The substructure of heavy meromyosin. The effect of Ca2+ and Mg2+ on the tryptic fragmentation of heavy meromyosin.

Heavy meromyosin, obtained by tryptic digestion of myosin, containing two main polypeptides whose masses were estimated as 81,000 and 74,000 dlatons from Na dodecyl-SO4 polyacrylamide gel electrophoresis, was further digested with trypsin. The Ca2+-activated ATPase activity remainded unchanged and the K+-EDTA activity increased while various smaller fragments were formed. The formation of some of these fragments is affected by Ca2+ or Mg2+ as first shown by Bálint et al. (Bálint, M., Schaefer, A., Biro, N. A., Menczel, L., AND Fejes, E. (1971) J. Physiol. Chem. Phys. 3, 455). On the basis of the time course of the appearance of fragments the following relationship emerges: see article. The 64K leads to 60K step is inhibited by divalent cations, while the breakdown of the 74K fragment is accelerated. The effect of Ca2+ was maximal at 0 similar to 0.1 muM, that of Mg2+ at 10 muM. The original light chains of myosin are not present in the heavy meromyosin serving as the starting material, but peptide material appears on electrophoresis in positions starting material, but peptide material appears on electrophoresis in positions where the light chains would be found. The fragments marked by an asterisk are considered to ba alpha-helical on the basis of their solubility at low ionic strength after precipitation with ethanol (Bálint et al.). The fact that alpha helical fragments are derived from the 60,000-dalton fragment indicateds that it is adjacent to the light meromyosin in the intact myosin while the 74,000- dalton fragment would be part of heavy meromysoin subfragment 1. Chromatography of Sephadex G-200 separates fractions with ATPase activity corresponding to heavy meromyosin and heavy meromyosin subfragment 1. Electrophoresis of these Sephadex fractions suggests that the main peptide constituting heavy meromysoin subfragment 1 is connected by noncobalent forces to a portion of the rod that is not immediately adjacent to it in the primary sequence. The significance of this finding is discussed in terms of the flexibility of the myosin head.     

Proteolytic fragmentation of myosin: location of SH-1 and SH-2 thiols.

The heavy chain fragmentation pattern of native myosin when digested by proteolytic enzymes is influenced by such conditions as the nature of the proteolytic agent, ionic strength and presence or absence of divalent cations. HMM and S-1 produced by digestion of 14CNEM-labelled myosin under various conditions were analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. Purified samples of these species were digested under controlled conditions by chymotrypsin and trypsin and a comparison of the observed heavy chain fragmentation patterns led to a sequential arrangement of the proteolytic fragments. The main features of this arrangement are the following: a 21K molecular weight tryptic peptide is found at the N-terminal side of myosin heavy chain. Adjacent to it is a 48K peptide, then a 19.5K peptide containing the two SH-1 and SH-2 thiols. These three peptides constitute the heavy chain of S-1. Adjacent to this S-1 heavy chain is a tryptic (and also chymotryptic) 40K peptide. The rest of the HMM heavy chain on the C-terminus is a sequence susceptible to both chymotrypsin and trypsin attack yielding an undefined number of small peptides.     

Photoaffinity labelling with an ATP analog of the N-terminal peptide of myosin.

Photoaffinity labelling of tryptic and chymotryptic heavy meromyosin with 3′O-3-[N-(4-azido-2-nitrophenyl) amino]propionyl-adenosine 5′-triphosphate (arylazido-β-alanine ATP) resulted in incorporation of radioactivity and inhibition of the ATPase activity. ATP prevented the reaction with the photoaffinity label, as shown by the lack of incorporation of ³H and intact ATPase activity. On the tryptic digestion of either type of photoaffinity labeled HMM the label was found in a 25K peptide identifiable with the N-terminus of the myosin heavy chain (Lu et al., Fed. Proc. $\text{37}$ 1695 1978). The results are discussed in the light of previous localization of the reactive thiol groups, SH-1 and SH-2 (Balint et al., Arch. Biochem. Biophys. $\text{190}$, 793 1978).     

Further characterization and thiophosphorylation of smooth muscle myosin

(i) Myosin from chicken gizzards was purified by a modification of an earlier procedure (M. N. Malik, 1978,Biochemistry17, 27–32). When this myosin, as well as that prepared by the method of A. Sobieszek and R. D. Bremel (1975,Eur. J. Biochem.55, 49–60), was analyzed by gradient slab gel using the discontinuous buffer system of Neville (1971,J. Biol. Chem.246, 6328–6334), a closely spaced doublet in the heavy chain and four light chains were observed as opposed to one heavy chain and two light chains with the method of Weber and Osborn (1969, J. Biol. Chem.244, 4406–4412). These findings raise the possibility of the existence of myosin isoenzymes in smooth muscle. (ii) The purified gizzard myosin was found to be free of kinase and phosphatase. Phosphorylation or thiophosphorylation of myosin was observed only by exogenously adding kinase. A maximum of 1.2 mol of ³²P/mol of myosin and 2.3 mol of ³⁵S/mol of myosin were obtained. The actin-activated ATPase activity depended upon the extent of thiophosphorylation of myosin; a four- to fivefold increase in the activity was observed when myosin was fully thiophosphorylated. Thiophosphorylated myosin was found to be more stable than phosphorylated myosin.     

Identification of two segments, separated by approximately 45 kilodaltons, of the myosin subfragment 1 heavy chain that can be cross-linked to the SH-1 thiol

The thiol-specific photoactivatable reagent 4-(2-iodoacetamido)benzophenone (BPIA) can be selectively incorporated into the SH-1 of myosin subfragment 1 (S1), and upon photolysis an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa or with the central 50-kDa region [Lu, R. C., Moo, L., & Wong, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392-6396]. Heavy chains with these two types of intramolecular cross-links and un-cross-linked heavy chain have different mobility on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels and therefore can be purified electrophoretically. Each type of heavy chain was cleaved with Staphylococcus aureus protease, chymotrypsin, or lysyl endopeptidase. The cleavage points were determined on the basis of the molecular weights of weights of peptides containing the N-terminus, which was identified with the use of an antibody. Locations of the cross-links were deduced by comparing the peptide maps of cross-linked and un-cross-linked heavy chains. The results indicate that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide, whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide. With use of the avidin-biotin system, it has been shown that SH-1 is located 13 nm from the head/rod junction [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1984) J. Mol. Biol. 178, 323-339]. Since BPIA spans less than 1 nm, our results show that two regions, separated by approximately 400 amino acid residues and located in the 25- and 50-kDa domains of S1, respectively, are also part of the head structure about 12-14 nm from the head/rod junction.     

The 204-kDa smooth muscle myosin heavy chain is phosphorylated in intact cells by casein kinase II on a serine near the carboxyl terminus

The heavy chain of smooth muscle myosin was found to be phosphorylated following immunoprecipitation from cultured bovine aortic smooth muscle cells. Of a variety of serine/threonine kinases assayed, only casein kinase II and calcium/calmodulin-dependent protein kinase II phosphorylated the smooth muscle myosin heavy chain to a significant extent in vitro. Two-dimensional maps of tryptic peptides derived from heavy chains phosphorylated in cultured cells revealed one major and one minor phosphopeptide. Identical tryptic peptide maps were obtained from heavy chains phosphorylated in vitro with casein kinase II but not with calcium/calmodulin-dependent protein kinase II. Of note, the 204-kDa smooth muscle myosin heavy chain but not the 200-kDa heavy chain isoform was phosphorylated by casein kinase II. Partial sequence of the tryptic phosphopeptides generated following phosphorylation by casein kinase II yielded Val-Ile-Glu-Asn-Ala-Asp-Gly-Ser*-Glu-Glu-Glu-Val. The Ser* represents the Ser(PO4) which is in an acidic environment, as is typical for casein kinase II phosphorylation sites. By comparison with the deduced amino acid sequence for rabbit uterine smooth muscle myosin (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737), we have localized the phosphorylated serine residue to the non-helical tail of the 204-kDa isoform of the smooth muscle myosin heavy chain. The ability of the 204-kDa isoform, but not the 200-kDa isoform, to serve as a substrate for casein kinase II suggests that these two isoforms can be regulated differentially.     

Crosslinking by thiol disulfide interchange of 5,5'-dithiobis(2-nitrobenzoic acid)-treated light chain and heavy chain of rabbit skeletal myosin

Interchain disulfide crosslinks between the heavy-chain fragment in heavy meromyosin and myosin light chain 2, generated by 5,5'-dithiobis(2-nitrobenzoic acid (Nbs2), are formed under appropriate ionic conditions at neutral pH as revealed by liberation of the chromogenic 2-nitro-5-thiobenzoic acid. The presence of the original or of a slightly digested light chain 2 reduces the rate of the reaction of heavy meromyosin with Nbs2-modified light chain 2 by 32 - 39%, if Ca2+ is present. Dodecyl sulfate/polyacrylamide gel electrophoresis in absence of reducing agents shows that Nbs2-modified light chain 2 attaches to the heavy chain in the region of the 21-kDa fragment of heavy meromyosin, which contains the essential thiol groups and which has been located at the subfragment 1/subfragment 2 junction of myosin [Balint, M., Wolf, I., Tarcsafalvi, A., Gergely, J. and Sreter, F. A. (1978) Arch. Biochem. Biophys. 190, 793-799]. Modification of thiol-1 groups with iodoacetamide as well as crosslinking the thiol-1 and thiol-2 groups by the bifunctional reagent p-N,N'-phenylenedimaleimide prior to incubation with Nbs2-modified light chain 2 has no substantial effect on the crosslinking reaction. This indicates that other thiol groups are involved in the binding of Nbs2-modified light chain 2 to the heavy chain. An examination of K+, Ca2+, Mg2+ and actin-activated Mg2+ ATPase activities of heavy meromyosin that had been crosslinked with Nbs2-modified light chain 2 shows only a slight change in comparison with intact heavy meromyosin, indicating that crosslinking had not altered significantly the hydrolytic site. Crosslinking of Nbs2-modified light chain 2 to light-chain-2-deficient heavy meromyosin restored the original light-chain-2-dependent Ca2+ sensitivity of the tryptic fragmentation of heavy meromyosin, suggesting that crosslinking takes place at the proper binding site for light 2.     

The two components of spectrin, filamin, and the heavy chain of smooth muscle myosin show no detectable homologies to one another by two-dimensional mapping of iodinated tryptic peptides.

The possible structural relationships among four high molecular weight mechanochemical proteins has been examined using two-dimensional mapping of the tryptic peptide fragments prepared from ¹²⁵I-labeled proteins (Elder et al., J. Biol. Chem. $\text{252}$:6510–6515 (1977)). Erythrocyte spectrin bands 1 and 2 protein, the heavy chain of smooth muscle (uterine) myosin, a filamin from human and rabbit were studied. The maps of the four proteins within each species differed considerably from each other, with no apparent homologies evident among them, whereas maps of the same individual protein between the two species showed a high degree of homology.     

Photochemical probes of the active site of myosin. Irradiation of trapped 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate labels the 50-kilodalton heavy chain tryptic peptide

3'-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP), an analog of ATP containing a photoreactive benzophenone moiety, was used as a probe of the ATP binding site of myosin subfragment 1 (SF1). The inactivation of SF1 NH+4-EDTA ATPase by the bifunctional thiol crosslinking system cobalt(II)/cobalt(III) phenanthroline complexes was enhanced by Bz2ATP to the same degree as by ATP. This treatment resulted in the stable trapping of Bz2ATP at the active site in nearly stoichiometric amounts in a manner exactly analogous to ATP (Wells, J.A., and Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970). Irradiation of SF1 containing trapped [3H]Bz2ATP gave approximately 50% covalent incorporation of the trapped nucleotide into the enzyme. Analysis of photolabeled SF1 by gel electrophoresis showed that all of the [3H]Bz2ATP was attached to the 95-kDa heavy chain fragment. No label was found in the light chains. Similar analysis of the same protein after limited trypsin treatment demonstrated that approximately 75% of the [3H]Bz2ATP was bound to the central 50-kDa peptide and its 75-kDa precursor from the heavy chain. The N-terminal 25-kDa tryptic peptide, shown to be photolabeled by other ATP analogs (Szilagyi, L., Balint, M., Sreter, F.A., and Gergely, J. (1979) Biochem. Biophys. Res. Commun. 87, 936-945; Okamoto, Y., and Yount, R.G. (1983) Biophys. J. 41, 298a), was not labeled (less than 1%) by Bz2ATP. These results demonstrate that portions of the 50 kDa-peptide of the heavy chain are within 6-7 A of the ATP binding site on SF1 and possibly contribute to nucleotide binding.     

A polyketide metabolite from the fungicolous Nodulisporium sp. SH-1

A new polyketide metabolite, nodulisporin A was isolated from the fungicolous Nodulisporium sp. SH-1. The structure of compound 1 was elucidated by analyses of NMR spectroscopic and mass spectrometry data in combination with chemical means. Compound 1 is an equilibrium mixture of a linear form and two hemiacetal forms at the C-8 carbon. Compound 1 was evaluated for cytotoxic and antimicrobial activities. The metabolite was weakly active against microorganisms and showed weak cytotoxicity against HL60 cells.     

Molt cycle-associated changes in calcium-dependent proteinase activity that degrades actin and myosin in crustacean muscle

The role of calcium-dependent proteinase (CDP) in the proecdysial atrophy of crustacean claw muscle has been investigated. During atrophy the molar ratio of actin to myosin heavy chain decreased 31%, confirming earlier ultrastructural observations that the ratio of thin:thick myofilaments declined from 9:1 to 6:1 (D. L. Mykles and D. M. Skinner, 1981, J. Ultrastruct. Res., 75, 314–325). The release of TCA-soluble material in muscle homogenates at neutral pH was stimulated by Ca²⁺ and completely inhibited by EGTA. The specific degradation of the major myofibrillar proteins (actin, myosin heavy and light chains, paramyosin, tropomyosin, troponin-T, and troponin-I) was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic activity was more than twofold greater in proecdysial muscle homogenates. Degradation of myofibrillar proteins was inhibited by EGTA, and the two inhibitors of cysteine proteinases, leupeptin and antipain, but not pepstatin, an inhibitor of aspartic proteinases. Unlike CDPs from vertebrate muscle, the CDP(s) in crab claw muscle degrades actin and myosin in addition to other myofibrillar proteins.     

Amino acid sequence of rabbit skeletal muscle myosin. 50-kDa fragment of the heavy chain

The amino acid sequence of the 50-kDa fragment that is released by limited tryptic digestion of the head portion of rabbit skeletal muscle myosin was determined by analysis and alignment of sets of peptides generated by digestion of the fragment at arginine or methionine residues. This fragment contains residues 205-636 of the myosin heavy chain; among the residues of particular interest in this fragment are N epsilon-trimethyllysine, one of four methyl-amino acids in myosin, and Ser-324, which is photoaffinity labeled by an ATP analogue (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995). Combination of this sequence with those of the 23- and 20-kDa fragments yields an 809-residue sequence that constitutes most of the heavy chain of chymotryptic S-1 of this myosin.     

The heavy chain of macrophage myosin is phosphorylated at the tip of the tail

Myosin purified from rabbit alveolar macrophages has been shown previously to be phosphorylated on the rod portion of the heavy chain and on the 20-kDa light chains (Trotter, J.A. (1982) Biochem Biophys. Res. Commun. 106, 1071-1077). Phosphorylation of the 20-kDa light chains by endogenous kinase activity is associated with a significant enhancement of the actin-activated MgATPase activity (Trotter, J.A., and Adelstein, R.S. (1979) J. Biol. Chem. 254, 8781-8785), whereas the function of heavy-chain phosphorylation is unknown. The isolated heavy chains of myosin purified from freshly harvested cells contain between 0.4 and 1.5 mol of PO4/mol of heavy chain, all esterified to serine residues. Using myosin phosphorylated by incubating living unstimulated macrophages in the presence of 32Pi, two-dimensional thin-layer mapping of tryptic peptides derived from heavy chains yields four phosphopeptides, which are phosphorylated to different extents. Limited trypsin digestion of similar radioactive myosin removes all radioactivity from the heavy chain while reducing its apparent molecular mass by less than 10 kDa. It is concluded that the heavy chain of macrophage myosin is phosphorylated on as many as four serines within 10 kDa of the tip of the tail.     

Measurement of the fraction of myosin heads bound to actin in rabbit skeletal myofibrils in rigor

Tryptic digestion of rabbit skeletal myofibrils at physiological ionic strength and pH results in cleavage of the myosin heavy chain at one site giving two bands (M_r = 200,000 and 26,000) on sodium dodecyl sulfate/polyacrylamide gels. Following addition of sodium pyrophosphate (to 1 mm) to dissociate the myosin heads from actin, tryptic proteolysis results in production of three bands, 160K², 51K and 26K, with a 74K band appearing as a precursor of the 51K and 26K species. Under these conditions, there is insignificant cleavage of heavy chain to the heavy and light meromyosins. Trypsin-digested myofibrils yield the same amount of rod as native myofibrils when digested with papain. These results indicate that actin blocks tryptic cleavage of the myosin heavy chain at a site 74K from the N terminus. From measurements of the amount of 51K species formed by digestion of rigor fibers at various sarcomere lengths, we estimate that at least 95% of the myosin heads are bound to actin at 100% overlap of thick and thin filaments. Hence all myosin molecules can bind to actin, and consequently both heads of a myosin molecule can interact simultaneously with actin filaments under rigor conditions.     

Large-scale purification and characterization of calmodulin from ram testis its metal-ion-dependent conformers

Calmodulin was isolated in large quantities from ram testis by a simple procedure involving sequentially ammonium sulfate fractionation, heat treatment, anion exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-200. Divalent cations (Mg²⁺ and/or Ca²⁺) were present throughout the purification which was entirely performed in the absence of chelators. The final yield was approx. 90 mg per kg testis. Ram testis calmodulin appears to be essentially identical to the brain homologous protein by the following criteria: ultraviolet absorption spectrum, amino acid composition showing a single residue of ?-N-trimethyl lysine, and tryptic peptide maps obtained by high performance liquid chromatography. Turkey gizzard myosin light-chain kinase, the activation of which is extremely specific for calmodulin (Walsh, M.P., Vallet, B., Cavadore, J.C. and Demaille, J.G. (1980) J. Biol. Chem. 255, 335–337), was indeed activated by ram testis calmodulin in the presence of calcium. The isolated protein migrated at different rates upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, depending on the absence or presence of divalent metals which probably induce different conformations. The relative migration rates were Ca²⁺ > Mn²⁺ > Mg²⁺ > EDTA. In the presenceof divalent metals, the observed doublet may be ascribed to the equilibrium between ion-free and ion-saturated forms, which exhibited different Stokes radii, as already suggested (Grab, D.J., Berzins, K., Cohen, R.S. and Siekevitz, P. (1979) J. Biol. Chem. 254, 8690–8696).     

Proximity relationships between sites on myosin and actin. Resonance energy transfer determination of the following distances, using a hybrid myosin: those between Cys-55 on the Mercenaria regulatory light chain, SH-1 on the Aequipecten myosin heavy chain, and Cys-374 of actin

Resonance energy transfer measurements have been made on hybrid myosins in order to map distances between sites on the regulatory light chain, heavy chain, and actin as well as to assess potential conformational changes of functional importance. Using scallop (Aequipecten) myosin hybrid molecules possessing clam (Mercenaria) regulatory light chains, we have been able to map the distance between Cys-55 on the regulatory light chain and the fast-reacting thiol on the myosin heavy chain (SH-1). This distance is shown to be approximately 6.4 nm, and it is not altered by the presence or absence of Ca2+, MgATP, or actin. Experiments performed at low ionc strength on heavy meromyosin (HMM) derived from these hybrid myosins gave results similar to those performed on the soluble parent myosin preparations. The distances between Cys-374 on actin and each of the above sites were also measured. Mercenaria regulatory light-chain Cys-55, within the hybrid myosin molecule, was found to be greater than 8.0 nm away from actin Cys-374. Scallop heavy-chain SH-1 is shown to be approximately 4.5 nm away from actin Cys-374, in broad agreement with earlier measurements made by others in nonregulatory myosins. The significance of our results is discussed with respect to putative conformational changes within the region of the heavy chain connecting SH-1 to the N-terminal region of the light chain.     

The primary structure of skeletal muscle myosin heavy chain: IV. Sequence of the rod, and the complete 1,938-residue sequence of the heavy chain

In the preceding paper [Maita, T., Miyanishi, T., Matsuzono, K., Tanioka, Y., & Matsuda, G. (1991) J. Biochem. 110, 68-74], we reported the amino-terminal 837-residue sequence of the heavy chain of adult chicken pectoralis muscle myosin. This paper describes the carboxyl terminal 1,097-residue sequence and the linkage of the two sequences. Rod obtained by digesting myosin filaments with alpha-chymotrypsin was redigested with the protease at high KCl concentration, and two fragments, subfragment-2 and light meromyosin, were isolated and sequenced by conventional methods. The linkage of the two fragments was deduced from the sequence of an overlapping peptide obtained by cleaving the rod with cyanogen bromide. The rod contained 1,039 amino acid residues, but lacked the carboxyl-terminal 58 residues of the heavy chain. A carboxyl-terminal 63-residue peptide obtained by cleaving the whole heavy chain with cyanogen bromide was sequenced. Thus, the carboxyl terminal 1,097-residue sequence of the heavy chain was completed. The linkage of subfragment-1 and the rod was deduced from the sequence of an overlapping peptide between the two which was obtained by cleaving heavy meromyosin with cyanogen bromide. Comparing the sequence of the adult myosin thus determined with that of chicken embryonic myosin reported by Molina et al. [Molina, M.I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488], we found that the sequence homology is 94%.     

Defining the "fast-reacting" thiols of myosin by reaction with 1, 5 IAEDANS.

The specificity of the fluorescent reagent N-iodoacetyl-N-(5-sulfo-1-naphthyl)ethylenediamine (1,5 IAEDANS) for a specific thiol group of myosin has been characterized by a comparison with iodoacetamide (IAA) and by observing maximal enhancement of the Ca²⁺-ATPase activity and inhibition of the K⁺-EDTA-ATPase activity of myosin. The stoichiometry of the [³H]1,5 IAEDANS bound to myosin indicates the presence of two fast-reacting thiols which correspond to the “SH₁” groups responsible for the catalytic properties of myosin. Moreover, it has been unequivocally demonstrated by gel electrophoresis that the fast-reacting thiol is located on the myosin heavy chain. A single radioactivity-labeled thiol peptide obtained from tryptic digests of myosin labeled with [³H]1,5 IAEDANS or iodo[1-¹⁴C]acetamide indicates strongly that the identical thiol was labeled by both reagents.     

The action of thiol reagents on the adenosine-triphosphatase activities of heavy meromyosin and L-myosin

1. The Ca²⁺-activated adenosine triphosphatase of heavy meromyosin is maximally stimulated by lower relative molar concentrations of phenylmercuric acetate than are required with myosin. 2. Stimulation of the Ca²⁺-activated adenosine triphosphatase of both heavy meromyosin and myosin by thiol reagents is markedly affected by ionic strength, the effects being greater with the former than with the latter. In particular, N-ethylmaleimide strongly inhibits the Ca²⁺-activated adenosine triphosphatase of heavy meromyosin at ionic strength below about 0·2. 3. The precise behaviour of the thiol reagents at low ionic strength is slightly modified by the age of the heavy meromyosin and myosin preparations. 4. Stimulation of the Mg²⁺-activated adenosine triphosphatase of heavy meromyosin by thiol reagents is relatively insensitive to ionic strength. 5. The adenosine triphosphatases of heavy meromyosin and myosin activated by potassium chloride in the absence of bivalent activators are inhibited by thiol reagents over the range of ionic strength at which stimulation occurs in the presence of calcium chloride as activator. 6. The modifying effects of potassium chloride and sodium chloride are qualitatively different when heavy-meromyosin adenosine triphosphatase is stimulated with phenylmercuric acetate. No such difference is observed when the enzyme is stimulated with N-ethylmaleimide.     

Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.