits the same ATPase ac vity as myosin S-1 (131, the fact that chemical modification of the single cysteine residues, located not at the catalytic site but on each alkali light chain of rabbit fast myosin, inhibits both actin binding and ATP hvdrolvsis (14) suggests that these light chains may participate in regulation of the actomyosin system-Using physiological conditions, recent work has dem-onstr^led a functional role for the Ca2, -binding light chain, LC_.` The extraction of I.C. from skinned skeletal muscle fiber with EDTA was found to be temperature dependent, and loss of one-third of the total I.C. markedly decreased the maximum velocity of shortening with little effect on isometric tension (15). A 50`i dissociation of LC_ from rabbit myosin, treated with EDTA, has been reported to cause a 15-20% reduction in actomvosin ATPase activity (16). In each case, subsequent reassc,cim-, n of the light chain reversed the effects of EDTA treatment.
It would appear logical to anticipate that correspondence between the molecular properties of rryosin and the physiology of contraction, may best be approached by comparing the biochemical and mechanical responses of muscles, homogeneous for a particular fiber type, in an experimental regimen designed to alter muscle function. In this respect, it is known that the pattern of muscular activity affects both the physiological and the biochemical properties of skeletal muscle (17)(18)(19). Also, chronic electrical stunulation favors the transformation of fast to slow muscle (17,20) and inactivity promotes the conversion of slow muscle to fast (21, 22).
We have approached this question b y determining mechanical properties and comparing them with certain structural ' and enzymatic features of-rayoain-from control and 6-week immobilized rats. Specifically, this study reports an integrated physiological and biochemical analysis of muscles containing primarily fast white (type IIb), fast red (type IIa), or slow red (type 1) fibers, from control and 6-week immobilized rat hind limb preparations. Since sample sue was limited by the casting procedure, a relatively simple method was developed for extracting myosin. The myosin light chain content was determined by one-dimensional and two-dimensionrl gel electrophoresis and compared with the ATPase actin ties. Results showed good correlation between the shorteni ig velocities and the Ca"-activated ATPase activity of each .,iuscle type. Fast witch white (type IIb) and the mixed fasi twitch red ( type Ila and t ype IIb) muscles could not be distinguished ph^siologi^ally. Six weeks of hindlimb immobilization caused the slow twitch soleus (type I) to become more "se a fast twitch muscle in terms of contractile properties and myosin Ca ATPase, without alteration in light chain pattern. Limited proteolysis demonstrated that the 1-D peptide pattern of immobilized soleus heavy chain was different from the control soleus and fast red myosin appeared to differ from fast white in terms of heavy chain structure, but the peptide pattern of these fast twitch muscles was unaffected by disuse.

MATERIALS AND METHODS
Tissues-Spragu o -Dawley 5-6-month-old female rats provided hind limb muscles of histochemically identi fied fiber . pe. The vastus lateralis and EDL provided fast twit , h : g ibers. The superficial region of the vastus lateralLs is 100 1, pure ft st white type IIb fibers (23): the deep r9d portion of the vastus latem li, is 7l1 t fast red type Ila fibers and 30`^ slow red-type I fibers (2:3). The EDL, used for physiological studies,-is (i i fast red-type Ila fibers and 40', fast white-type IIb fibers (24). The coleus provided slow red muscle, being composed of 85% slow twitch-type I fibers and 15 '^ fast red-type Ila fibers (24).
Cmcling-As an experimental model, muscle disuse and inactivity were produced by simple immobilization of rat hind limbs using plaster casts (25). Immoinilization by casting maintains nerve arz' blood supply intact and causes muscular atrophy without the comph,:ations of contracture introduced by tenotomy (26).
Myofibril Preparation-Selected muscles (SVL, DVL, EDL, and sole-,is) were rapidly removed from the Fiind limbs of parallel groups of control or 6-week immobilized rats. The muscle tissue was cleaned of fat, fascia, and tendons and thoroughly minced. The mince was blended in 0.1 M KCI, 5 mm MgCI,, 5 mm ethylene glycol his (f3aminoethyl ether)-N,N,N',N',-tetraacetic acid, I mm Na p yrophosphate, 25 mm imidazole, pH 7.0, using a Sorvall Omni-Mixer. The myofibrils were pelleted by c entrifugation at 800 x g for 10 min and washed three times in an imidazole buffer (25 mm, pH 7.0) by centrifugation and resuspension. All procedures were performed at 0°C , in order to ensure relaxed myofibrils. The three times-washed myofibrils were either frozen overnight at -70 'C prior to mvoF in extraction or imme-'ately solubdized in isoelectric focusing buffer containing 9.5 M urea (1:2, v/v) and another 1 g of solid urea was added per 3 ml of total volume, prior to freezing. Myofibrdlar proteins were resolved by isoelectric focusing, followed by two-dimensional gel electrophoresis, exactly as described by Whalen et Gl. (27).
Myosin Extraction-The regimen necessary for maintenance of rats in hind limb plaster casts. combined with the resulting muscular atrophy, limited the sample size available, especiall y of the small soleus muscle. Myosin, prepared from slow twitch cat and rabbit soleus by conventional procedures, has been reported as unacceptably contaminated by other muscle proteins (7, 20)• We have, prepared myosin by a procedure that incorporates two rounds of actomyosin dissociation, each followed by high speed centrifugation to remove actin (20) and a final ammonium sulfate precipitation step (28). In this manner, myosin was prepared of a purity suitable for the electrophoretic and enzymatic characterization of the three fiber types.
Myofibrils were first prepared in order to maximize the yield of myosin (7). Since proteolytic degradation was considered a problem during the extraction of cat coleus myos i n (20), all extraction buffers contained 0.1 mm phenvlmethanesulfonvl fluoride (final concentration). The presence of this protease -nhibitor also enhances the resolution of contractile proteins by gel electrophoresis (29). The washed myofibrils were resuspended in buffer I (0.3 m KCI, 5 mm MgCl,, 5 mm ATP, 2 mm sodium pyrophosphate, 0.5 mm dithiothreitol, 0.15 M KH:PO,, pH 6.6). The actomyosin was dissociated by gently stirring the myofibrils on ice for 20 min. The extracted myofi-briLs were centrifuged at 165,W0 x g for 4 h to pallet actin (20). The myosin was precipitated front supernatant by dial ysis overnight against low salt buffer (5 mm KH_PO,, 20 mm KCI, adjusted to pH 6.3). The precipitated myosin was collected by low speed centrifugation and resuspended in buffer II (0.6 M KCI, 5 mm MgCl2, 5 mm ATP, 0.5 mm dithiothreitol, 2 mm sodium pyrophosphate, 0.15 M KH_PO,, adjusted to pH 6.6). The ATP dissociation, high speed centrifugation. and overnight dialysis were repeated as above. The precipitated myosin was washed three times with low salt buffer to remove nucleotides. The final myosin pellet was resuspended in buffer III (0.5 M KCI, 5 mm MgCI_, 5 mm ATP, 5 mm dithiothreitol, I miv EDTA, 32.5 me KEPO,, 17.5 mm KH_PO,, pH 7.01. Ammonium sulfate fractionation was performed as described by Offer  Enzvrne Assays-Purified myosin was resuspended in high salt buffer (0.5 M KCI, 1 mm dithiothreitol, 0.05 M Tris•CI , pH 7.51 and dialyzed overnight against the same buffer to remove ammonium sulfate. After centrifugation at 40,000 x g for 15 min to clarify the preparation, the myosin was used for enzymatic analysis. Potassium-EDTA-activated ATPase was measured in a reaction mixture (0.5 ml) containing I mm EDTA, 0.5 M KCI, 5 mm ATP, 50 mm Tris • Cl , pH 7.5. Calcium -activated ATPase was measured in a reaction mixture (1.0 ml) containing 10 mm CaCh, 5 mm ATP, 50 mm Trls•CI pH 7.5. In each assay, the estimations were performed in triplicate with 02 mg of protein/ml of reaction mixture, and controls lacking enzyme were routinely included. The reaction was started by the addition of ATP and incubation was at 25 °C for 10 min. The reaction was stopped by the addition of 0.2 ml of ice-cold 15% (w/v) trichloroacetic acid/ml of reaction mixture. After cooling on ice for 10 min, protein was removed by centrifugation and a 0.3-m1 sample of the supernatant was taken for inorganic phosphate determination (32). The results were obtained using assay conditions in which a linear relationship between activity and enzyme concentration was maintained.
P-otein Deterrrtinatiom-The fluorescamine method of Bohlen et al. (33) was used, with bovine serum albumin as standard.
Gel Electrophoresis-Polyacrylamide gel ele-trophoresis was carried out in the presence of sodium dodecyl sulfate according to Weber and Osborn ( 341, using 12.5`;• acrylamide separating gels or 7.5% crosslinked gels under the electrophoretrc conditions of Laemmli (35). Myosin samples were prepared for SDS-polyacrylamide electrophoresis as described b y Weeds (7).
Molecular weight of the myosin light chains was determined by comparison with the mobility of marker proteins (aldolase, carbonic anhydrase, trypsin, hemoglobin, and cytochrome c) run on 12.5% cross-linked SDS-polyacrylamide gels in phosphate buffer (34).
The subunit composition of myosin light chains was estimated by scanning densitometry (5). The concentration of myosin heavy chains and light chains could not be estimated from densitometric tracings owing to problems encountered when proteins of widely varying molecular weight are analyzed on separate gels (36. 37). Therefore, the percentage of total light chains in myosin was determined by the method of Fenner et al. (38). Using this method, both heavy and light chains gave clearl y stained bands following electrophoresis on 6% cross-linked SDS-polyacrylamide gels. The bands, stained with Coomassie brilliant blue, were cut out and the dye was eluted by maceration in 25% pyridine in water (v/v). In our hands, the concentration of dye was proportional to absorbance up to 2.2 absorbance units and the absorbance of the eluted dye was proportional to load up to 75 pg of standard protein. The dye elution procedure was applied to myosin extracted, as described above, from each fiber type. The concentration of eluted dye was determined for each sample by running three different myosin concentrations over the linear range for both heavy and light chains using 6% gels. The percentage of total light chains in myosin was thus calculated and the stoichiometry of the light chains was determined using the formula of Lowry and Risby (5), assuming a value of M, = 470,000 for myosin.
Two-dimensional .-lectrophoresis was performed using the tech-niqu^ of O'Farrell ('9). The urea extracts were placed at -70 °C and electrophoreaie was carried out within 2 weeks of sample preparation to minimize storage artifacts.
Polyacrylamide Gel Electrophoresis of Native Myosin-Electrophoresis was carried out at 4 °C in a Pharmacia apparatus (GE-24LS) with recirculation of buffer between the anodic and cathodic chambers. Running buffer was 0 . 04 N tetrasodium pyrophosphate, pH 8.86, 10`4 glycerol, 2 ma cysteine. Crude extracts of myosin were prepared using coleus, DVL, SVL, and EDL. The muscles were rapidly dissected, cut into small pieces, and swirled with 3 volumes of Guba-Straub's buffer (0.3 M KCI, 0.1 M KH 2 PO., 1 mm EDTA, pH 6.5) for 15 min ( 40). The fragments were pelleted by low speed centrifugation and an equal volume of buffer ( 80 mm tetrasodium pyrophosphate, pH 8.86) containing Wt glycerol was added to the supernatants. Samples were stored at -20 °C prior to electrophoresis. Gels (6 x 0.5 cm), 3.2% acrylamide, 0.115` methylene bisacrylamide were prepared in 0.04 M tetra.odium pyrophosphate, pH 8.86, with 0.4`t tetramethylethylene diamine, 0.2% ammonium persulfate. Crude muscle extracts, 20-80 Al, were loaded on the gels. Electrophoresis was performed at a constant voltage of 50 V for between 22 and 24 h. ATPase activity in the gels was confirmed using the method described by Hoh et al. (41), in which the phosphate released after incubation in substrate was precipitated as calcium phosphate. The myosin isoenzymes were visualized by staining with Coomassie brilliant blue, as described for the regular polyacrylamide gel electrophoresis.
Limited proteolysis of heavy chains and single dimension peptide mapping was accomplished by exploiting the ability of protease to produce reproducible breakdown products from proteins complexed with sodium dodecyl sulfate (42). Purified myosin was electrophoresed on 5 rM separating gels at 3 mA/gel for 3 h, using a "iris-glycir-_ system in the presence of SDS (35). After rapid staining and des.aining for about 15 min, , he zones corresponding to the heavy chants were cut from the gets and equilibrated in buffer (0.125 M Tris-H(1, pH 6.8, 0.16% SDS, 1 mar EDTA, 1 mm dithiothreitol). Proteolvsis of the heavv chains was accomplished by treating the sample, during electrophoresis, with Staphylococcus aureus protease (43). The SDS gel electrophoresis was performed using 7-cut separating and 2-cm stacking gels of 12 and 5 1; acrylarttide concentration, respectively. A gel slice containing about 56 pg of heav y chains was placed on top of the stacking gel and electrophoresis was allowed to proceed at 3 mA/tube for 90 min until the marker dye just entered the stacking gel. Staphylococcus protease (10 µl, 0.2 mg/ml of solution in 0.125 to Tris -HCI (pH 6.8), 0.14 SDS, 1 mm EDTA, 20'i gl ycerol) was pipetted onto the top of the st. ^king gel and the run was continued at the same current for 4.5 h. 'I he gets were stained with Coomassie brilliant blue and destained electrophoretically, and the proteolvtic cleavr.ge products were recorded by densitometric scanning using a Gilford recording spectrophotometer.
Physiological Studies-The contractile properties of the fast type q b SVL, the fast t ype Ila and IIb EDL, and the slow type I coleus were determined in vivo at 22 °C using the isometric and isotonic measuring system described elsewhere (44,45). A detailed description of the methods used and the experimental results has been published (44,45). Selected physiological data are included in this paper to enable comparison with the biochemical studies on myosin.

Structural Characterization of Myosin from Rat Skeletal
Muscle of Defined Fiber Type-Myosin extracted from SVL (100% type IIb fibers), DVL (70% type IIa fibers), or soleus (85`X, type I fibers) myofibrils resulted in clearly resolved light chain patterns after SDS electrophoresis.
Slow .,uncle myosin contains two light chain species and fast wl.ite myosin three (Fig. 1, a and c) in agreement with chick slow and rabbit fast myosin (5, 6). One-dimensional gels resolved four components (LC,., LC,r, LC., and LC.,) in fast red DVI, myosin (Fig. lb). However, electrophoresis for longer time resolved two components in the rather broad LC_ (5,5'dithiobis (nitro benzoic acid) light chain) region of the gel, indicating a total of five protein light chain species associated with the DVL sample. The impression that DVL may contain both slow and fast myosin light chains was confirmed by mit.ing experiments. Equal quantities of purified soleus and DVL myosins separated into a total of four bands upon SDS electrophoresis. These bands correspond to the sum of the contributing light chains, viz. LC,., LC,r, LC-2 + LC.f, LC,,f, and rat fwit (type IIb or IIa) and slow (type I) myosin contain a total of three and two light chain species, respectively.

Molecular Weights, Enzyme Activity, and Stoiehiometry of Rat Myosin Light
Chains-Analysis of apparent molecular weight of fast twitch red DVL myosin, and a mixture of fast twitch white SVL plus slow twitch soleus myosin, indicates that fast red DVL represents an admixture of fast and slow myosin light chains ( Table 1). The slow myosin light chains (LC. and LC2.), observed in the fast red DVL (Figs. lb, 4c, and 5c), are undoubtably due to the 30% type I fiber contamination of this sample. Further support for this can be found by examining the light chain composition of a fast twitch muscle containing approximately 60% type IIa and 40% type IIb fibers and, importantly, no type I fibers. A two-dimensional analysis of myofibrils from the EDL (Fig. 5e) shows three light chains (LC f, LC_,, and LC.0, a pattern clearly different from the DVL (Fig. 50. The presence of fast type IIa fibers in rat soleus (24) is

Stoichiornetry of Rat Myos, n Light Chains in Various
Fiber Types-Percentage of total light chains in myosin was determined by eluting stained heavy and light chain bands from the same gel with pyridine (38) and quantitating the proteins spectrophotometrically (Table II). This method has been used for heart myosin and resulted in 15.5% total light chains in bovine heart (46) and 9.8% total light chains in canine heart (38), compared with 14.7`r r total light chains in chicken heart, measured by a densitometric method (5). The values we report for the percentage of total lig;tt chains in various rat myosins are comparable with those reported for rabbit and chicken (5). Applying the equation of Lowey and Risby (5) and using molecular weights calculated for individual rat myosin light chains (Table I)

Electrophoresis of Myosin under Vondissociating Condi-
tions-Mvosin from rat soleus separated as a single slower migrating component (Fig. Sal compared with the three faster migrating species present in fast twitch myosin (Fig. 3, b, c,  and d). Even under conditions of heavy loading, we were unable to demonstrate a faster migrating, minor species, previously reported for rat soleus (40,47). Myosin from the fast twitch muscles was resolved into three isoenz y-matic components FMI, FM2, and FM3, in order of decreasing mobility (48). Mixing experiments demonstrated the separate identity of the slow and fast isomyosins. The three fast isomyosins in type IIb SVI, and type IIa/IIb EDL appeared to be identical,

Relative stoichiometry of rat m yosin light chains
The moles of light chain in myosin of each fiber type were estimated by the relationship (per cent total light chains in rat myusin) (per cent mass) (molecular weight of myosin) /(molecular weight of light chain) (5).  The per cent mass of each myosin light chain was obtained from densitometer traces of SV'i gels of myosin from representative fiber types. Each value represents the average of four to six preparations ± S.E., from pooled tis.iue. The total sum of light chains in each el is taken as 100ri.. The moles of light chain were calculated for each myosin-type by the method of Lowey and ltisby (5). The values for the percentage of total light chains in myosin were calculated by quantifying the stained heavy and light chains after eluting the dye from 6`b polvacrylamide gels (38), and represents an average of seven to nine gels stained with Coomassie brilliant blue at different pro ein loads for each fiber tvpe.  since they were superimposable when the crude muscle extracts were mixed prior to electrophoresis. Also, FM3 and FM2 were present in roughly equivalent proportions, with FM representing a minor component (Fig. 3, c and d). Type Ila/I DVL myosin alsc showed three components, but the slowest migrating FM3 was a broad, heavily staining band in this fast twitch muscle (Fig. 3b). The major nature of FM3 was unlikely to be the result of contamination with slow twitch fibers, since mixing soleus and DVI, extracts resulted in four isomyosins, SM, FM 1, FM2, and FM3, after pyrophosphate electrophoresis (data not shown). The protein species, separated from the crude muscle extracts by pyrophosphate gel electrophoresis, were shown to have ATPase activity and thus to represent isomyosins by the presence of a white precipitate of calcium phosphate of identical mobility to the stained bands following incubation of the gels in substrate (41).

Effect of 6-week Immobilization on Rat Hind Limb Fast
Twitch and Slow Twitch Fibt.-s-The calcium-activated and the EDTA-activated m yosin ATPase level of the fast twitch DVL and SVL were unaffected by disuse (Table III).
The single (Fig. 1) and double (Fig. 4) dimensional gel patterns for myosin purified from fast twitch muscles were characteristic of the fiber type, but were unaffected by 6 weeks of immobilization. The appearance of additional spots in the elect rophoretograms of myofibrillar proteins with identical molecular weights but different isoelectric points (arrow, Fig.  5h) are probably due to light chain phosphorylation (49). The fast twitch red DVL myosin is clearly resolved into five species of light chain that are unaffected by disuse (Fig. 4, c and d). The presence of slow twitch light chains in rat DVL, suggested by one-dimensional electrophoresis, is confirmed by resolving mixtures of soleus and DVL myosin. The higher molecular weight LQ of rat DVL precisely superimposes upon the larger of the two soleus light chains (Fig. 4d). The light chains of fast twitch SVL (Fig. 5g) could not be distinguished from those present in fast twitch DVL (Fig. 5c) or EDL (Fig. 5e).
Immobilization caused a 20`k increase in rat soleus calciumactivated myosin ATPase, with no alteration in the distribution or stoichiometry of the myosin light chains. Myosin, purified from control and immobilized rat soleus, was mixed in equal amount and resolved by two-dimensional electrophoresis. The two species of light chain from each sample proved to be superimposable, thereby confirming that coleus responded to disuse by an increase in ATPase unaccompanied by structural changes in the myosin light chains.
The possibility of differential extraction of myosin light chains from the various fiber types was tested by resolving total myofibrillar proteins by two-dimensional electrophoresis. This approach also enabled the effects of disuse to be monitored, with respect to the majority of the contractile proteins (27). The presence of myosin light chains, actin and the two major forms of tropomyosin (alpha-TM and beta-TM) were resolved in each of the myofibrillar preparations (Fig. 5).
Myofibrils of slow twitch soleus (Fig. 5a) confirm the presence of the two myosin light chains characteristic of slow twitch fibers. No alteration in the pattern of myofibrillar proteins was observed 6 weeks after immobilization (Fig. 5b).
Myofibrils of fast red DVL demonstrate five species of   bl myosin light chains, thereby confirming the presence of slow twitch fibers in the DVL muscle. The contamination with slow twitch light chain is not observed in either fast twitch EDL (Fig. 5e) or fast twitch SVL (Fig. 5g).
None of the myofibrillar preparations demonstrated structural alterations in any contractile proteins following immobilization.
Myosin Heavy Chains-Since immobilization resulted in an increased coleus myosin ATPase activity, but identical light chain pattern, compared with normal slow red muscle, it was of interest to determine if these changes were associated with alteration in the heavy chain primary structure (43,50). As a first step toward analyzing molecular alterations, the myosin heavy chains were subjected to digestion by S. aureus protease in the presence of SDS. The proteolytic cleavage products of the entire myosin heavy chain were analyzed by one-dimensional gel electrophoresis. Densitometric scans of the proteolytic pattern of heavy chains indicate unambiguous and reproducible differences between slow twitch red soleus and fast twitch white SVL (Fig. 6, a and b). The pattern Flo. 5. Two-dimensional electrophoretic comparison of rat skeletal myofibrillar proteins prepared from control and 6week immobilized rat muscle. Washed myofibrils were prepared from muscles of selected fiber type and solubilized in i-oelectric focusing buffer. The apparent molecular weights of rat myosin light chains (LC) are given in Table I  obtained after proteolysis of fast twitch red DVL -myosin proved to be more complex and variable from run to run , data not shown), presumably reflecting both admixture of fast twitch and slow twitch fibers in this muscle and structural differences betwee.-fast red and fast white myosin heavy chains (24,61). In agreement with previous enzyme measurement and light chain analysis, the proteolytic cleavage patterns of fast white myosin extracted from normal animals was indistinguishable, within experimental precision, from patterns from 6-week immobilized rats (Fig. 6b). The densitometric profiles of immobilized and control soleus myosin heavy chain peptides (Fig. 6a) displayed clear differences, particularly in peptldc-of high molecular weight.
Although the Ca"-activated myosin ATPase activity was increased by immobilization in slow but not fast twitch muscles, all muscles showed an elevated maximal shortening velocity (Table III). The per cent increase was considerably higher in the soleus (67% increase) than in the predominantly type IIa EDL (35% increase) or the IOC <; type IIb SVL (20% increase). The isometric twitch duration (contraction time and one-half relaxation time) was prolonged by disuse in the fast EDL and SVL, while inactivity shortened the twitch duration of the slow solcus.

DISCUSSION
Muscle fiber structure satisfies two main functional requirements, speed of contraction, which is correlated with myosin ATPase activity (51) and is higher in fast white fibers than in slow re 1, and force generation. Fast twitch type Ila and type IIb fibers appear to be indistinguishable by physiological criteria; rat EDL and SVL have similar contraction times, one-half relaxation times, and maximal shortening velocities (Table III). The myosin ATPase-specific activities of fast red and fast white fibers can be calculated from the data in Table  III. If we accept that rat coleus muscle contains 85% type I fibers and 15% type IIa fibers (24), and we assume that the specific activity of type IIa myosin ATPase is the same as type IIb, then the specific activity of type I myosin ATPase may be calculated as 0.234 µmol of P • mg -' • min -' (using values in Table III) from the relationship: per cent type IIa fibers x specific activity of tyr° IIa ATPase + per cent type I fibers x specific activity of type I fibers = measured soleus C82* -ATPase specific activity. Similar logic applies to the DVL. The measured activity of 0.734 agrees well vith the theoretical calculated value. 0.7(0.955) + 0.3(0.:..x4) = 0.739. The concept that there is no detectable difference between myosin ATPase activities of fast red and fast white fibers receives further suppo.-t from the observation ( Table IV) that rat EDL, comprising 60% type IIa and 40% type IIb fibers, has a myosin ATPase activity comparable with rat SVL (Table  III), which is comprised of 100% type IIb fibers. The maximal shortening velocity of rat slow red coleus is about one-third of the corresponding values for fast white SVL and mixed fast red/fast white EDL (Table III), and this physiological parameter of muscle function correlates well with the Ca"-activated myosin ATPase activities of these fiber types. However, the relationship between fiber t yping and myosin ATPase activity is not straightforward. The soleus of cat and guinea pig is considered pure slow red muscle homogeneous for type I fibers on the basis of both histochemical fiber typing (24,59) and the distribution of mitochondria (60), while rabbit and rat soleus are rated inhomogeneous slow twitch muscles (59,60). Van Winkle et al. (56) However, the histochemically homogeneous cat soleus has a myosin ATPase activity more comparable with the mixed fiber rat soleus than with the relatively pure slow twitch rabbit coleus (Table IV). This disparity in myosin ATPase activities in the slow twitch soleus from various species cannot be reconciled with the myosin light chain pattern, since LC, is a doublet in the cat and the rabbit (7, 56), but only a single polypeptide in the rat (21) and guinea pig (61). The light chain stoichiometry of rat soleus myosin, calculated in the present study, corresponds to values published for rat (62) and rabbit (63). The presence of LC:,f was not normally detected in myosin purified from rat slow twitch soleus muscle. However, rat coleus myofibrils stored in the freezer for 5 months prior to myosin extract ton revealed a protein species that migrated on Laemmli-type SDS gels :ith a mobility identical with LC 3f. Paradoxically, this component was not present in immobilized rat solem myofibrils similarly stored. It would appear that this band is mu;: likely to represent a storage artifact, than myosin of the fast type phenotype (64).
Rat fast twitch white SVL and fast twitch red DVL muscles cannot be distinguished or, the basis of myosin ATPase or the distribution and molecular weights of the light chains. The relative abundance of fast twitch light chains (Table II) in the rat SVL compares with other mammalian fast twitch muscles (5, 61, 62), except for the low level of LC3 f in the rat. It is not clear if this represents a true decrease in alkali 2 chains in the rat or an extraction problem, since it was only by examining total cellular pre teins that Keller and Emerson (65) were able to demonstrate the presence of LC 3f in an embryonic system. Quantitation fro,n two-dimensional gels of total myofibrillar protein extracts would be needed to resolve this question. However, the distribution of light chains of fast muscle can be correlated with the proportion of individual isoenzymatic species. The individual fast isomyosins have been shown to differ in light chain composition, viz. FM1 comprises (LC 2f) 2 (LC3f )2, FM2 is LC,f(LC 2f ) 2 LC3f, and FM3 corresponds to (LC,f )2 LC2f (40,48,50). The fact that the isomyosin profile of rat SVL shows equivaler-t proportions of FM2 and FM3, with FM1 present as a mkior component, would correlate with the small proportion of LCf3 in this muscle. Rat SVL myosin is similar to rabbit fast myosin (66,67), containing 2 mol of LC2/ mol and LC, in 2-fold molar excess over LC3, but the alkali light chains are present in 1.6 times molar excess in the rabbit compared with the rat. The reduced content of essential light chains in rat compared with rabbit SVL does not affect the myosin ATPase activity (Table N). Rat fast twitch red myosin differs from rat fast twitch white myosin in light chain stoichiometry and in the percentage of total light chains, and these features may be reflected in the differential staining of these fiber types by immunofluorescent antibodies prepared against the alkali light chains (68).
Hind limb immobilization failed to alter either the light chain distribution or stoichiometry in any of the fiber types examined. However, the maximal shortening velocity was significantly increased after 6 weeks of disuse in both the slow twitch and the fast twitch muscles (Table III), but only in the immobilized coleus was there an increased myosin ATPase activity. Clearly, elevated ATPase activity and increased mechanical V... is accomplished without alteration in the myosin light chain pattern. Recent work on skinned fibers has demonstrated no correlation between V,,,., and the ratio of the myosin light chains in fast twitch or slow twitch fibers (69). Furthermore, although fibers from fast twitch and slow twitch muscles could clearly be distinguished on the basis of their light chain patterns and V,,,.., the relative proportions of the light chains present were independent of tension development, muscle stiffness, or unloaded shortening velocity (69).

Rat Muscle Myosin and Alteration with Disuse
The myosin from different types of muscle can be distinguished on the basis of the pattern of peptides generated by preteolytic digestion of the heavy chains. This statement is true for both slow twitch and fast twitch muscles of the chicken (43), and in this manner, guinea pig fast twitch red masaater was distinguished from fast twitch white tensor fasciae latae (61). This latter d istinction could not be made unambiguously in the present study, presumably because rat DVL is an admixture of both slow red and fast red fibers (24).
Further work is needed to determine the sites and extent of changes in amino acid sequence that may be brought about in the rat coleus myosin heavy chain by short terns immobilization. Although short term immobilization of rat slow twitch soleus resulted in increased V..., higher ATPase activity, and altered pattern of heavy chain proteolytic cleavage products, long tenn immobilization can cause the synthesis of fast type light chains in cat soleus (22), suggesting that the response to disuse may be temporally related. The increased Vm., in fast twitch SVL and EDL suggests that mechanical measurement offers a more sensitive indicator of alteration in muscle function with short tern immobilization than does biochemical analysis.