The Proteolytic Substructure of Light Meromyosin LOCALIZATION OF A REGION RESPONSIBLE FOR THE LOW IONIC STRENGTH INSOLUBILITY OF MYOSIN*

10 mM 2-mercaptoethanol, at 20 "C for 20 min; the digestion was stopped with 0.5 mM phenylmethylsulfonyl fluoride. The digests were dialyzed against a solution containing 40 mM NaCI and 5 mM Na phosphate, pH 6.5, and centrifuged at 75,000 X g for 1 h. Rod was obtained from the pellets after ethanol treatment. Fragments of LMM were produced by digesting a suspension of conventional LMM (6 mg/ml in 30 mM KCI, 30 mM Tris-HCI, pH 8.0, and 10 mM EDTA) with trypsin, using an enzyme-to-substrate ratio of 1:120, at 20 "C for 16 min, and stopping the digestion with soyhean trypsin inhibitor. The digests were dialyzed overnight at 4 "C against 10 mM KC1 and 20 mM Na phosphate, pH 6.5, and centrifuged at 75,000 X g for 1 h. The pellet, designated insoluble fraction, was washed twice with the dialysis buffer, redissolved in 0.5 M KCI, and Ivophylized. The washings, combined with the supernatant, designated the soluble fraction, were lyophylized after the KC1 concentra-B

C and N over the arrows indicate removal of residues from the COOH and NH, terminus, respectively.
The positions of the peptides along the myosin heavy chain have been established by comparison with the published primary structures of rabbit skeletal (Elzinga, M., Behar, K., Walton, G., and Trus, B. L. (1980) Fed. Proc. 33 (40,41). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ T o whom correspondence should be sent at 20 Staniford Street, tion with their locations along the myosin heavy chain, suggest that a relatively small stretch of peptide (chain weight, 5,000 Da) located about 100 residues from the COOH terminus of myosin heavy chain is responsible for the insolubility of LMM at low ionic strength.
The myosin molecule consists of two heavy chains and four light chains. The NH2-terminal half of each heavy chain is folded into a globular head, while the remainder participates in a rod-like coiled-coil a-helical structure. Limited enzymatic proteolysis occurs chiefly in two regions, one at the head-rod joint and the other within the rod. Cleavage in the first region results in subfragment-1 (S-1) and rod, while cleavage in the second region produces HMM' and LMM (1). Subfragment-2 (S-2), which connects S-1 and LMM, can be produced by further digestion of HMM or rod, S-1 or HMM can be further degraded into well defined fragments whose connectivities have been established (2)(3)(4)(5). Some functionally important amino acid residues and binding regions for actin, light chains, and an ATP-analog have been located in these fragments (6)(7)(8)(9)(10)(11)(12)(13)(14)(15). However, little is known about the substructure of LMM. As previously reported, after prolonged tryptic digestion of LMM, three distinct bands, uiz. LF-1, LF-2, and LF-3, appear on electrophoresis under nondenaturing conditions (16). It was thought that the three fragments had the same COOH terminus and differed at the NH, termini (16). The present work defines the precise relation of the fragments to each other and determines their positions within the LMM structure by means of SDS-PAGE and NH2-and COOH-terminal analysis.
During this work we found that LMM preparations are often heterogenous, containing peptides LMM-A, LMM-B, and LMM-C; they differ slightly in their M,, as deduced from their mobilities on SDS-PAGE; they are identical in their NH,-terminal sequences, but differ in their COOH termini. Tryptic digestion of LMM yields three peptides with chain weights of 63,000, 47,000, and 30,000 Da, which are soluble at low ionic strength, corresponding to LF-1, LF-2, and LF-3, respectively, described earlier (16). In addition, there is a fragment with chain weight of 56,000 Da that is insoluble at low ionic strength, which we named LMM-D. The results of 13214 Insolubility-determining Region in Light Meromyosin NH,-terminal sequence determinations indicate that LMM-D is formed by the removal of residues from the NH, terminus of LMM while LF-1 is formed by clipping at the COOH terminus of LMM. LF-2 and LF-3 result from successive degradation at the COOH terminus of LMM-D. Therefore, a short segment near the COOH terminus of LMM appears responsible for the low ionic strength aggregation of LMM and it may also play a role in the assembly of myosin into filaments.

MATERIALS AND METHODS
Myosin was prepared as described previously (17) from rabbit white muscle except that the final column purification step was omitted.
LMM was ohtained by digesting myosin with trypsin (Sigma, Type III), at 20 'C in a solution containing 0.5 M KCI, 30 mM Tris-HCI, pH 8.2, 10 mM 2-mercaptoethanol, and 1 mM CaCI2, for 10 min with an enzyme-to-substrate ratio of 1:500 by weight. LMM made by this procedure will he referred to as conventional LMM. Myosin concentration was about 25 mg/ml and the digestion was stopped by adding soyllean trypsin inhibitor (Sigma, Type I S ) , twice the weight of tr.ypsin. After dialyzing overnight at 4 "C against 15 volumes of 10 mM Na phosphate, pH 6.5, the digests were centrifuged for 1 h a t 75,000 X g. LMM was ohtained from the pellets after ethanol treatment according to Szent-Gyorgyi et al. (18). Myosin rod was prepared based on the procedure of Weeds and Pope (19). Myosin (25 mg/ml) was digested with chymotrypsin (Sigma, Type 11) with an enzyme-to-substrate ratio of 1:400 by weight in 0.12 M NaCI, 10 mM Na phosphate, pH 7.0, 1 mM EDTA, and 10 mM 2-mercaptoethanol, a t 20 "C for 20 min; the digestion was stopped with 0.5 mM phenylmethylsulfonyl fluoride. The digests were dialyzed against a solution containing 40 mM NaCI and 5 mM Na phosphate, pH 6.5, and centrifuged a t 75,000 X g for 1 h. Rod was obtained from the pellets after ethanol treatment.
Fragments of LMM were produced by digesting a suspension of conventional LMM (6 mg/ml in 30 mM KCI, 30 mM Tris-HCI, pH 8.0, and 10 mM EDTA) with trypsin, using an enzyme-to-substrate ratio of 1:120, a t 20 "C for 16 min, and stopping the digestion with soyhean trypsin inhibitor. The digests were dialyzed overnight a t 4 "C against 10 mM KC1 and 20 mM Na phosphate, pH 6.5, and centrifuged at 75,000 X g for 1 h. The pellet, designated insoluble fraction, was washed twice with the dialysis buffer, redissolved in 0.5 M KCI, and Ivophylized. The washings, combined with the supernatant, designated the soluble fraction, were lyophylized after the KC1 concentra- trypsin at an enzyme-to-substrate ratio of 1:120 (w/w), in a solution containing 30 mM KC1,20 mM Tris-HCI, pH 8.0, and 10 mM EDTA, at 25 "C. Aliquots were taken a t various times and 20 and 30 pg of protein were placed on the gel for LMM and rod digests, respectively. tion of the mixture had been raised to 0.5 M. The lyophilized proteins were redissolved in distilled water (about 7-10 mg of protein/ml) and dialyzed against a solution containing 0.5 M KCI, 10 mM Na phosphate, pH 7.0, 1 mM EDTA and 5 mM 2-mercaptoethanol.
Edman degrada ions were carried out on a Beckman sequencer 89OC using progri m 122974; 2 mg of Polybrene was added to the sample before it I as applied to the cup (21). Anilinothiazolinones were converted to srresponding phenylthiohydantoins and identified by two independe It methods: thin layer chromatography (22) and amino acid analys~ ; after regeneration of free amino acids by hydrolysis in 56.6% hyc riodic acid a t 150 "C for 4 h (23). Amino acid analyses were perf lrmed on a Beckman analyzer 119 CL.
Carbox-ypeptida e A and B were purchased from Worthington and the digestions were carried out in 50 mM Na phosphate, pH 7.6, and 0.5 M NaC1. Aliquots were taken at various times and the reaction was stopped by acidifying the solution to pH 2 followed by centrifugation. The supernatant was applied to the amino acid analyzer to identify the free amino acid released.
Protein concentrations were determined by biuret (24) or microhiuret (25) method, using bovine serum albumin as the standard.

RESULTS'
Heterogeneity of LMM-SDS-gel electrophoresis of conventional LMM shows two bands, LMM-B and LMM-C, 72 and 68 kDa subunit mass, respectively (Fig. 1A). Even when myosin is digested with trypsin under very mild conditions, at an enzyme-to-substrate ratio of 1:1000, two bands appear on SDS-PAGE: the band of 72 kDa, which is obviously equivalent to a LMM-B chain, and the band of 78 kDa, designated LMM-A; the latter is not present if the digestion is carried out at an enzyme-to-substrate ratio of 1500 ( LMM-C, is also seen after the rod is digested with trypsin for 2 min (Fig. 2). LMM-A may be regarded as the longest form of LMM. As shown by the sequential appearance of LMM-B and LMM-C on digesting an LMM preparation, LMM-A is the precursor of LMM-B and LMM-C (Fig. 1B).
Tryptic Digestion of LMM and Rod-LMM-A present in the rod preparation quickly disappears as LMM-B and LMM-C are formed when trypsin is added (Fig. 2). Changes with time in the band pattern of tryptic digests of LMM indicate that LF-1 and LMM-D are formed first, and are then further degraded into smaller fragments, uit. LF-2 and LF-3. The fragments in tryptic digests of LMM and rod, LF-1, LMM-D, LF-2, and LF-3 have chain masses of 63, 56, 47, and 30 kDa, respectively (Fig. 2). Dimers of LF-1, LF-2, and LF-3 would have masses corresponding to those of the fragments proviously studied under nondenaturing conditions and identically designated as LF-1, LF-2, and LF-3 (16). and LMM-C, and some LF-1 (Fig. 3). Since purified LF-1 is fully soluble a t low ionic strength, we suggest that the appearance of LF-1 in the precipitate is due to the formation of hybrid dimers (see "Discussion").
The Behavior of the LMM Fragments under Nondenaturing Conditions-Gel filtration on a Sephadex G-200 column led to the separation of three fragments from the soluble fraction ( Fig. 4 A , in Miniprint), uiz. LF-1, LF-2, and LF-3, each containing a single peptide (Fig. 5, A and B, channels 1, 3,  and 4). LMM-D was purified by gel filtration of the insoluble fraction (Fig. 4B, in miniprint); it is also apparently homogenous under both nondenaturing and denaturing conditions (Fig. 5, A and B, channel 2). On SDS-PAGE, the migration velocities, in increasing order, are LMM-B, LMM-C, LF-1, LMM-D, LF-2, and LF-3. Under nondenaturing conditions, the mobility of LMM-D is reduced it becomes slower than LF-1 (Fig. 5, A and B, channel 6 ) and co-migrates with LMM-C (Fig. 5, A and B, channel  7). It should be noted that conventional LMM appears as a single band under nondenaturing conditions, although on SDS-gel electrophoresis, two peptides can be distinguished ( Fig. 1 and Fig. 5, A and B, channel 5).
NH2-terminal and COOH-terminal Sequence Determination of LMMs and the Fragments-To throw further light on the relationship among the various kinds of peptides, NH2-and COOH-terminal sequence analyses were carried out. The same NH?-terminal sequence, uiz. Gly-Lys-Gln-Ala-Phe-Thr-Gln-Gln-Ile-Glu-Glu-Leu-Lys-Arg-Gln . . . , was obtained (Table I) for all our LMM preparations, whether they contained mostly LMM-A (Fig. 1, channel ZZZ) or a mixture of LMM-B and LMM-C (Fig. 1, channel 0. The same NH2terminal sequence was found for LMM-C isolated from the insoluble fraction of the tryptic digests of LMM. These results indicate that the NH2-terminus of LMM-A remains intact and it is the residues in the COOH-terminal region that are removed as LMM-A is degraded to LMM-B and finally to LMM-C. The NHZ-terminal region of LF-1 is identical with that of LMM which shows that a segment is removed from the COOH-terminal region of LMM-C when LF-1 is formed. LMM-D, LF-2, and LF-3 have the same NH2-terminal se-

quence, Asn-Phe-Asp-Lys-Ile-Leu-Ala-Glu-Trp-Lys-His-
Lys-Tyr-Glu-Glu . . . , which however differs from that of LMM. Thus, these fragments are formed as a result of clipping both from the NH2-terminal and the COOH-terminal regions of LMM.
When a preparation of LMM containing mostly LMM-A was treated with CpA, no amino acid was released. However, Arg was released when CpB was used and Leu and Lys were also released when both CpA and CpB were used. Thus, the COOH-terminal sequence of LMM-A is . . . Lys-Leu-Arg.  Using the same technique, . . . Leu-Lys was identified as the COOH terminus of LMM-C; only Lys was released from LF-1 or LF-2 and Arg from LMM-D or LF-3 (Table I).

DISCUSSION
Some peptides in tryptic digests of the rod and LMM are identical. Additional ones in the digests of rod correspond to the S-2 which is at the NH2-terminal portion of the rod, and its fragments. Thus the 58-kDa peptide is presumably the long S-2 (19,26,27), while the 37-kDa peptide can be identified as the short form of S-2 (28,29). It has been shown that it is the COOH terminal end of long S-2 that is removed when short S-2 is formed (30). The fact that each LMM fragment, uiz. LF-1, LF-2, LF-3, and LMM-D, isolated by column chromatography under nondissociating conditions shows only one band on SDS-PAGE suggests that the two chains that make up LMM fragments are very similar, if not identical. The molecular weights of the dimers of LF-1, LF-2, and LF-3 calculated from the apparent chain weights based on the mobilities on SDS-PAGE are 126,000, 94,000, and 60,000, which agrees with the previously reported values for LF-1, LF-2, and LF-3, respectively (Table 11, in Miniprint).
The interpretation of the digestion pattern of LMM appears, a t first glance, difficult because of the existence of heterogeneity in LMM preparations, their homogeneity on a nondenaturing gel notwithstanding. We have shown here that the chains denoted as LMM-B and LMM-C result from the sequential degradations of LMM-A at the carboxyl terminus. On the basis of information on the NH2-and COOH-terminal sequences alone, the following relationship among the fragments can be deduced: LMM-A + LMM-B + LMM-C + LF-1 + LF-2 + LF-3 and LMM-D + LF-2 + LF-3. The amino acid sequence of the rod portion of nematode myosin has recently been deduced from the DNA sequence (31). Based on the homology between these two species, the common NH2 terminus of LMM-A, LMM-B, LMM-C, and LF-1 is placed a t residue number 467 which would give 466 and 632 amino acids for S-2 and LMM, respectively (Fig. 6). The common NH, terminus of LMM-D, LF-2, and LF-3 is placed at residue number 611, suggesting a stretch of about 144 residues between the NH2 termini of the two groups of peptides. The peptides are aligned with the nematode sequence so as to obtain the greatest number of identical residues. It should be emphasized that the identity of the NH2-terminal sequences within each of the two groups of fragments and the differences between the two groups of fragments rest on direct sequence data and are independent of the homology assignment between rabbit and nematode myosin. If one takes into account the molecular weight difference between LMM-B and LMM-D and the temporal changes in the intensities of the peptide bands in digests of rod and LMM (Fig. 2), it appears that LMM-D is derived from LMM-B directly by the removal of a stretch of amino acids from the NH2 terminus. The same applies to the relationship between LF-1 and LF-2; the fact

S-2-C 37k
that LF-1 and LF-2 differ about 17 kDa in their chain weights suggests that only a small segment, if any, was removed at the COOH terminus (Fig. 7). The most likely relationship among the fragments is represented by the following scheme. the COOH and NH, termini, respectively. Comparison of the COOH-terminal sequence of LMM-A with the recently published sequence of the COOH-terminal region of the rabbit heavy chain (32,33) indicates that even the longest form of LMM has lost 14 amino acids from the original COOH terminus of myosin heavy chain (Fig. 7). Elzinga et al. (32) have reported that in the LMM preparation they used, 38 residues from the COOH terminus of heavy chain were missing. The COOH-terminal sequence of LMM-C, Leu-Lys, taken in conjunction with the molecular weight of the peptide and the known sequence of the LMM portion (32,33), suggests that it has lost 129 residues from the COOH terminus of rod. The fact that one of'the cleavage sites, that between 969 and 970 (COOH terminus of LMM-C), is located at a region where a stable coiled-coil a-helical structure is interrupted (31) suggests that susceptibility to proteolytic enzyme is related to the conformational characteristics.
The fact that LMM-C is insoluble, while LF-1, whose NHZterminal sequence is identical to that of LMM-C, but lacks a segment at the COOH terminus, is soluble at low ionic strength leads to the conclusion that the proteolytic removal of a peptide at the COOH terminus, following the formation of LMM-C, renders the fragments soluble. Or, conversely, the presence of a small region (5 kDa, the difference in chain mass of LMM-C and LF-1) close to the COOH terminus of the myosin heavy chain is responsible for the self-association property of the myosin rod and LMM (Fig. 7 ) , and, by inference, for filament formation in uiuo. (The same conclusion can be drawn based on the analogous situation: LMM-D and LF-2 have identical NH,-terminal sequence, however, only LF-2 which lacks a segment at the COOH terminus is soluble a t low ionic strength). This is further borne out by the fact the smaller fragment, LMM-D, that contains this COOH-terminal region is insoluble, while LF-1, which contains a larger portion of the rod but lacks the segment near the COOH terminus, is soluble a t low ionic strength. Although some LF-1 is found in the insoluble fraction, it can be explained by postulating the existence of "hybrid" LMM-C/LF-1 dimers or LMM molecules with one of the chains nicked.
The existence of such hybrids would suggest that the presence of the relevant region in one of the two chains is sufficient to produce the insolubility at low ionic strength.
We have noted in comparing the order of migration velocities of the fragments in nondenaturing and SDS gel, that LMM-D (56 kDa) migrates more slowly than LF-1 (63 kDa) under nondenaturing conditions. The reversed order of mobilities suggests that the COOH-terminal region responsible for the insolubility has a lower density of net negative charge.
At the time this paper was completed, a report appeared (34) in which the authors also reached the conclusion that a relatively small stretch of the heavy chain is responsible for the insolubility of LMM fragments. They, however, have not been able to decide whether the location of the solubility determining region was close to the S-2/LMM joint or to the COOH terminus of the heavy chain. Our results clearly show it is located near the COOH terminus. 11.0 11.5 11.1 10.5 9.9 " " "-" "-