DEVELOPMENTAL
Structural
BIOLOGY
46, 317-325 (1975)
and Functional Comparison
Changes
with Adult
of Myosin
during
Development
Fast, Slow and Cardiac Myosin
F. A. SR~TER, M. BALINT
AND
J. GERGELS’
Department of Muscle Research, Boston Biomedical Research Institute: Department of Neurology, Massachusetts Genera! Hospital; and Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02114 Accepted
May 20, 1975
ATPase (Ca*+ and K’ activated) activity of myosin prepared from muscles of 3-4 week rabbit embryos (EM) is slighly lower than that of adult fast muscle myosin (FM), but in contrast to the less active adult slow muscle myosin (SM) is stable on exposure to pH 9.2. Studies of the time course, by means of Na dodecyl-SO, polyacrylamide gel electrophoresis. of changes in the pattern of polypeptides released by tryptic digestion show that in this regard EM is closest to SM. The light chain complement of EM appears identical with that of FM rather than of SM or cardiac myosin (CM) by the criteria of coelectrophoresis and removal by 5,5’-dithio-2.dinitrobenzoate treatment of LC2 except that the relative amount of LC, is less in EM than in FM. The staining pattern of light meromyosin (EMM) paracrystals prepared from EM is distinct from either the FM, SM or CM LMM staining pattern. These studies suggest that different genes are involved in the coding for embryonic and adult heavy chains. INTRODUCTION
Fast muscle myosin molecules synthesized in very young chick myotubes have essentially the same ATPase activity and, as judged by SPAGE’, the same light chain complement (LC,, LC, and LC,) as adult fast muscle myosin (FM) except that the fastest light chain (LC,) is always present in smaller amounts than in adult myosin; furthermore, negatively stained paracrystals of light meromyosin (LMM) prepared from tryptic digests of chick embryonic ’ Correspondence to: J. Gergely, Boston Biomedical Research Institute, Dept. of Muscle Research, 20 Staniford Street, Boston, MA 02114. This work was supported by grants from the National Institutes of Health (HL-5949, HD-06203), the National Science Foundation (GB-43484, GB-41383) and the Muscular Dystrophy Associations of America, Inc. Postdoctoral Research Fellow, American Heart Association, Massachusetts Affiliate. Abbreviations: LC,, light chain 1; LC,, light chain 2; LC,, light chain 3; HMM. heavy meromyosin; LMM, light meromyosin; EM, embryonic muscle; SM. slow myosin; FM. fast myosin; CM, cardiac myosin: SDS, sodium dodecyl sulfate; DTNB, 5,5’dithiobis-(2-nitrobenzoic acid): EDTA, ethylenediamine tetraacetic acid; DTT, dithiothreitol; SPAGE, SDS polyacrylamide gel electrophoresis. 317 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
breast muscle myosin (EM) show the same subperiodicity as those of adult FM type LMM. These facts suggested that avian embryonic myosin is rather like adult FM (Sr&er et al., 1972), except that as the muscle fibers mature there is an increase in the LC, content. On the other hand, according to Perry and his coworkers (Holland and Perry, 1969; Perry, 1970; Trayer et al., 1968; Trayer and Perry, 1966) embryonic avian and mammalian myosin closely resemble myosin extracted from slow adult muscle (SM) on the basis of immunological tests, lower ATPase activity and the lack of 3-methylhistidine. The latter is present in FM from adult muscles but not in SM. From these findings they concluded that myosin exists in two isozymic forms (embryonic or slow and adult or fast), the transformation taking place in the last stages of intra-uterine life and/or during the first few days of postpartum life, presumably in response to changes in innervation and physiological activity. The present work was undertaken to compare the properties of embryonic rabbit myosin with adult-fast and slow skele-
318
DEVELOPMENTAL BIOLOGY
tal and cardiac mammalian (rabbit) myosin. The ATPase activity of EM is, with respect to its alkaline stability, similar to but somewhat lower in its absolute value than that of FM. The light chain complement of embryonic myosin contains all three subunits described for FM (Lowe! and Risby, 1971; Sarkar et al.. 1971) and is similar to avian EM in that the fastest moving band is present in a smaller amount. The fragments produced by mild tnptic digestion from EM resemble those from CM and SM. The negatively stained LMM paracrystals from EM are quite different from either slow or fast adult types (Griffith, 19721. These results, together with previous work on the methylation of histidine (Trayer et al., 1968; HUSzar, 1972) suggest that different genes are involved in coding for the heavy chains of the myosin molecule in the embryo and in the adult. We obtained no evidence for the existence of qualitatively different light chains in rabbit EM.
VOLUME 46. 1975
croscopic examination of negatively stained paracrystals (Nakamura et al., 1971). Tppitic digestion. The digestion of myosin with trypsin was carried out in a medium containing 0.5 M KCl. 0.03 M Tris-HCl, pH 8.2 at 20°C. The trypsin myosin ratio is indicated in the legends (l:lOO-1:200 w/w). Samples were taken at various intervals and the digestion was stopped by addition of soyabean trgpsin inhibitor (Sigma) in a 2:l w/w ratio to trypsin. Following dialysis for 16 hr against 15 vol of 0.01 M phosphate, pH 6.5 the samples were centrifuged at 100,OOOg.The supernatant was used for ATPase assays and SDS-gel electrophoresis and will be referred to as HMM. The sediment was used for preparation of LMM as described earlier (Nakamura et al., 19711. Ethanol treatment of LMM was carried out essent ially according Szent-GyGrgyi et al. (19601. The samples were incubated with 3 vol of ethanol at room temperature and then centrifuged in a Sorvall centrifuge EXPERIMENTAL PROCEDURES (35,000g for 10 min). The sediment was dried by passing a stream of N, over it, Myosin and LMM. Rabbit embryos (3-3 dissolving it in 0.02 M phosphate, pH 6.5 week old), newborn, lo-day old and adult in the original volume and dialysis against rabbits were used. In embryos and newborn rabbits only the superficial muscles of the 20 vol of 0.02 M phosphate (pH 6.5) at 4°C for 16-18 hr. The samples were then cenhindlegs and back were used to avoid contamination with slow muscles found in trifuged and the supernatant used for SDS the deeper regions close to the bones. In gel electrophoresis. 6-week-old and adult animals the slowATPase assay. Prior to ATPase activity twitch muscles (soleus. semitendinosus. measurements the digests were dialyzed against a solution containing 0.5 M KC1 intertransversarius and crureusl were identified, excised and studied separately. The and 0.025 M Tris maleate, pH 7.0. Ca*+activated ATPase was measured in a medissected muscles were immediately frozen 0.050 M Tris-HCl. pH in liquid N,. For myosin preparations the dium containing frozen samples were thawed in an ice cold 7.6, 10 mM CaCl,. 2.5 mM ATP and 0.2 mg For K+-activated ATPase solution containing 0.05 M phosphate + 1 of protein/ml. mM EDTA. pH 7.0, cut into pieces and Ca”+ was replaced with 5 mM EDTA and 0.6 M KCl. Reaction volume 2 ml, 2 min homogenized with a Polytron (Brinkmann incubation, 25°C. Alkali lability of the Instr.) for 3 .< 5 sec. Previously published enzyme (Gergely et al., 19651 was deterprocedures were used for the preparation and purification of myosin (Sreter et al., mined by preincubation at pH 9.2 as de1972) including treatment with RNase to scribed in the legend to Table I. Protein was determined by a miremove contaminating RNA, and for the concentration preparation of LMM and the electron mi- crobiuret method (Itzhaki and Gill. 1964).
SR~TER, B~LINT AND GERGEL\
SDS gel electrophoresis was carried out as reported earlier (Sarkar et al.. 1971) except that 100 mm gels were used in 115-120 x 5 mm glass tubes. For carboxymethylation of samples used in SDS-gel electrophoresis the protein iO.:3-3 mg) was incubated in 5 ml of a solution containing 6M guanidine HCl. 50 mM P-mercaptoethanol and 0.2 M Tris-HCl. pH 8.2. for 2 hr at 10” under constant stirring. Na iodoacetate was then added to a final concentration of 0.12 M and the incubation continued for 30 min. The reaction mixture was dialyzed against 200 vol of a solution containing 10 mM Na phosphate. pH 7. at 2-4°C. DTNB treatment was carried out according to Weeds and Lowey (1971) and a portion of the dialyzed solution was applied to the gel. RESULTS
Tofptic digestion. As shown by the appearance of a protein soluble at low ionic strength, the rate of digestion is the same for fast and embryonic myosin (Fig. 1) but much slower for slow and cardiac myosin: TABLE
1
F.4x.1. SLOII.. CAHDI.K .\Nn EMBRYONIC Mvos~s ATPase ACTIVIT’~~ OF RABBIT MUSCLE Ca” Activated Without With 10 min preincubation. pH 9.2
Fast Slow Cardiac Embryonic 1 day old 10 day old
0.90 0.24 0.49 0.65 0.80 0.89
Ap,~~molmg-‘min-L 0.96 0.08 0.40 0.71 0.80 0.9”
K+. EDTA Act ivated
1.88 1.10 1.00 1.16 1.76 1.79
‘I In the experiments with preincuhation at pH 9.2 myosin solutions containing 0.2 mg of protein per ml. ‘25 mM Tris. 10 mM CaCI, were kept at ‘/S”C for 10 min. At the end of this period the pH was adjusted to 7.6 by adding Tris and HCI to a final concentration of 50 mM as required. The ATPase assay was then started hy adding 2.5 mM ATP. For other details of the ATPase assays see Experimental Procedures.
M\,osin during Decelopment
319
FIG. 1. Formation ot t’ragmentx wluhle at low ionic strength nn tryptic digestion ot’ mywin. IlIyosin was digested with trypbin ti)r the time3 indicated and the HMhl fraction was prepared aa described under Experimental Procedure>. The protein concentration is given a> ‘( ot the original myosin. Key: 0. emhryonic: Cl. fast: A. slow; -, cardiac.
although in the case cut’slow and cardiac myosin the trypsin:myosin ratio was doubled. The difference in the rate of digestion of slow and cardiac myosins. on the one hand, and fast myosin. on the other, was reported earlier (Gergely et al.. 1965). The ATPase activities of EM are somewhat lower than those of FM, but the Ca’+-ATPase of FM, similarly to that ot FM and in contrast to that of SM. shows no alkaline lability (Table I). It should be noted that of all types ot’ myosin. EM showed the greatest increase in specific activity on purification so that some of the reported lower ATPase acti\,ities t’or EM (Perry. 1970) may be due to the presence of contaminating proteins. The Cal+activated ATPase activities of HMM digests from either FM or EM considerabl! increased as the digest ion proceeded; there were only very slight increases in digests ot SM and CM (Fig. 2). and on extended tryptic digestion their ATPase activity eventually decreased. Tryptic digestion ot’ myosin has been shown to produce internal peptide bond cleavages within the polypeptide chain in the HMM portion (Bhlint et al.. 1974: Lowey. 1961,. The SDS-gel pattern of the
320
DEWLOPMENTAL BIOLOGY
low-salt soluble fragments (HMM) produced during digeston of FM, SM and EM (Fig. 3) indicates that while embryonic myosin shares a number of properties with adult FM, in some respects its behavior is more closely related to that of CM (Fig. 4) and SM. Fragments with molecular weights over 81.000 are present during the first 16 min of digestion in the case of EM and SM, while in the case of FM digests peptides with these high molecular weights are present only in the first 4 min of digest ion. Among the peptides with masses over 100,000 dalton, in digests of FM, SM and
TIME
OF
DIGESTION,
CM the pattern is dominated by a peptide of a mass about 150,000 dalton, while in EM a peptide band closer to 110,000 dalton is stronger. These large fragments give rise, with time, to a complex pattern of smaller fragments with characteristic differences among the various types of muscle. Thus a striking difference between HMM’s from FM, SM, E’M and CM is that the 81,000 and 74,000 dalton fragments found in FHMM are not seen in those of the other types of HMM. FM differs from SM and EM also with respect to there being more material in the 51,000-30.000 dalton region in FM digests. Electrophoretograms of HMM from EM show a band in the 22,000 dalton region not present in the other digests. Cardiac HMM is closer to F-HMM with respect to the 51,000-30,000 dalton region which contains more fragments than the corresponding region in gels of S-HMM and E-HMM. Light chains. Coelectrophoresis experiments show that the light chains of EM (Fig. 5) are indistinguishable from those of adult FM. As in the case of chicken EM (Srkter et al., 1972) LC, seems to be present in smaller amounts than in adult FM. The two bands indicated by LC, are two forms of the same polypeptide; the fast one disappears on reduction by DTT (S&-
MIN
Frc. 2. Ca2+ activated ATPase activity of HMM prepared after various durations of tryptic digestion. For details of digestion and ATPase assay, see Experimental Procedures. Key: 0. embn.onic: Cl, fast: A, slow; V. cardiac.
-
I
** ci +a
Q?
2
4
VOLUME 46, 1975
Ilr w a
-
m (I
+
8
F
16
32
2
4
8
s
16
2
4
8
16
32
MIN
E
FIG. 3. Electrophoresis of fragments of fast. slow and embryonic myosin after various times of digestion. SDS polyacvlamide gels (6%). Trypsin protein ratio 1:200 for fast and embryonic and 1:lOO for slow muscle myosin. Amount of protein applied, 10 pg.
SRBTER, BALINT AND GERGEL~
Myosin
during Development
tinguishable from the adult type (Srkter et al., 1972).
321 fast muscle
lO-5x M.W.
DISCUSSION
I .o .0 .6
.2
I z
,‘2
“\ 4
“\
4 8
16
/’ 32 MIN
FM;. 1. Electrophoresis of fragments of cardiac myosin. SDS polyacnlamide !6?1 gels. Trypsin pmtein ratio 1: 100.
ter et al., 1972; Roy et al., 19751, presumably of an internal disulfide bond (Griffith, 1972). The identity of the LC, of EM with that of FM is further supported by the fact that DTNB treatment, which removes the LC, of FM but not of SM or CM (Weeds and Lowey. 1971) also removes the LC, of EM. Light merom>,osin. The staining patterns of rabbit embryonic LMM paracrystals (Fig. 6c and d) differ from those of both fast and slow muscle (adult) type LMM. In addition to featureless spindle-shape aggregates we have encountered two types of striated paracrystals. Both are dominated by narrow heavily stained lines separated by about 43 nm. the same periodicity as that found in adult LMM paracrystals. However, one type of embryonic paracrystal shows practically no structure within the lightly stained regions separated by the heavily stained lines. The other shows some, not well defined, increase in staining in the middle of the repeat. These patterns are clearly distinguishable from those characteristic of fast (Fig. 6a) or slow muscle (Fig. 6b) LMM. These findings on rabbit embryonic LMM are in contrast to our earlier ones showing that chick embryo LMM is indis-
The present studies carried out on the rabbit, while confirming the view based on studies of chicken muscle that in man! properties EM is closely related to the myosin of adult FM (BAlint et al., 1974). also show significant differences suggesting that the heavy chain of EM, at least in the rabbit, may be the product of a gene not active in the adult of the species. The rate of the appearance on tryptic digestion of fragments soluble at low ionic strength was the same with FM and EM. and both rates were higher than those with CM and SM. The ATPase activity of rabbit EM, both in terms of its magnitude and its stability with respect to alkali exposure, is of the type found in adult FM and distinct from that of CM and SM. The gel electrophoretic pattern of the tryptic fragments showed similarities between SM and EM and. to a lesser extent.
LC,-
e
LC, {z
q-
LC,-
am1
F
!
S
FIG. 5. SDS polyacrylamide ( 10’7) gel electrophoresis of adult and embryonic rabbit skeletal myosin. Amount of each protein applied. 15 fig. Key: F, fast: S, slow; E, embryonic myosin; F + E and S + E coelectrophoresis of embryonic myosin with fast and slow myosin. respectively.
SR~TER,
B~LINT
AND GERGEI.~
CM. Thus in digests of EM and SM fragments between 100,000 and 150,000 dalton persist longer than in digestion of FM and CM. Fragments in the 75,000-80,000 dalton region are characteristic of FM digests, but are absent from digests of EM, SM and CM. In FM digests the formation of the 75,000-80,000 dalton fragments clearly precedes that of the smaller ones. The band pattern of the EM and CM digests indicates the formation of short-lived intermediates between the 100,000 dalton and -60.000 dalton fragments. The absence of 75.000-80,000 dalton fragments in SM digests suggests the possibility of a direct breakdown of the - 150,000 dalton fragment into smaller ones, although even in this case the exclusion of the very transient formation of the larger intermediates would require more work. Jean et al. ( 1973) have earlier pointed out that tryptic digests of SM and FM of adult rat differ from each other. The presence of a fragment having a mass of 170.000 daltons was characteristic of SM digests, and a fragment of 88.000 daltons is characteristic of FM digests. The 88.000 dalton fragment of Jean et al. (1973) probably corresponds to our 80.000 dalton fragment since there ma) be a species difference in the precise mode of tryptic breakdown of myosin. In recent studies in which HMM isolated after a very brief tvptic digestion was subjected to further tryptic treatment we attempted to locate the tryptic fragments within the HMM structure (Balint et al.. 1975). The gel pattern obtained on the digestion of intact myosinas in this work-rather than HMM may be complicated by the presence of additional low ionic strength soluble pept ides resulting from the fragmentation of the LMM portion of myosin (Balint et al.. 19681. The question whether the major fragments obtained by digestion of myosin correspond exactly to those obtained with HMM as
323
Mxosin during Development
starting material and whether subtle differences among apparently corresponding peptides from different types of myosin do exist can only be answered by more work. A feature of embryonic myosin distinguishing it from adult FM and SM and from CM becomes manifest on electron microscopic examination of negativel! stained LMM paracrystals. We have previously reported differences between FM and SM with respect to the finer details of the staining pattern of LMM paracrystals (Nakamura. et al. 1971). LMM paracrystals ot EM clearly differ from their counterpart from SM or FM with respect to the staining pattern. These differences suggest that the rod portion of EM has distinct features, or at least that the region in which trypsin action leads to the separation of HMM and LMM is different. which could account for the new type of staining pattern. The region whose tryptic digestion leads to the separation of LMM from HMM is regarded as a possible “hinge” (Burke et al., 1973; Huxley, 1969) within the myosin molecule that may play a role in determining the movement of the myosin heads in oiuo. Thus, if the differences in LMM staining pattern reveal differences in the hinge region, this may suggest that in the embryo there exists a myosin with functional properties differing from those in adult myosin. Studies of the light chain complement ot embryonic myosin reported in this paper confirm our previous conclusions drawn from the chick embryonic myosin. strongl! suggesting that the light chains of myosin are identical with those of fast adult muscle myosin light chains. The finding that the chain moving at the highest speed in SDS gels, LC,, is present in smaller amounts in EM is similar to our previous findings with chick CM. The presence of LC, in smaller amounts suggesting. under some conditions, its total absence (Dow and Stracher, 1971) may have been under-
FIG. 6. Comparison of staining pattern of embryonic paracrystals. Key: a, fast; b, slow; c and d. embryonic LMM.
and adult
skeletal
muscle
light
meromyosin
324
DEVELOPMENTAL
BIOLOGY
lying previous reports indicating that EM is similar to SM and contains only LC I and LC,. The fact that, as shown in the present experiments, LC, of embryonic myosin can be dissociated with DTNB further confirms the similarity of adult FM, EM light chains and their mode of attachment to the heavy chains. Taken together with previous reports on chemical differences residing in the subfragment-l portion of myosin between EM and adult myosin (Huszar. 1972) the reported studies have shown that while many similarities between embryonic and adult myosins of all three types (fast. slow and cardiac) exist. in view of the differences one has to consider it likely that a gene not expressed in the adult governs the production of the heavy chain of embryonic myosin. In view of our inability to show detectable qualitative differences between embryonic and adult F light chains it would seem that the same genes control these light chains. However, the quantitative aspects of their operation, viz. the control of the amount of LC, formed (Dow and Stracher, 1971; Sr&er et al., 1972) appear to be different in the embryonic muscles. The precise relation of the number and kind of heavy chains on the myosin molecules has not been finally settled. While there is a reasonable consensus regarding the presence of two heavy chains and four light chains per myosin molecule, it is not clear whether every myosin molecule must contain one LC, and one LC, moiety or whether myosin molecules containing two LC,‘s or two LC*‘s may exist (Frank and Weeds, 1974; Sarkar et al., 1971; Weeds and Frank, 1972). This t’urther raises the question of heterogeneity of heavy chains and leads to the question whether or not there are two kinds of heavy chains corresponding to LC, and LC,. If this were the case the heavy chain corresponding to LC, would also be present in smaller amounts in EM. Further studies on this problem are clearly required, particularly in the light of
VOLUME
46, 1975
a recent report (Strahs and Holtzer, 1973) that LC,-at least in muscle culturesmay originate in fibroblasts. REFERENCES BALINT. M., SR~TER, F. A.. WOLF, B.. NAGI., B.. and GERCELI.. J. (1974). The substructure of heavy
meromyosin.
Fed. Proc. 33, 1370.
BRUNT, M., SR~TER, F. A., WOLF, I., NAGY, B.. and GERGELI.. J. (1975) The substructure of heav!
meromyosin. The effect of CaZ’ Mg2+ on the tryptic fragmentation of heavy meromyosin. J. Biol. Chem. 250.6168-6177 B~LINT, M., QZILAGYI, G., FEKETE, M., BLAZSO. M., and BIRO, N. A. (1968). Studies on protein complexes of muscle by means of proteolysis V. Fragmentation of light meromyosin by trypsin. J. Mol. Biol. 37. 317-330. BARIL. E. F.. LOVE. D. S.. and HERRhIANk. H. (1966). Investigation of rryosin heterogeneity during chromatography on diethylaminoethyl cellulose. J. Sol. Chem. 241, 82%330. BURKE, M., HIMMELFARB, S., and HAFZRINGTON, W. F. (1973). Studies on the “hinge” region of myosin. Biochemistry 12,701-710. Dow. J.. and STR.KHEH. A. (19711. Identification ot the essential light chains of myosin. BOC. Nat. Acad. Sci. U.S.A. 69, 1107-1110 FRANK, G., and WEEDS, A. G. (1974). The amino acid sequence of the alkali light chains of rabbit skeletal musc!e myosin. Eur. J. Biochem. 44, 315-331. GERGEI.~. J.. PR~CAY. A.. SCHOLTZ. J.. SEIDEI.. J.. SR~TER. F. A., and THOhlPsON. M. (1965). In “Comparative Studies on 1Vhite and Red Muscle”(H. Kumagai and S. Ebashi, eds.), pp. 145-159. Igaku Shoin. Ltd., Tokyo. GRIFFITH. I. P. i 1972). The eftect of cross-links on the mobility of proteins in dodecyl sulfate polyacrylamide gels. Biochem. J. 126, 553-560. HOLLAND. D. I., and PERRY. S. V. 11969). The adenosine triphosphatase and calcium ion transporting activities of the sarcoplasmic reticulum ot’developing muscles. Biochem J. I I-i, 161-170. HUSZAR. G. (19X,. Developmental changes of the primary structure and histidine methylation in rabbit skeletal muscle myosin. Nature (New Biol.) 240, 260-264. HCIXLE~. H. E. (1969,. The mechanism of muscle contraction. Science 164. 1356-1366. ITZHAKI, R.. and GILL D. M. 11964). A micro-biuret method for estimating protein. Anal. Riochem. 9. 401-407. JEAN, D. H., GUTH, L., and ALBERS. R. W. (1973). Neural regulation of the structure of myosin. Erp. Neural. 38, 458-471. LOWEI, S. ( 1964). Myosin substructure: isolation of a helical subunit trom heavy meromvosin. Science
SRBTER, B~LINT AND GERCELV 145, 597-59s. LOWE\. S.. and RISBY. D. (19711. Light chains from fast and slow muscle myosins. Nature (London) 234. 81m2i5. NAKAMYRA. A., SR~TER. F. A.. and GERGELY. J. (19741. Comparative studies of light meromyosin paracrystals derived from red, white and cardiac muscle myosins. J. Gel/. Biol. 49, 88:3-898. PERRY. S. \‘. (1970,. Biochemical adaptation during development and growth in skeletal muscle. In “Physiology and Biochemistry of Muscle as a Food” (E. J. Briskey, R. G. Cassens, and B. B. March), pp. 537-553. The University of Wisconsin Press. SARKAR S., MUKHERJEE, S. P.. and SULTIN (1975). Intramolecular disulphide forms of LC, lightchain of rabbit skeletal myosin in native head and purified subunits. Abst. Biophys. Sot. p. 33a. SAR~~M. S. (197’). Stoichiometry and sequential removal of light chains ot’myosin in the mechanism ot muscle contraction. Co/d Spring Harbor Symp. &mzt. Biol. 37, 14-17. SARKAR, S., SR~ER, F. A.. and GERGELY, J. i1971,. Light chains of myosins from tast. slow and cardiac muscles. Proc. Nat. Acad. Sci. C!S.A. 68, 946-950. S~TER, F. A.. HOLTZER J., GERGELY, J., and HOLTZER, H. ( 1972). Some properties of embryonic myosin. J. Cell. Biol. 55, 586-59-1.
Myosin
during Dellelopment
325
SR~TER. F. A.. SALMONS. S.. ROMSI.L. F. C. A.. and GERGELY, d. (1975). The effect of changed activity
pattern on some biochemical characteristics of muscle. In “Exploratory Concepts in Muscle Disease and Related Disorders” IA. Milhorat. ed.) Excerpta Medica Internatl. Congress Series Vol. 333,338-313. STRAHS. K.. and HOLTZER, H. (1974). Effects of cytochalasir-B and colcimide on cells in muscle cultures. Abstract. Amer. Sot. Cell. Biol. Ann. Meeting. No. 671. SZENT-GY~RCU, A. G., COHEN, C.. and PHILPO-IT, D. E. (1960). Light meromyosin fraction I: A helical model for myosin. J. Mol. Biol. 2, 133-142. TR.AYEH. I. P.. HARRIS. C. I., and PEHHI.. S, V. (19681. %methyl histidine and adult and fetal forms ot skeletal muscle myosin. Nature (London) 217, 4X-153. TRAYER I. P.. and PERRY, S. V. (1966). The myosin ot developing skeletal muscle. Biochem. Z. 335, a;-100. WEEDS. A. G., and FRANK. G. (1972). Structural studies on the light chains of myosin. Cold Spring Harbor S,vmp. &ant. Bio/. 37, S-14. LVEEW. A. G.. and LOU.EY. S. (1971) Substructure of the myosin molecule II. The light chains ot myosin. J. A4ol. Bio/. 61. 701-7”s.
Structural
BIOLOGY
46, 317-325 (1975)
and Functional Comparison
Changes
with Adult
of Myosin
during
Development
Fast, Slow and Cardiac Myosin
F. A. SR~TER, M. BALINT
AND
J. GERGELS’
Department of Muscle Research, Boston Biomedical Research Institute: Department of Neurology, Massachusetts Genera! Hospital; and Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02114 Accepted
May 20, 1975
ATPase (Ca*+ and K’ activated) activity of myosin prepared from muscles of 3-4 week rabbit embryos (EM) is slighly lower than that of adult fast muscle myosin (FM), but in contrast to the less active adult slow muscle myosin (SM) is stable on exposure to pH 9.2. Studies of the time course, by means of Na dodecyl-SO, polyacrylamide gel electrophoresis. of changes in the pattern of polypeptides released by tryptic digestion show that in this regard EM is closest to SM. The light chain complement of EM appears identical with that of FM rather than of SM or cardiac myosin (CM) by the criteria of coelectrophoresis and removal by 5,5’-dithio-2.dinitrobenzoate treatment of LC2 except that the relative amount of LC, is less in EM than in FM. The staining pattern of light meromyosin (EMM) paracrystals prepared from EM is distinct from either the FM, SM or CM LMM staining pattern. These studies suggest that different genes are involved in the coding for embryonic and adult heavy chains. INTRODUCTION
Fast muscle myosin molecules synthesized in very young chick myotubes have essentially the same ATPase activity and, as judged by SPAGE’, the same light chain complement (LC,, LC, and LC,) as adult fast muscle myosin (FM) except that the fastest light chain (LC,) is always present in smaller amounts than in adult myosin; furthermore, negatively stained paracrystals of light meromyosin (LMM) prepared from tryptic digests of chick embryonic ’ Correspondence to: J. Gergely, Boston Biomedical Research Institute, Dept. of Muscle Research, 20 Staniford Street, Boston, MA 02114. This work was supported by grants from the National Institutes of Health (HL-5949, HD-06203), the National Science Foundation (GB-43484, GB-41383) and the Muscular Dystrophy Associations of America, Inc. Postdoctoral Research Fellow, American Heart Association, Massachusetts Affiliate. Abbreviations: LC,, light chain 1; LC,, light chain 2; LC,, light chain 3; HMM. heavy meromyosin; LMM, light meromyosin; EM, embryonic muscle; SM. slow myosin; FM. fast myosin; CM, cardiac myosin: SDS, sodium dodecyl sulfate; DTNB, 5,5’dithiobis-(2-nitrobenzoic acid): EDTA, ethylenediamine tetraacetic acid; DTT, dithiothreitol; SPAGE, SDS polyacrylamide gel electrophoresis. 317 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
breast muscle myosin (EM) show the same subperiodicity as those of adult FM type LMM. These facts suggested that avian embryonic myosin is rather like adult FM (Sr&er et al., 1972), except that as the muscle fibers mature there is an increase in the LC, content. On the other hand, according to Perry and his coworkers (Holland and Perry, 1969; Perry, 1970; Trayer et al., 1968; Trayer and Perry, 1966) embryonic avian and mammalian myosin closely resemble myosin extracted from slow adult muscle (SM) on the basis of immunological tests, lower ATPase activity and the lack of 3-methylhistidine. The latter is present in FM from adult muscles but not in SM. From these findings they concluded that myosin exists in two isozymic forms (embryonic or slow and adult or fast), the transformation taking place in the last stages of intra-uterine life and/or during the first few days of postpartum life, presumably in response to changes in innervation and physiological activity. The present work was undertaken to compare the properties of embryonic rabbit myosin with adult-fast and slow skele-
318
DEVELOPMENTAL BIOLOGY
tal and cardiac mammalian (rabbit) myosin. The ATPase activity of EM is, with respect to its alkaline stability, similar to but somewhat lower in its absolute value than that of FM. The light chain complement of embryonic myosin contains all three subunits described for FM (Lowe! and Risby, 1971; Sarkar et al.. 1971) and is similar to avian EM in that the fastest moving band is present in a smaller amount. The fragments produced by mild tnptic digestion from EM resemble those from CM and SM. The negatively stained LMM paracrystals from EM are quite different from either slow or fast adult types (Griffith, 19721. These results, together with previous work on the methylation of histidine (Trayer et al., 1968; HUSzar, 1972) suggest that different genes are involved in coding for the heavy chains of the myosin molecule in the embryo and in the adult. We obtained no evidence for the existence of qualitatively different light chains in rabbit EM.
VOLUME 46. 1975
croscopic examination of negatively stained paracrystals (Nakamura et al., 1971). Tppitic digestion. The digestion of myosin with trypsin was carried out in a medium containing 0.5 M KCl. 0.03 M Tris-HCl, pH 8.2 at 20°C. The trypsin myosin ratio is indicated in the legends (l:lOO-1:200 w/w). Samples were taken at various intervals and the digestion was stopped by addition of soyabean trgpsin inhibitor (Sigma) in a 2:l w/w ratio to trypsin. Following dialysis for 16 hr against 15 vol of 0.01 M phosphate, pH 6.5 the samples were centrifuged at 100,OOOg.The supernatant was used for ATPase assays and SDS-gel electrophoresis and will be referred to as HMM. The sediment was used for preparation of LMM as described earlier (Nakamura et al., 19711. Ethanol treatment of LMM was carried out essent ially according Szent-GyGrgyi et al. (19601. The samples were incubated with 3 vol of ethanol at room temperature and then centrifuged in a Sorvall centrifuge EXPERIMENTAL PROCEDURES (35,000g for 10 min). The sediment was dried by passing a stream of N, over it, Myosin and LMM. Rabbit embryos (3-3 dissolving it in 0.02 M phosphate, pH 6.5 week old), newborn, lo-day old and adult in the original volume and dialysis against rabbits were used. In embryos and newborn rabbits only the superficial muscles of the 20 vol of 0.02 M phosphate (pH 6.5) at 4°C for 16-18 hr. The samples were then cenhindlegs and back were used to avoid contamination with slow muscles found in trifuged and the supernatant used for SDS the deeper regions close to the bones. In gel electrophoresis. 6-week-old and adult animals the slowATPase assay. Prior to ATPase activity twitch muscles (soleus. semitendinosus. measurements the digests were dialyzed against a solution containing 0.5 M KC1 intertransversarius and crureusl were identified, excised and studied separately. The and 0.025 M Tris maleate, pH 7.0. Ca*+activated ATPase was measured in a medissected muscles were immediately frozen 0.050 M Tris-HCl. pH in liquid N,. For myosin preparations the dium containing frozen samples were thawed in an ice cold 7.6, 10 mM CaCl,. 2.5 mM ATP and 0.2 mg For K+-activated ATPase solution containing 0.05 M phosphate + 1 of protein/ml. mM EDTA. pH 7.0, cut into pieces and Ca”+ was replaced with 5 mM EDTA and 0.6 M KCl. Reaction volume 2 ml, 2 min homogenized with a Polytron (Brinkmann incubation, 25°C. Alkali lability of the Instr.) for 3 .< 5 sec. Previously published enzyme (Gergely et al., 19651 was deterprocedures were used for the preparation and purification of myosin (Sreter et al., mined by preincubation at pH 9.2 as de1972) including treatment with RNase to scribed in the legend to Table I. Protein was determined by a miremove contaminating RNA, and for the concentration preparation of LMM and the electron mi- crobiuret method (Itzhaki and Gill. 1964).
SR~TER, B~LINT AND GERGEL\
SDS gel electrophoresis was carried out as reported earlier (Sarkar et al.. 1971) except that 100 mm gels were used in 115-120 x 5 mm glass tubes. For carboxymethylation of samples used in SDS-gel electrophoresis the protein iO.:3-3 mg) was incubated in 5 ml of a solution containing 6M guanidine HCl. 50 mM P-mercaptoethanol and 0.2 M Tris-HCl. pH 8.2. for 2 hr at 10” under constant stirring. Na iodoacetate was then added to a final concentration of 0.12 M and the incubation continued for 30 min. The reaction mixture was dialyzed against 200 vol of a solution containing 10 mM Na phosphate. pH 7. at 2-4°C. DTNB treatment was carried out according to Weeds and Lowey (1971) and a portion of the dialyzed solution was applied to the gel. RESULTS
Tofptic digestion. As shown by the appearance of a protein soluble at low ionic strength, the rate of digestion is the same for fast and embryonic myosin (Fig. 1) but much slower for slow and cardiac myosin: TABLE
1
F.4x.1. SLOII.. CAHDI.K .\Nn EMBRYONIC Mvos~s ATPase ACTIVIT’~~ OF RABBIT MUSCLE Ca” Activated Without With 10 min preincubation. pH 9.2
Fast Slow Cardiac Embryonic 1 day old 10 day old
0.90 0.24 0.49 0.65 0.80 0.89
Ap,~~molmg-‘min-L 0.96 0.08 0.40 0.71 0.80 0.9”
K+. EDTA Act ivated
1.88 1.10 1.00 1.16 1.76 1.79
‘I In the experiments with preincuhation at pH 9.2 myosin solutions containing 0.2 mg of protein per ml. ‘25 mM Tris. 10 mM CaCI, were kept at ‘/S”C for 10 min. At the end of this period the pH was adjusted to 7.6 by adding Tris and HCI to a final concentration of 50 mM as required. The ATPase assay was then started hy adding 2.5 mM ATP. For other details of the ATPase assays see Experimental Procedures.
M\,osin during Decelopment
319
FIG. 1. Formation ot t’ragmentx wluhle at low ionic strength nn tryptic digestion ot’ mywin. IlIyosin was digested with trypbin ti)r the time3 indicated and the HMhl fraction was prepared aa described under Experimental Procedure>. The protein concentration is given a> ‘( ot the original myosin. Key: 0. emhryonic: Cl. fast: A. slow; -, cardiac.
although in the case cut’slow and cardiac myosin the trypsin:myosin ratio was doubled. The difference in the rate of digestion of slow and cardiac myosins. on the one hand, and fast myosin. on the other, was reported earlier (Gergely et al.. 1965). The ATPase activities of EM are somewhat lower than those of FM, but the Ca’+-ATPase of FM, similarly to that ot FM and in contrast to that of SM. shows no alkaline lability (Table I). It should be noted that of all types ot’ myosin. EM showed the greatest increase in specific activity on purification so that some of the reported lower ATPase acti\,ities t’or EM (Perry. 1970) may be due to the presence of contaminating proteins. The Cal+activated ATPase activities of HMM digests from either FM or EM considerabl! increased as the digest ion proceeded; there were only very slight increases in digests ot SM and CM (Fig. 2). and on extended tryptic digestion their ATPase activity eventually decreased. Tryptic digestion ot’ myosin has been shown to produce internal peptide bond cleavages within the polypeptide chain in the HMM portion (Bhlint et al.. 1974: Lowey. 1961,. The SDS-gel pattern of the
320
DEWLOPMENTAL BIOLOGY
low-salt soluble fragments (HMM) produced during digeston of FM, SM and EM (Fig. 3) indicates that while embryonic myosin shares a number of properties with adult FM, in some respects its behavior is more closely related to that of CM (Fig. 4) and SM. Fragments with molecular weights over 81.000 are present during the first 16 min of digestion in the case of EM and SM, while in the case of FM digests peptides with these high molecular weights are present only in the first 4 min of digest ion. Among the peptides with masses over 100,000 dalton, in digests of FM, SM and
TIME
OF
DIGESTION,
CM the pattern is dominated by a peptide of a mass about 150,000 dalton, while in EM a peptide band closer to 110,000 dalton is stronger. These large fragments give rise, with time, to a complex pattern of smaller fragments with characteristic differences among the various types of muscle. Thus a striking difference between HMM’s from FM, SM, E’M and CM is that the 81,000 and 74,000 dalton fragments found in FHMM are not seen in those of the other types of HMM. FM differs from SM and EM also with respect to there being more material in the 51,000-30.000 dalton region in FM digests. Electrophoretograms of HMM from EM show a band in the 22,000 dalton region not present in the other digests. Cardiac HMM is closer to F-HMM with respect to the 51,000-30,000 dalton region which contains more fragments than the corresponding region in gels of S-HMM and E-HMM. Light chains. Coelectrophoresis experiments show that the light chains of EM (Fig. 5) are indistinguishable from those of adult FM. As in the case of chicken EM (Srkter et al., 1972) LC, seems to be present in smaller amounts than in adult FM. The two bands indicated by LC, are two forms of the same polypeptide; the fast one disappears on reduction by DTT (S&-
MIN
Frc. 2. Ca2+ activated ATPase activity of HMM prepared after various durations of tryptic digestion. For details of digestion and ATPase assay, see Experimental Procedures. Key: 0. embn.onic: Cl, fast: A, slow; V. cardiac.
-
I
** ci +a
Q?
2
4
VOLUME 46, 1975
Ilr w a
-
m (I
+
8
F
16
32
2
4
8
s
16
2
4
8
16
32
MIN
E
FIG. 3. Electrophoresis of fragments of fast. slow and embryonic myosin after various times of digestion. SDS polyacvlamide gels (6%). Trypsin protein ratio 1:200 for fast and embryonic and 1:lOO for slow muscle myosin. Amount of protein applied, 10 pg.
SRBTER, BALINT AND GERGEL~
Myosin
during Development
tinguishable from the adult type (Srkter et al., 1972).
321 fast muscle
lO-5x M.W.
DISCUSSION
I .o .0 .6
.2
I z
,‘2
“\ 4
“\
4 8
16
/’ 32 MIN
FM;. 1. Electrophoresis of fragments of cardiac myosin. SDS polyacnlamide !6?1 gels. Trypsin pmtein ratio 1: 100.
ter et al., 1972; Roy et al., 19751, presumably of an internal disulfide bond (Griffith, 1972). The identity of the LC, of EM with that of FM is further supported by the fact that DTNB treatment, which removes the LC, of FM but not of SM or CM (Weeds and Lowey. 1971) also removes the LC, of EM. Light merom>,osin. The staining patterns of rabbit embryonic LMM paracrystals (Fig. 6c and d) differ from those of both fast and slow muscle (adult) type LMM. In addition to featureless spindle-shape aggregates we have encountered two types of striated paracrystals. Both are dominated by narrow heavily stained lines separated by about 43 nm. the same periodicity as that found in adult LMM paracrystals. However, one type of embryonic paracrystal shows practically no structure within the lightly stained regions separated by the heavily stained lines. The other shows some, not well defined, increase in staining in the middle of the repeat. These patterns are clearly distinguishable from those characteristic of fast (Fig. 6a) or slow muscle (Fig. 6b) LMM. These findings on rabbit embryonic LMM are in contrast to our earlier ones showing that chick embryo LMM is indis-
The present studies carried out on the rabbit, while confirming the view based on studies of chicken muscle that in man! properties EM is closely related to the myosin of adult FM (BAlint et al., 1974). also show significant differences suggesting that the heavy chain of EM, at least in the rabbit, may be the product of a gene not active in the adult of the species. The rate of the appearance on tryptic digestion of fragments soluble at low ionic strength was the same with FM and EM. and both rates were higher than those with CM and SM. The ATPase activity of rabbit EM, both in terms of its magnitude and its stability with respect to alkali exposure, is of the type found in adult FM and distinct from that of CM and SM. The gel electrophoretic pattern of the tryptic fragments showed similarities between SM and EM and. to a lesser extent.
LC,-
e
LC, {z
q-
LC,-
am1
F
!
S
FIG. 5. SDS polyacrylamide ( 10’7) gel electrophoresis of adult and embryonic rabbit skeletal myosin. Amount of each protein applied. 15 fig. Key: F, fast: S, slow; E, embryonic myosin; F + E and S + E coelectrophoresis of embryonic myosin with fast and slow myosin. respectively.
SR~TER,
B~LINT
AND GERGEI.~
CM. Thus in digests of EM and SM fragments between 100,000 and 150,000 dalton persist longer than in digestion of FM and CM. Fragments in the 75,000-80,000 dalton region are characteristic of FM digests, but are absent from digests of EM, SM and CM. In FM digests the formation of the 75,000-80,000 dalton fragments clearly precedes that of the smaller ones. The band pattern of the EM and CM digests indicates the formation of short-lived intermediates between the 100,000 dalton and -60.000 dalton fragments. The absence of 75.000-80,000 dalton fragments in SM digests suggests the possibility of a direct breakdown of the - 150,000 dalton fragment into smaller ones, although even in this case the exclusion of the very transient formation of the larger intermediates would require more work. Jean et al. ( 1973) have earlier pointed out that tryptic digests of SM and FM of adult rat differ from each other. The presence of a fragment having a mass of 170.000 daltons was characteristic of SM digests, and a fragment of 88.000 daltons is characteristic of FM digests. The 88.000 dalton fragment of Jean et al. (1973) probably corresponds to our 80.000 dalton fragment since there ma) be a species difference in the precise mode of tryptic breakdown of myosin. In recent studies in which HMM isolated after a very brief tvptic digestion was subjected to further tryptic treatment we attempted to locate the tryptic fragments within the HMM structure (Balint et al.. 1975). The gel pattern obtained on the digestion of intact myosinas in this work-rather than HMM may be complicated by the presence of additional low ionic strength soluble pept ides resulting from the fragmentation of the LMM portion of myosin (Balint et al.. 19681. The question whether the major fragments obtained by digestion of myosin correspond exactly to those obtained with HMM as
323
Mxosin during Development
starting material and whether subtle differences among apparently corresponding peptides from different types of myosin do exist can only be answered by more work. A feature of embryonic myosin distinguishing it from adult FM and SM and from CM becomes manifest on electron microscopic examination of negativel! stained LMM paracrystals. We have previously reported differences between FM and SM with respect to the finer details of the staining pattern of LMM paracrystals (Nakamura. et al. 1971). LMM paracrystals ot EM clearly differ from their counterpart from SM or FM with respect to the staining pattern. These differences suggest that the rod portion of EM has distinct features, or at least that the region in which trypsin action leads to the separation of HMM and LMM is different. which could account for the new type of staining pattern. The region whose tryptic digestion leads to the separation of LMM from HMM is regarded as a possible “hinge” (Burke et al., 1973; Huxley, 1969) within the myosin molecule that may play a role in determining the movement of the myosin heads in oiuo. Thus, if the differences in LMM staining pattern reveal differences in the hinge region, this may suggest that in the embryo there exists a myosin with functional properties differing from those in adult myosin. Studies of the light chain complement ot embryonic myosin reported in this paper confirm our previous conclusions drawn from the chick embryonic myosin. strongl! suggesting that the light chains of myosin are identical with those of fast adult muscle myosin light chains. The finding that the chain moving at the highest speed in SDS gels, LC,, is present in smaller amounts in EM is similar to our previous findings with chick CM. The presence of LC, in smaller amounts suggesting. under some conditions, its total absence (Dow and Stracher, 1971) may have been under-
FIG. 6. Comparison of staining pattern of embryonic paracrystals. Key: a, fast; b, slow; c and d. embryonic LMM.
and adult
skeletal
muscle
light
meromyosin
324
DEVELOPMENTAL
BIOLOGY
lying previous reports indicating that EM is similar to SM and contains only LC I and LC,. The fact that, as shown in the present experiments, LC, of embryonic myosin can be dissociated with DTNB further confirms the similarity of adult FM, EM light chains and their mode of attachment to the heavy chains. Taken together with previous reports on chemical differences residing in the subfragment-l portion of myosin between EM and adult myosin (Huszar. 1972) the reported studies have shown that while many similarities between embryonic and adult myosins of all three types (fast. slow and cardiac) exist. in view of the differences one has to consider it likely that a gene not expressed in the adult governs the production of the heavy chain of embryonic myosin. In view of our inability to show detectable qualitative differences between embryonic and adult F light chains it would seem that the same genes control these light chains. However, the quantitative aspects of their operation, viz. the control of the amount of LC, formed (Dow and Stracher, 1971; Sr&er et al., 1972) appear to be different in the embryonic muscles. The precise relation of the number and kind of heavy chains on the myosin molecules has not been finally settled. While there is a reasonable consensus regarding the presence of two heavy chains and four light chains per myosin molecule, it is not clear whether every myosin molecule must contain one LC, and one LC, moiety or whether myosin molecules containing two LC,‘s or two LC*‘s may exist (Frank and Weeds, 1974; Sarkar et al., 1971; Weeds and Frank, 1972). This t’urther raises the question of heterogeneity of heavy chains and leads to the question whether or not there are two kinds of heavy chains corresponding to LC, and LC,. If this were the case the heavy chain corresponding to LC, would also be present in smaller amounts in EM. Further studies on this problem are clearly required, particularly in the light of
VOLUME
46, 1975
a recent report (Strahs and Holtzer, 1973) that LC,-at least in muscle culturesmay originate in fibroblasts. REFERENCES BALINT. M., SR~TER, F. A.. WOLF, B.. NAGI., B.. and GERCELI.. J. (1974). The substructure of heavy
meromyosin.
Fed. Proc. 33, 1370.
BRUNT, M., SR~TER, F. A., WOLF, I., NAGY, B.. and GERGELI.. J. (1975) The substructure of heav!
meromyosin. The effect of CaZ’ Mg2+ on the tryptic fragmentation of heavy meromyosin. J. Biol. Chem. 250.6168-6177 B~LINT, M., QZILAGYI, G., FEKETE, M., BLAZSO. M., and BIRO, N. A. (1968). Studies on protein complexes of muscle by means of proteolysis V. Fragmentation of light meromyosin by trypsin. J. Mol. Biol. 37. 317-330. BARIL. E. F.. LOVE. D. S.. and HERRhIANk. H. (1966). Investigation of rryosin heterogeneity during chromatography on diethylaminoethyl cellulose. J. Sol. Chem. 241, 82%330. BURKE, M., HIMMELFARB, S., and HAFZRINGTON, W. F. (1973). Studies on the “hinge” region of myosin. Biochemistry 12,701-710. Dow. J.. and STR.KHEH. A. (19711. Identification ot the essential light chains of myosin. BOC. Nat. Acad. Sci. U.S.A. 69, 1107-1110 FRANK, G., and WEEDS, A. G. (1974). The amino acid sequence of the alkali light chains of rabbit skeletal musc!e myosin. Eur. J. Biochem. 44, 315-331. GERGEI.~. J.. PR~CAY. A.. SCHOLTZ. J.. SEIDEI.. J.. SR~TER. F. A., and THOhlPsON. M. (1965). In “Comparative Studies on 1Vhite and Red Muscle”(H. Kumagai and S. Ebashi, eds.), pp. 145-159. Igaku Shoin. Ltd., Tokyo. GRIFFITH. I. P. i 1972). The eftect of cross-links on the mobility of proteins in dodecyl sulfate polyacrylamide gels. Biochem. J. 126, 553-560. HOLLAND. D. I., and PERRY. S. V. 11969). The adenosine triphosphatase and calcium ion transporting activities of the sarcoplasmic reticulum ot’developing muscles. Biochem J. I I-i, 161-170. HUSZAR. G. (19X,. Developmental changes of the primary structure and histidine methylation in rabbit skeletal muscle myosin. Nature (New Biol.) 240, 260-264. HCIXLE~. H. E. (1969,. The mechanism of muscle contraction. Science 164. 1356-1366. ITZHAKI, R.. and GILL D. M. 11964). A micro-biuret method for estimating protein. Anal. Riochem. 9. 401-407. JEAN, D. H., GUTH, L., and ALBERS. R. W. (1973). Neural regulation of the structure of myosin. Erp. Neural. 38, 458-471. LOWEI, S. ( 1964). Myosin substructure: isolation of a helical subunit trom heavy meromvosin. Science
SRBTER, B~LINT AND GERCELV 145, 597-59s. LOWE\. S.. and RISBY. D. (19711. Light chains from fast and slow muscle myosins. Nature (London) 234. 81m2i5. NAKAMYRA. A., SR~TER. F. A.. and GERGELY. J. (19741. Comparative studies of light meromyosin paracrystals derived from red, white and cardiac muscle myosins. J. Gel/. Biol. 49, 88:3-898. PERRY. S. \‘. (1970,. Biochemical adaptation during development and growth in skeletal muscle. In “Physiology and Biochemistry of Muscle as a Food” (E. J. Briskey, R. G. Cassens, and B. B. March), pp. 537-553. The University of Wisconsin Press. SARKAR S., MUKHERJEE, S. P.. and SULTIN (1975). Intramolecular disulphide forms of LC, lightchain of rabbit skeletal myosin in native head and purified subunits. Abst. Biophys. Sot. p. 33a. SAR~~M. S. (197’). Stoichiometry and sequential removal of light chains ot’myosin in the mechanism ot muscle contraction. Co/d Spring Harbor Symp. &mzt. Biol. 37, 14-17. SARKAR, S., SR~ER, F. A.. and GERGELY, J. i1971,. Light chains of myosins from tast. slow and cardiac muscles. Proc. Nat. Acad. Sci. C!S.A. 68, 946-950. S~TER, F. A.. HOLTZER J., GERGELY, J., and HOLTZER, H. ( 1972). Some properties of embryonic myosin. J. Cell. Biol. 55, 586-59-1.
Myosin
during Dellelopment
325
SR~TER. F. A.. SALMONS. S.. ROMSI.L. F. C. A.. and GERGELY, d. (1975). The effect of changed activity
pattern on some biochemical characteristics of muscle. In “Exploratory Concepts in Muscle Disease and Related Disorders” IA. Milhorat. ed.) Excerpta Medica Internatl. Congress Series Vol. 333,338-313. STRAHS. K.. and HOLTZER, H. (1974). Effects of cytochalasir-B and colcimide on cells in muscle cultures. Abstract. Amer. Sot. Cell. Biol. Ann. Meeting. No. 671. SZENT-GY~RCU, A. G., COHEN, C.. and PHILPO-IT, D. E. (1960). Light meromyosin fraction I: A helical model for myosin. J. Mol. Biol. 2, 133-142. TR.AYEH. I. P.. HARRIS. C. I., and PEHHI.. S, V. (19681. %methyl histidine and adult and fetal forms ot skeletal muscle myosin. Nature (London) 217, 4X-153. TRAYER I. P.. and PERRY, S. V. (1966). The myosin ot developing skeletal muscle. Biochem. Z. 335, a;-100. WEEDS. A. G., and FRANK. G. (1972). Structural studies on the light chains of myosin. Cold Spring Harbor S,vmp. &ant. Bio/. 37, S-14. LVEEW. A. G.. and LOU.EY. S. (1971) Substructure of the myosin molecule II. The light chains ot myosin. J. A4ol. Bio/. 61. 701-7”s.
