Photocross-linking from dinitrophenylated SH1 in myosin head. II. Cross-linked site on 50-kDa fragment

We reported in the preceding paper [Muno, D., et al. (1987) J. Biochem. 101, 661-669] that the dinitrophenyl group exclusively introduced to SH1 on the 20-kDa fragment of myosin subfragment 1 was cross-linked to the 50-kDa fragment by irradiation, and that limited trypsinolysis of the cross-linked S1 generated an 83-kDa peptide, a cross-linking product between the 20- and 50-kDa fragments. This paper will deal with the location of the cross-linked residue on the 50-kDa fragment. When the 83-kDa fragment labeled at SH2 with a fluorogenic SH reagent was subjected to bromocyanolysis, a main fluorescent band, which implied a cross-linked peptide, appeared in the position with an apparent molecular mass of 18.5-kDa on SDS-PAGE. On the other hand, another cross-linked peptide was obtained from a complete tryptic digest of a 83-kDa fragment rich fraction. Amino acid sequence analysis of the two cross-linked peptides revealed that the DNP moiety attached at SH1 was cross-linked with a residue in the segment of the heavy chain spanning the 485-493 region from the N-terminus of the heavy chain.     

Biosynthesis of rat alpha 1-macroglobulin. Identification of an intracellular precursor

Alpha 1-macroglobulin was purified from rat plasma by gel filtration (Sephacryl S-300) and ion exchange chromatography (DE52). Analysis of the purified alpha 1-macroglobulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two polypeptides: a light chain which could be resolved into a double band (36/38 kDa) and a heavy chain (160 kDa). Under non-reducing conditions complexes of 200 and 400 kDa could be demonstrated. Antibodies were raised against both chains of alpha 1-macroglobulin which did not cross-react with either rat alpha 2-macroglobulin or rat alpha 1-inhibitor 3. It was shown that in the medium of [35S]methionine-labeled hepatocytes the two subunits of alpha 1-macroglobulin are linked by disulfide bridges. Intracellularly, however, a high molecular mass polypeptide (185 kDa) could be immunoprecipitated with either the antiserum to the heavy or the light chain of alpha 1-macroglobulin, indicating the existence of a polyprotein precursor. Also in a cell-free translation system alpha 1-macroglobulin was synthesized as a polyprotein consisting of heavy and light chains (162 kDa). In a pulse-chase experiment using tunicamycin to block N-glycosylation, alpha 1-macroglobulin secretion was totally inhibited. This finding reflects the importance of the oligosaccharide side chains for the proteolytic processing to the two subunits and/or secretion of alpha 1-macroglobulin.     

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.     

Reconstitution of human factor VIII from isolated subunits

Human factor VII heterodimers were fractionated into component heavy and light chains using an anti-light chain specific monoclonal antibody immunosorbant. Neither the light chain nor the heavy chain alone possessed activity. Factor VII activity was reconstituted by recombining the subunits in the presence of Mn2+ or Ca2+. Reconstitution of activity also showed ionic strength dependence suggesting the importance of hydrophobic and electrostatic interactions. All factor VIII heavy chains (93 to 210 kDa) recombined with the 83 kDa light chain as judged by retention of all reconstituted heterodimeric forms by the monoclonal immunosorbant. Maximum specific activity (3 units/micrograms) was obtained at a 1:1 molar ratio of light chain:heavy chain. The presence of von Willebrand factor enhanced the rate of factor VIII reconstitution as much as 5-fold. This effect was both ionic strength-dependent and dose-dependent up to a 25-fold weight excess of von Willebrand factor over factor VIII.     

Nucleotide-induced change of the interaction between the 20- and 26-kilodalton heavy-chain segments of myosin adenosinetriphosphatase revealed by chemical cross-linking via the reactive thiol SH2

When myosin subfragment 1 (S-1) reacts with the bifunctional reagents with cross-linking spans of 3-4.5 A, p-nitrophenyl iodoacetate and p-nitrophenyl bromoacetate, the 20-kilodalton (20-kDa) segment of the heavy chain is cross-linked to the 26-kDa segment via the reactive thiol SH2. The well-defined reactive lysyl residue Lys-83 of the 26-kDa segment was not involved in the cross-linking. The cross-linking was completely abolished by nucleotides. Taking into account the recent report that SH2 is cross-linked to a thiol of the 50-kDa segment of S-1 using a reagent with a cross-linking span of 2 A [Chaussepied, P., Mornet, D., & Kassab, R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2037-2041], present results suggest that SH2 of S-1 lies close to both the 26- and 50-kDa segments of the heavy chain. The data also encourage us to confirm our previous suggestion that the ATPase site of S-1 residues at or near the region where all three segments of 26, 50, and 20 kDa are contiguous [Hiratsuka, T. (1984) J. Biochem. (Tokyo) 96, 269-272; Hiratsuka, T. (1985) J. Biochem. (Tokyo) 97, 71-78].     

Purification of fusion ferritin from recombinant E. coli using two-step sonications

Fusion ferritin, combined by heavy chain ferritin (21 kDa) and light chain ferritin (19 kDa), was expressed in recombinant E. coli. The fusion ferritin was easily purified by two-step sonications as well as gel filtration chromatography. SDS-gel electrophoresis showed a single band of 38 kDa with heavy and light chains. MALDI-TOF MS gave a molecular weight of fusion ferritin was 38 kDa. The specific activity and yield of purified fusion ferritin are 0.41 Fe³⁺ mg mg^–1 of protein and 66%. Those values are larger than the previous ones of 0.2 Fe³⁺ mg mg^–1 (Kim et al. 2001).     

Localisation of light chain and actin binding sites on myosin

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.     

A new, smaller actin-activatable myosin subfragment 1 which lacks the 20-kDa, SH1 and SH2 peptide

The actin-dependent ATPase activity of myosin is retained in the separated heads (S1) which contain the NH2-terminal 95-kDa heavy chain fragment and one or two light chains. The S1 heavy chain can be degraded further by limited trypsin treatment into characteristic 25-, 50-, and 20-kDa peptides, in this order from the NH2-terminal end. The 20-kDa peptide contains an actin-binding site and SH1 and SH2, two thiols whose modification dramatically affects ATPase activity. By treating myosin filaments with trypsin at 4 degrees C in the presence of 2 mM MgCl2, we have now obtained preferential cleavage at the 50-20-kDa heavy chain site without any cleavage at the head-rod junction and hinge region in the rod. Incubation of these trypsinized filaments at 37 degrees C in the presence of MgATP released a new S1 fraction which lacked the COOH-terminal 20-kDa heavy chain peptide region. This fraction, termed S1'(75K), has more than 50% of the actin-activated Mg2+-ATPase activity of S1 and the characteristic Ca2+-ATPase and K+-EDTA ATPase activities of myosin. These results show that SH1 and SH2 are not essential for ATPase activity and that binding of actin to the 20-kDa region is not essential for the enhancement of the Mg2+-ATPase activity.     

Photocross-linking from SH1 of the myosin heavy chain to light chains through the dinitrophenyl group attached to SH1

Trinitrobenzene selectively dinitrophenylates SH1, a specific thiol in the myosin heavy chain which contains 1 mol of this cysteinyl residue. When the SH1-DNP-myosin thus obtained was irradiated with a mercury lamp, a cross-linked product was formed with a molecular weight of about 220K daltons. It was shown that this product was composed of both heavy and light chains by fluorescence labeling of the heavy chain at SH2, another specific thiol, and immune reaction using an anti-light chain antibody, respectively.     

Cross-linking of three heavy-chain domains of myosin adenosinetriphosphatase with a trifunctional alkylating reagent

The chemotherapeutic alkylating reagent tris(2-chloroethyl)amine (TCEA) was used as a trifunctional cross-linking reagent with a cross-linking span of 5 A for myosin subfragment 1 (S-1). When S-1 was incubated with TCEA, all three domains of 20, 26, and 50 kDa in the S-1 heavy chain were cross-linked via the highly reactive sulfhydryl group SH1 (Cys-707) on the 20-kDa domain. The cross-linking was accelerated by nucleotides. The present observation is consistent with the proposal that SH1 is close to both the 26- and 50-kDa domains of S-1 and that movement within S-1 associated with the nucleotide binding occurs around SH1 as well as around another reactive thiol, SH2 & Wong, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392-6396; Hiratsuka, T. (1987) Biochemistry 26, 3168-3173].     

The interaction and photolabeling of myosin subfragment 1 with 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate

The photoprobe 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (Bz2ATP) was used to characterize the nucleotide-binding site of myosin subfragment 1 (SF1). Improved synthesis and purification of Bz2ATP are reported. 1H NMR and ultraviolet spectroscopy show that Bz2ATP is a 60:40 mixture of the 3'(2')-ribose isomers and that the epsilon M261 is 41,000 M-1 cm-1. Bz2ATP is hydrolyzed by SF1 comparably to ATP in the presence of actin or K+, NH4+, or Mg2+ ions; and the product, Bz2ADP, has a single binding site on SF1 (K'a = 3.0 X 10(5) M-1). [3H]Bz2ATP was photoincorporated into SF1 with concomitant loss of K+-EDTA-ATPase activity. Analysis of photolabeled SF1 showed that the three major tryptic peptides (23, 50, and 20 kDa) of the heavy chain fragment and the alkali light chains were labeled. The presence of ATP during irradiation protected only the 50-kDa peptide, indicating that the other peptides were nonspecifically labeled. If Bz2ATP was first trapped on SF1 by cross-linking the reactive thiols, SH1 and SH2, with p-phenylenedimaleimide, only the 50-kDa tryptic peptide was labeled. These results confirm and extend previous observations that [3H]Bz2ATP trapped on SF1 by cobalt(III) phenanthroline photolabeled the same 50-kDa peptide (Mahmood, R., and Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959). Thus, the 50-kDa peptide is labeled with or without thiol cross-linking, indicating that the relative position of SH1 and SH2 does not affect the labeling pattern.     

Two smooth muscle myosin heavy chains differ in their light meromyosin fragment

Smooth muscle myosin heavy chains [SM1, approximately 205 kilodaltons (kDa), and SM2, approximately 200 kDa] were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Peptide maps of the two heavy chains showed unique patterns. Limited proteolytic cleavage of purified swine stomach myosin was performed by using a variety of proteases to produce the major myosin fragments which were resolved on SDS gels. A single band was obtained for heavy meromyosin in the soluble fraction following chymotrypsin digestion. However, a variable number of bands were observed for light meromyosin fragments in the insoluble fraction after chymotrypsin digestion. Peptide mapping indicated that the two bands observed after short digestion times with chymotrypsin had relative mobility and solubility properties consistent with approximately 100- and 95-kDa light meromyosin (LMM) fragments. These results indicate that the region of difference between SM1 and SM2 lies in the LMM fragment.     

Functional analysis of tail domains of Acanthamoeba myosin IC by characterization of truncation and deletion mutants

Acanthamoeba myosin IC has a single 129-kDa heavy chain and a single 17-kDa light chain. The heavy chain comprises a 75-kDa catalytic head domain with an ATP-sensitive F-actin-binding site, a 3-kDa neck domain, which binds a single 17-kDa light chain, and a 50-kDa tail domain, which binds F-actin in the presence or absence of ATP. The actin-activated MgATPase activity of myosin IC exhibits triphasic actin dependence, apparently as a consequence of the two actin-binding sites, and is regulated by phosphorylation of Ser-329 in the head. The 50-kDa tail consists of a basic domain, a glycine/proline/alanine-rich (GPA) domain, and a Src homology 3 (SH3) domain, often referred to as tail homology (TH)-1, -2, and -3 domains, respectively. The SH3 domain divides the TH-3 domain into GPA-1 and GPA-2. To define the functions of the tail domains more precisely, we determined the properties of expressed wild type and six mutant myosins, an SH3 deletion mutant and five mutants truncated at the C terminus of the SH3, GPA-2, TH-1, neck and head domains, respectively. We found that both the TH-1 and GPA-2 domains bind F-actin in the presence of ATP. Only the mutants that retained an actin-binding site in the tail exhibited triphasic actin-dependent MgATPase activity, in agreement with the F-actin-cross-linking model, but truncation reduced the MgATPase activity at both low and high actin concentrations. Deletion of the SH3 domain had no effect. Also, none of the tail domains, including the SH3 domain, affected either the K(m) or V(max) for the phosphorylation of Ser-329 by myosin I heavy chain kinase.     

Isolation and distribution of myosin isoenzymes in chicken pectoralis muscle

The preparation of rabbit antibodies uniquely specific for the alkali 1 (A1) and alkali 2 (A2) light chains of chicken pectoralis myosin has led to the direct isolation of two homodimeric species of myosin: A1-myosin and A2-myosin, molecules which contain the same light chain on each head. The existence of a heterodimeric species, containing both A1 and A2 light chains, was also inferred. The three types of alkali light chain isoenzymes occur in approximately equal amounts in adult chicken pectoralis muscle.The specificities of the antibodies were determined by modified Farr and solid phase radioimmunoassays, as well as by antibody-affinity chromatography. The determinants in myosin that are recognized by the purified antibodies appear to be confined to the N-terminal sequences of the alkali light chains. As a result of this narrow specificity, these immunological reagents can be used to characterize the distribution of A1 and A2 within the myosin molecule, and to localize the individual light chains within the muscle.By labeling the antibodies with a fluorescent marker we have shown that A1 and A2 are present within each myofibril, as well as within the same fiber (Lowey et al., 1979a). Moreover, by using goat anti-rabbit immunoglobulin to enhance the visualization of the primary antibodies against the light chains, we have demonstrated in the electron microscope that A1 and A2 co-exist along the length of each myofilament. This observation suggests that whatever functional differences may exist among the alkali light chain isoenzymes, they must operate within the constraints of a single filament.     

The disappearance of the dependence of actin-myosin interaction on the phosphorylation of myosin light chains in the "freezing" of the structure of heavy meromyosin by a bifunctional reagent

Using glycerinated muscle fibers, free of myosin, tropomyosin and troponin, a study was made of the structural state of F-actin modified by N-(iodoacetyl)-N'-(1-naphthyl-5-sulfo)-ethylendiamine (1.5-IAEDANS) and by rhodaminyl--phalloin at decoration of thin filaments with a proteolytic fragment of myosin--heavy meromyosin containing phosphorylated and dephosphorylated myosin light chains. The heavy meromyosin used has three SH-groups of heavy chain SH1, SH2 and SH chi modified by bifunctional reagent N,N'-n-phenylmaleimide (SH1-SH2, SH2-SH chi). At decoration of thin filaments with heavy meromyosin, some changes in polarized fluorescence of rhodaminyl--phalloin and 1.5-IAEDANS independent of phosphorylation of myosin light chains were found. Fluorescence anisotropy of the fiber was found to depend primarily on the character of heavy chain of SH-group modification. The ability of heavy chains to change their conformations is supposed to play an important role in the mechanism of myosin system modulation of muscle contraction.     

Glutamic acid-88 is close to SH-1 in the tertiary structure of myosin subfragment 1

The thiol-specific photoactivatable reagent benzophenone iodoacetamide (BPIA) can be selectively incorporated into the most reactive thiol, SH-1, of myosin 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 region or with the central 50-kDa region [Lu et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392]. Comparison of the peptide maps of cross-linked and un-cross-linked S1 heavy chains indicates 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 [Sutoh & Lu (1987) Biochemistry 26, 4511]. In this report, S1 was labeled with radioactive BPIA, photolyzed in the absence of nucleotide, and then degraded with proteolytic enzymes. Peptides containing cross-links were isolated by liquid chromatography and subjected to amino acid sequence analyses. The results show that Glu-88 is the major site and Asp-89 and Met-92 are the minor sites involved in cross-linking with SH-1 (Cys-707) via BPIA. These residues are very near the reactive lysine residue (Lys-83) but relatively remote in the primary structure from the putative nucleotide binding region.     

Liver cell myosin: isolation and properties

We have purified myosin from isolated rabbit liver cells that had been previously shown to be well separated from blood vessels and connective tissue (Okamoto, Y. et al. (1983) J. Biochem. 94, 645-653). It comprises a 200-kDa heavy chain and light chains of 24-kDa, 22-kDa, and 17-kDa. In the light chain composition and in the mobility in PPi-PAGE, liver cell myosin differs from the myosin in liver blood vessels. The light chains of liver cell myosin were phosphorylated by myosin light-chain kinase from chicken gizzard and the Mg2+-ATPase activity of phosphorylated myosin was activated 10-fold by F-actin.     

Nucleotide-induced movements in the myosin head near the converter region

Structural changes in subfragment 1 of skeletal muscle myosin were investigated by cross-linking trypsin-cleaved S1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In the absence of nucleotide the alkali light chains are cross-linked to the 27 kDa heavy chain fragment; the presence of MgATP reduces the efficiency of this reaction. On the other hand, MgATP promotes the cross-link formation between the N-terminal 27 kDa and C-terminal 20 kDa fragments of the heavy chain. The chemical cleavage of the cross-linked heavy chains fragments with N-chlorosuccinimide and hydroxylamine indicates that the cross-links are formed between the regions spanning residues 131-204 and 699-809. These results indicate that the two regions of the heavy chain that are relatively distant in nucleotide-free skeletal S1 [Rayment et al. (1993) Science 261, 50-58] can potentially interact upon addition of nucleotide.     

Myosin from human erythrocytes

We have purified myosin from human erythrocytes using methods similar to that for other cytoplasmic myosins with a yield of about 500 micrograms/100 ml of packed cells. It consists of a 200-kDa heavy chain and light chains of 26- and 19.5 kDa and therefore differs from the isozyme in platelets which has light chains of 20- and 15 kDa. At low ionic strength, the myosin forms short bipolar filaments like those of platelet myosin. Eight of eight monoclonal antibodies to platelet myosin also bind to erythrocyte myosin. Like most myosins, it has a high ATPase activity in the presence of Ca2+ or EDTA, but is inhibited by Mg2+. Myosin light-chain kinase transfers 1 phosphate from ATP to the 20-kDa light chain, and this stimulates the actin-activated ATPase. Thus, myosin may play a role in shape changes in the erythrocytes.     

Isoforms of myosin and actin in human, monkey and rat myometrium. Comparison of pregnant and non-pregnant uterus proteins

Using several electrophoretic procedures, we have compared the forms of myosin and actin in pregnant and non-pregnant uterus of woman, monkey (Macaca fascicularis) and rat. On non-dissociating gels, native myosin of the three species migrates as a single band, of identical mobility independently of the physiological state. Remigration of this band in dissociating conditions shows that it is constituted of two heavy chains of respectively 201 kDa and 205 kDa; the relative proportions of these two bands are different for the three animal species but do not vary during pregnancy. Using two-dimensional gel electrophoresis, we found that the 17-kDa light chain of purified uterus myosin exists under two isoelectric forms, the more acidic one becoming progressively predominant at the end of pregnancy in the human as in the monkey uterus, while we observed no changes in the rat. In two-dimensional gel electrophoresis, actin of human, monkey and rat uterus is present under three isoforms, the most basic one (the gamma form) increasing early in pregnancy in the two primate species but being always the most abundant form in the rat. The ATPase activity of human uterus myosin was found to be similar for the protein extracted from both pregnant and non-pregnant uterus. The changes observed in the 17-kDa light chain and in the actin isoforms might nevertheless participate in the modifications of contractility of the uterus during pregnancy of the primates.