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Tryptic peptides and chymotryptic peptides from a polypeptide chain in All component of adult chicken hemoglobin were isolated and purified by using column chromatography and paper chromatography. Furthermore amino acid sequence analyses of these peptides were performed mainly by redigestion with enzyme, the PTC method and the DNP method. From the result of these experiments, the primary structure of this a polypeptide chain was determined. That is, a polypeptide chain in All component from adult chicken hemoglobin consisted of 141 amino acids. In comparison with the primary structure of a polypeptide chain from adult human hemoglobin, amino acid sequence exchanges were found at 35 positions, and with a polypeptide chain from horse hemoglobin, amino acid sequence exchanges were found at 40 positions.

Hemoglobin was prepared from blood of adult animals of slow loris, and furthermore a and β polypeptide chains of the globin were separated by column chromatography. These two kinds of polypeptide chains were respectively digested with trypsin. The tryptic peptides thus obtained were isolated and purified by column chromatography and paper chromatography, and then each amino acid composition was analyzed.

An abnormal human hemoglobin was discovered in a woman living at Atago in Nagasaki and was designated Hb Atago. In the thin-layer starch gel electrophoresis with Tris-EDTA-borate buffer, pH 8.6, the main component of this abnormal hemoglobin migrated towards the anode a little behind Hb F, and the minor abnormal component migrated to the same site as non-hemoglobin protein did. These abnormal components were isolated by CM-cellulose column chromatography. The hybridization method proved that the α chain of Hb Atago had the abnormality. The abnormal α chain was isolated by CM-cellulose column chromatography in 8 M urea. The tryptic peptide map of the aminoethylated abnormal α chain showed that the abnormality existed in αT9. The abnormal αT9 was isolated and purified by high voltage paper electrophoresis. The abnormal chymotryptic peptides from αT9 were purified by high voltage paper electrophoresis and paper chromatography. The amino acid compositions of these peptides were analyzed. As a result, it was found that in Hb Atago, a tyrosine residue was substituted for the aspartic acid residue at α85 in the normal human hemoglobin.

An abnormal human hemoglobin was found in one family living in Nagasaki and was designated Hb-Nagasaki. It migrated faster than the normal human hemoglobin towards the anode in thin-layer starch gel electrophoresis with tris-EDTA-borate buffer, pH 8.6. The observation suggested that the abnormality occurred in the (S chain. This abnormal ft chain was isolated and digested with trypsin and pepsin, respectively. The abnormal tryptic peptide and the abnormal peptic peptide thus obtained were isolated and purified by high voltage paper electrophoresis and paper chromatography, and the amino acid compositions of these peptides were determined. As a result, it was found that in Hb-Nagasaki, a glutamic acid was substituted for the lysine residue at β 17 in the normal hemoglobin.

Countercurrent distribution separated rhesus monkey hemoglobin into α and β polypeptide chains, which were respectively oxidized with performic acid, dissolved in 8 M urea to be denatured at 60° for 45 min, and then digested with pepsin at 37° for 2 hours. Peptic peptides thus obtained from each polypeptide chain were isolated and purified by column- and by paper chromatography, and the amino acid composition of each peptide was analyzed. Sequence analysis was carried out by using PTC and DNP methods, if necessary. From the results thus obtained, the present authors established the primary structure of rhesus monkey hemoglobin, taking account of the amino acid sequence in all the tryptic peptides from the α and β polypeptide chains previously reported.

The a polypeptide chain of adult hemoglobin of the rhesus monkey was digested with trypsin. All the tryptic peptides thus obtained were isolated and purified by column and paper chromatography and amino acid sequence analyses of these peptides were performed mainly by partial hydrolyses with enzymes, the DNP method, and the PTC method. The comparison of the results with the known sequences of the corresponding peptides from the a polypeptide chain of human hemoglobin showed that there were four differences in amino acid sequences of these peptides between rhesus monkey and humans.

All component prepared from adult chicken hemoglobin was separated into α and β polypeptide chains by column chromatography. Of the two chains, the a polypeptide chain was digested with trypsin. The soluble tryptic peptides thus obtained were isolated and purified by column chromatography and paper chromatography. On the other hand, the a polypeptic chain in AH component was digested with chymotrypsin. The chymotryptic peptides thus obtained were isolated and purified by column chromatography and paper chromatography the same way as in the case of tryptic peptides. The amino acid compositions of these tryptic and chymotryptic peptides were analyzed.

Globin prepared from adult hemoglobin of the rhesus monkey was separated into α and β polypeptide chains by the countercurrent distribution method. The α and β polypeptide chains were digested with trypsin. All the tryptic peptides were isolated and purified by column and paper chromatography. The amino acid compositions and the N-terminal amino acid of these tryptic peptides were analyzed. According to the results thus obtained, the α and β polypeptide chains of adult hemoglobin of the rhesus monkey are respectively composed of 141 and 146 amino acid residues just as those of human hemoglobin. However, it is presumed that there exist at least four amino acid substitutions in the a polypeptide chain and eight in the β polypeptide chain.

Tryptic and peptic peptides of the L-4 light chain of chicken skeletal muscle myosin were isolated. The amino acid sequences of all the tryptic peptides were determined. The alignment of the tryptic peptides in the protein was deduced from their homology with the primary structure of the A2 (L-4) light chain of rabbit skeletal muscle myosin. The compositions of the peptic peptides confirmed the alignment. Comparing the whole sequence of the L-4 light chain thus established with that of the A2 light chain of rabbit skeletal muscle myosin, 24 amino acid substitutions, were recognized.

The light chain fraction was separated from myosin extracted from chicken cardiac muscle. Two light chain components, L-1 and L-2 in the fraction were isolated by chromatography on a column of DEAE-cellulose (DE-52) in the presence of 4 M urea. After performic acid oxidation, the L-1 chain was digested with trypsin and the resulting peptides were isolated. The amino acid sequences of the peptides were established. The ordering of these tryptic peptides in the L-1 chain was deduced from the amino acid compositions and the partial sequences of peptic peptides from S-carboxymethylated L-1 chain. Comparing the whole sequence of the L-1 chain thus established with that of alkali light chain of rabbit skeletal muscle myosin, 67 amino acid substitutions and two insertions were recognized.

A 64-year-old man with a 5-year history of progressive systemic sclerosis (PSS) was hospitalized because of melena. Radiological and endoscopic examinations showed an ulcerative lesion with sharply demarcated and raised margins in the fornix of the stomach. Tumor markers--serum carcinoembryonic antigen (CEA, 11.3 mg/ml) and neuron-specific enolase (NSE, 38.9 ng/ml) were elevated. Histological examination of endoscopic biopsy specimens (and of necropsy specimens) showed proliferation of atypical small round cells. Immunohistological examination of these cells showed they were positive for epithelial membranous antigen (EMA), and neuron-specific enolase (NSE), but negative for UCHL1, leukocyte common antigen (LCA), anti-leukocyte B-cell (MB1), and anti-leukocyte T-cell (MT1) antigens. Based on these histological and immunohistological tests, a definite diagnosis of small cell carcinoma of the stomach with PSS was established. Our case is a rare combination of PSS and gastric small cell carcinoma. We also reviewed the literature for the association between PSS and gastric cancer in Japanese patients.

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%.

We have previously demonstrated that the two heads of chicken gizzard heavy meromyosin (HMM) in a rigor complex with rabbit skeletal F-actin could be cross-linked by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Here, we report the location of the cross-linked sites in the amino acid sequence of the HMM heavy chain. One of the cross-linked residues was identified as Glu-168 by sequencing the CN1.CN6 cross-linked peptide containing residues 1-77 (CN1) and 164-203 (CN6). This site is located close to the ATP-binding site of HMM. Since the other site was further into the amino acid sequence of CN1, another cross-linked peptide corresponding to residues 53-66 and 145-182 was isolated from the 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-treated acto-tryptic gizzard HMM digested further by other proteolytic enzymes. The amino acid sequence of this peptide and its cyanogen bromide fragment indicated that the cross-linking occurred between Glu-168 and Lys-65. Our results suggests that these two amino acid side chains are in contact with each other in the acto-gizzard HMM rigor complex and participate in the electrostatic interaction between the two HMM heads bound to F-actin. Based on the head-to-head contact, we propose a three-dimensional model for the attachment of gizzard HMM heads to F-actin.

The interaction between the heavy and the regulatory light chains within chicken gizzard myosin heads was investigated by using a zero-length chemical cross-linker, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC). The chicken gizzard subfragment 1 (S-1) used was treated with papain so that the heavy chain was partly cleaved into the NH2-terminal 72K and the COOH-terminal 24K fragments and the regulatory light chain into the 16K fragment. S-1 was reacted with EDC either alone or in the presence of ATP or F-actin. In all cases, the 16K fragment of the regulatory light chain formed a covalent cross-link with the 24K heavy chain fragment but not with the 72K fragment. The 38K cross-linked peptide, which was the product of cross-linking between the 16K light chain and the 24K heavy chain fragments, was isolated and further cleaved with cyanogen bromide and arginylendopeptidase. Smaller cross-linked peptides were purified by reverse-phase HPLC and then characterized by amino acid analysis and sequencing. The results indicated that cross-linking occurred between Lys-845 in the heavy chain and Asp-168, Asp-170, or Asp-171 in the regulatory light chain. The position of the cross-linked lysine was only three amino acid residues away from the invariant proline residue mapped as the S-1-rod hinge by McLachlan and Karn [McLachlan, A. D., & Karn, J. (1982) Nature (London) 299, 226-231]. We propose that the COOH-terminal region of the regulatory light chain is located in the neck region of myosin and that this region and the phosphorylation site of the regulatory light chain together may play a role in the phosphorylation-induced conformational change of gizzard myosin.

Subfragment-1 was prepared from adult chicken pectoralis myosin by limited digestion with alpha-chymotrypsin, and an amino-terminal 23 kDa fragment of the heavy chain was obtained by digesting the subfragment-1 with trypsin. The 205-residue sequence of the fragment was determined by sequencing its cyanogen bromide, tryptic, and chymotryptic peptides. The amino-terminal alpha-amino group of the fragment was acetylated, and two methylated lysines; epsilon-N-monomethyllysine and epsilon-N-trimethyllysine were recognized at the 35th and 130th positions, respectively, as in rabbit skeletal myosin. Comparing the 205-residue sequence of the skeletal myosin with those of cardiac, and gizzard myosins from chicken, considerable differences are recognized, especially in the amino-terminal region, but strong homologies are observed around the reactive lysine residue, around the epsilon-N-trimethyllysine residue, and around the consensus sequence of GXXGXGKT for nucleotide-binding proteins. On the other hand, only 12 amino acid substitutions are recognized between adult and embryonic skeletal myosins, allowing for the post-translational methylation.

The complete amino acid sequence of the 50 kDa fragment of subfragment-1 from adult chicken pectoralis muscle myosin was determined. It contained 431 residues including an epsilon-N-trimethyllysine at position 346. The 431-residue sequence corresponds to the sequence of residues 206 to 639 of chicken embryonic breast muscle myosin heavy chain which was predicted from the nucleotide sequence of the cDNA by Molina et al. [Molina, M. I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488]. Comparing the two sequences, 23 amino acid substitutions and three deletions/insertions are recognized.

The amino acid sequence of the 197-residue 22 kDa fragment from chicken pectoralis muscle was determined to be as follows: K-K-G-S-S-F-Q-T-V-S-A-L-F-R-E-N-L-N-K-L- M-A-N-L-R-S-T-H-P-H-F-V-R-C-I-I-P-N-E-T-K-T-P-G-A-M-E-H-E-L-V-L-H-Q-L-R- C-N-G-V- L-E-G-I-R-I-C-R-K-G-F-P-S-R-V-L-Y-A-D-F-K-Q-R-Y-R-V-L-N-A-S-A-I-P-E-G-Q- F-M-D-S- K-K-A-S-E-K-L-L-G-S-I-D-V-D-h-T-Q-Y-R-F-G-H-T-K-V-F-F-K-A-G-L-L-G-L-L-E- E-M-R-D- D-K-L-A-E-I-I-T-R-T-Q-A-R-C-R-G-F-L-M-R-V-E-Y-R-R-M-V-E-R-R-E-S-I-F-C-I- Q-Y-N-V-R-S-F-M-N-V-K-H-W-P-W-M-K-L-F-F-K, where h stands for 3-N-methylhistidine. The amino acid sequences of the 22 kDa fragment and its equivalent fragment from chicken ventricle and gizzard muscle myosins were also determined by our group. Predicted secondary structures of these 22 kDa fragment regions and of the reported chicken embryo myosin revealed some possible structural differences.(ABSTRACT TRUNCATED AT 250 WORDS)

We examined whether the gizzard MHC gene is expressed in other smooth muscle tissues and, if so, whether there exist any smooth muscle MHC isoforms at the mRNA level. Northern blot analysis showed that the gizzard MHC gene was also expressed in the aorta and jejunum, but not in the pectoralis muscle or in fibroblasts. This indicates that striated muscle and non-muscle MHC isoforms are encoded in genes distinct from the smooth muscle MHC gene. Further, nuclease S1 mapping showed that the aortic smooth muscle MHC mRNA was distinct from the gizzard mRNA in the 5′-terminal coding region. Both of these mRNA species are expressed in the jejunum. These observations suggest that there exist at least two chicken smooth muscle MHC isoforms, vascular-type and intestinal-type, and that these isoforms are generated from a single-copy gene, probably by an alternative mRNA processing mechanism.

The functional activities of myosin head are located in a 95 kilodalton (kDa) heavy chain which can be divided into three fragments of 23 kDa, 50 kDa, and 20 kDa. ATP hydrolysis sites were suggested to be located in the 23 kDa and 50 kDa fragments, and actin binding sites were in the 50 kDa and 20 kDa fragments. In this study, we obtained electron microscopic images of the myosin molecule bound with antibodies directed to the 23 kDa and 50 kDa fragments. We determined that the antigenic sites for 23 kDa fragment are located at 140-180 A from the head-rod junction of myosin, and those for 50 kDa fragment at 160 A from the junction and at the tip of the head itself. The relationship between the spatial locations and the primary structures is discussed.

alpha and beta chains from adult hemoglobin of the slender loris (Loris tardigradus) were isolated by Amberlite CG-50 column chromatography. After S-aminoethylation, both chains were digested with trypsin and the amino acid sequences of the tryptic peptides obtained were analyzed. Further, the order of these tryptic peptides in each chain was deduced from their homology with the primary structures of alpha and beta chains of human adult hemoglobin. Comparing the primary structures of the alpha and beta chains of adult hemoglobin of the slender loris thus obtained with those of adult hemoglobin of the slow loris, 4 amino acid substitutions in the alpha chains and 2 in the beta chains were recognized.

The amino acid sequence of the regulatory light chain of foot muscle myosin from surf-clam (Spisula sachalinensis) was determined by conventional methods. It was: xS-D-D-K-K-A-K-A-A-T-S-S-V-L-T-K-F-T-Q-N-Q-I-Q-E-M-K-E-A-F-T-M-I-D-Q-N-R -D-G-L- I-D-V-S-D-L-K-E-M-Y-S-N-L-G-T-A-P-Q-D-S-V-L-Q-A-M-V-K-E-A-P-Q-M-N-F-T-G- F-L-S-L- F-S-E-K-M-S-G-T-D-P-E-E-T-L-R-N-A-F-Q-M-F-D-S-D-N-T-G-Y-I-P-E-E-Y-M-K-D- L- L-E-N-M-G-D-N-F-S-K-D-E-V-R-Q-T-W-K-E-A-P-I-A-G-G-K-V-D-Y-N-A-F-V-S-K-I- K- G-K-E-Q-D-D-A. The alpha-amino group of the light chain was blocked, and a typical calcium binding structure was recognized at the 33rd to 44th residues, as in other myosin regulatory light chains. The sequences of regulatory light chains from various muscle myosins were arranged according to the well known four-domain structure, and structural homologies were obtained for each of the domains. Based on the homologies, the relationships between the structure, function, and molecular evolution were discussed.

The aminoethylated beta polypeptide chain in AII component from the hemoglobin of adult chicken was digested with trypsin [EC 3.4.21.4] and the resulting peptides were separated and purified by ion exchange chromatography, paper chromatography, and gel filtration. Eighteen tryptic peptides, which were nonoverlapping, accounted for all of the amino acid residues in the beta polypeptide chain. The amino acid sequences of the tryptic peptides were established by a combination of enzymatic digestion and subtractive Edman degradation.

Frequencies of mutations between reconstructed ancestor and descendant sequences of codons for metazoan globin chains show that natural selection guided protein evolution. Mutations which improved haemoglobin function were accepted at an accelerated rate in the first vertebrates. Rates decelerated after functional opportunities had been exploited.

Amino acid sequences of the regulatory light chains of striated adductor muscle myosin from Ezo giant scallop (Patinopecten yessoensis) and akazara scallop (Chlamys nipponensis akazara) were determined. Tryptic peptides of each light chain were isolated and sequenced. The alignment of the tryptic peptides in each chain was deduced from the amino acid compositions and the partial sequences of peptic peptides of the Ezo giant scallop light chain. The light chains both consist of 156 residues. Heterogeneous residues, glutamic acid and aspartic acid were observed at the 155th position in the sequence of the akazara scallop light chain. A comparison of the Ezo giant scallop light chain with the glutamic acid-containing and aspartic acid-containing akazara light chains revealed 6 and 7 amino acid substitutions, respectively. When the presented sequences were compared with those of the regulatory light chains of gizzard, cardiac and skeletal muscle myosins, a strong homology was observed in the calcium binding region, but there were considerable heterogeneities in the N- and C-terminal regions.

Tryptic peptides of the alpha and beta chains from silvery marmoset (Callithrix argentatus) and cotton-headed tamarin (Saguinus oedipus) hemoglobins were isolated and sequenced, respectively, by conventional methods. The ordering of the peptides in each chain was deduced from the homology of their sequences with those of human adult hemoglobin. The primary structures thus deduced are compared with those of other primate hemoglobins, and the rate of evolution in New World monkey hemoglobins is discussed.

Three kinds of ATP analogues were synthesized. These ATP analogues can be classified into two conformations, i.e. syn and anti forms with respect to the N-glycosidic bond between adenine and ribose groups of ATP. 3'-O-(N-Methylanthraniloyl)-2-azidoadenosine 5'-triphosphate (MantN2(3)ATP) is recognized as the anti form, as ATP, and the other two, 3'-O-(N-methylanthraniloyl)-8-azidoadenosine 5'-triphosphate (MantN8(3)ATP) and 1,N6-etheno-8-azidoadenosine 5'-triphosphate (epsilon N8(3)ATP) are both syn forms. Mant and etheno groups are both fluorescent which allows detection of their binding to proteins. The photochemical binding of azido groups in ATP analogues to the myosin active site, examined in the presence and absence of ATP, showed that all the analogues bound to the site of myosin ATPase. These analogues also acted as substrates of the ATPase and were hydrolyzed in the active site, as judged by competitive inhibition of the ATPase and by their ATPase activities. Of these analogues, MantN2(3)ATP is very similar to ATP in divalent-cation dependence of its hydrolysis rate and in its ability to trap ADP in the active site with vanadate, while the other two are different from ATP in these respects. The photochemical binding sites of ATP analogues were localized by gel electrophoresis of trypsinized myosin ATPase with photocross-linked ATP analogues and/or by isolating the modified peptides. MantN2(3)ATP was found in the 23-kDa fragment which has a structure common to ATP-binding proteins, i.e. Gly-Xaa-Xaa-Gly-Xaa-Gly-Lys-Thr. Mant N8(3)ATP was found in a region of the 20-kDa fragment where actin is reported to attach.

A fluorescent fragment of Mr = 23,800 was obtained by the papain digestion of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene diamine (abbreviated as IAEDANS)-modified chicken gizzard myosin. The fragment was isolated by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine-HCl followed by anion exchange chromatography on a QAE Sephadex A-50 column. This fragment contained 203 amino acid residues which could be assigned as a COOH-terminal part of the S-1 heavy chain based on the homology with the known sequence of rabbit skeletal myosin fragment. The amino acid sequence was K-G-M-F-R-T-V- G-Q-L-Y-K-E-Q-L-T-K-L-M-T-T-L-R-N-T-N-P-N-F-V-R-C-I-I-P-N-H-E-K-R-A- G-K-L-D-A-H-L-V-L-E-Q-L-R-C-N-G-V-L-E-G-I-R-I-C-R-Q-G-F-P-N-R-I-V-F-Q- E-F-R-Q-R-Y-E-I-L-A-A-N-A-I-P-K-G-F-M-D-G-K-Q-A-C-I-L-M -I-K-A-L-E-L- D-P-N-L-Y-R-I-G-Q-S-K-I-F-F-R-T-G-V-L-A-H-L-E-E-E-R-D-L-K- I-T-D-V-I-I-A- F-Q-A-Q-C-R-G-Y-L-A-R-K-A-F-A-K-R-Q-Q-Q-L-T-A-M-K-V-I-Q-R-N-C-A -A-Y-L-K-L-R-N-W-Q-W-W-R-L-F-T-K-V-K-P-L-L-Q-V-T-R. The cysteine residue which was modified with IAEDANS was of the SH1 type (Cys-65). Pro-197 was suggested to be the NH2-terminal boundary of the alpha-helical coiled-coil rod sequence of gizzard myosin, based on the homology with the nematode sequence reported by MacLachlan and Karn (Proc. Natl. Acad. Sci. U.S. 80, 4253-4257 (1983)). Three different COOH-terminal peptides (Val-Lys-Pro-Leu-Leu-Gln-Val-Thr-Arg, Val-Lys-Pro-Leu-Leu-Gln, and Val-Lys-Pro-Leu-Leu) were isolated from the tryptic digest of this fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

Smooth muscle myosin from scallop (Patinopecten yessoensis) adductor muscle contains two kinds of regulatory light chains (regulatory light chains a and b), and myosin having regulatory light chain a is suggested to be suitable for inducing "catch contraction" rather than myosin having regulatory light chain b (Kondo, S. & Morita, F. (1981) J. Biochem. 90, 673-681). The amino acid sequences of these two light chains were determined and compared. Regulatory light chain a consists of 161 amino acid residues, while regulatory light chain b consist of 156 amino acid residues. Amino acid substitutions and insertions were found only in the N-terminal regions of the sequences. The structural difference between the two light chains may contribute to the functional difference between myosins having regulatory light chains a and b.

The adult Grand Galago (Galago crassicaudatus) was found to have two hemoglobin components (Hb I and Hb II) which were separated by carboxymethyl cellulose column chromatography. The alpha and beta chains of each component were isolated. The tryptic peptides of the alpha and beta chains were each isolated and sequenced by the conventional method. The alignment of these peptides in each chain was deduced from the homology of their sequences with that of human adult hemoglobin. The alpha chains from Hb I and Hb II were considered to be identical. On the other hand, there was only one amino-acid difference between the two beta chains at the 125th residue from the N-terminus.

We have studied the primary structures of myosins from chicken muscles in order to clarify the relationship between structure and function of muscle myosin. The primary structures of the various kinds of light chains from chicken muscle myosins have been determined. We also report the primary structure of the 23K fragment of subfragment-1 (S-1) component from the heavy chain of chicken fast skeletal muscle myosin. In addition, antibody was prepared against the 23K fragment. The antibody was found to inhibit the Mg2+-ATPase activity and the initial Pi burst of the ATPase in the S-1 component. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. These results are also discussed.

The sequence of the NH2-terminal 808 amino acid residues of chicken pectoralis muscle myosin head was determined. Three characteristic 20-, 23-, and 50-kDa fragments were isolated from a digest of myosin subfragment 1 (S1) by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine hydrochloride, followed by anion-exchange chromatography on a QAE-Sephadex A-50 column in the presence of 8 M urea. The fragments were sequenced completely by conventional methods. Peptides overlapping the 23- and 50-kDa fragments and also overlapping the 50- and 20-kDa fragments were obtained by cleaving S1 with cyanogen bromide. Comparison of the 23-kDa and 50-kDa sequences with that of the overlapping peptide indicated that no additional amino acid exists between the 23- and 50-kDa fragments and that 5 amino acids exist between the 50- and 20-kDa fragments of S1. Methylated amino acid residues were found at four positions: epsilon-N-monomethyllysine at position 35, epsilon-N-trimethyllysine residues at 130 and 550, and 3-N-methylhistidine at 754.

The amino acid sequence of the essential light chain (abbreviated as SHLC) of adductor muscle myosin from Ezo giant scallop (Patinopecten yessoensis) was determined by conventional methods. The light chain was composed of 156 amino acid residues with proline and lysine as its amino and carboxyl termini, respectively. Comparing this sequence with that of the SHLC from bay scallop (Aquipecten irradians), only 5 amino acid substitutions were recognized. The sequence homology between scallop and squid SHLCs was 53.7%. On the other hand, a partially fragmented SHLC "modified SHLC" reported by Konno and Watanabe (J. Biochem. 98, 141-148 (1985) was prepared by chymotryptic digestion of the scallop myosin in the presence of EDTA, and was assigned as the carboxyl-terminal 106-residue peptide of the SHLC. This may suggest that the regulatory light chain covers the amino-terminal region of the SHLC in the myosin molecule.

Chicken gizzard myosin was modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine (IAEDANS) in the presence of ATP and in 0.15 M KCl, where the myosin assumed 10S conformation. From the tryptic digest of the modified myosin, a fluorescent fragment (24 kilodaltons) was isolated by gel filtration on a Sephadex G-100 column followed by chromatography on a CM 52 column. The amino acid sequence of the fragment was analyzed by conventional methods, and was: (S,Z)K-P-L-S-D-D-E-K-F-L-F-V-D-K-N-F-V-N-N-P-L-A-Q-A-D-W-S-A-K-K- L-V-W-V-P-S-E-K-H-G-F-E-A-A-S-I-K-E-E-K-G-D-E-V-T-V-E-L-Q-E-N-G-K-K- V-T-L-S-K-D-D-I-Q-K-M-N-P-P-K-F-S-K-V-E-D-M-A-E-L-T-C-L-N-E-A-S-V-L- H-N-L-R-E-R-Y-F-S-G-L-I-Y-T-Y-S-G-L-F-C-V-V-I-N-P-Y-K-Q-L-P-I-Y-S-E-K-I- I-D-M-Y-K-G-K-K-R-H-E-M-P-P-H-I-Y-A-I-A-D-T-A-Y-R-S-M-L-Q-D-R-E-D-Q- S-I-L-C-T-G-E-S-G-A-G-K-T-E-N-T-K-K-V-I-Q-Y-L-A-V-V-A-S-S-H-K-G-K. The amino-terminus was blocked, and the fragment was assigned as an amino-terminal part of the heavy chain of gizzard myosin. Position 127 was occupied by epsilon-N-trimethyllysine. Trp-130 of rabbit skeletal myosin heavy chain, which was reported to cross-link to an azide derivative of ATP by Okamoto and Yount (Proc. Natl. Acad. Sci. U.S. 82, 1575-1579 (1985], was replaced by glutamine in gizzard myosin. Cys-93 of the fragment is the amino acid residue whose reaction with IAEDANS alters the ATPase activity of gizzard myosin (Onishi, H. (1985) J. Biochem. 98, 81-86).

Antibody was prepared against the 25,000-dalton tryptic fragment of subfragment-1 from skeletal muscle myosin. The antibody was found to inhibit the Mg2+-ATPase activity and the initial P1-burst of the ATPase. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. The acto-S-1 ATPase activity was also inhibited by the antibody. These results suggest that there is a site in the 25K fragment region responsible for the transition of the myosin-ATP complex to another high energy complex.

In the rigor complex between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), the direct contact between two HMM heads was demonstrated by using a zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]maleimide (EDC) [Onishi, H., Maita, T., Matsuda, G., & Fujiwara, K. (1989) Biochemistry (preceding paper in this issue)]. Here, the 60K peptide which was a product of the EDC cross-linking between two 24K heavy chain (tryptic) fragments of HMM was further fragmented with cyanogen bromide, and the location of the cross-linking sites on the amino acid sequence of the HMM heavy chain was investigated. The result showed that one site resided within the 77-residue peptide region (residues 1-77) on one head of HMM, whereas the other site belonged to the 40-residue peptide region (residues 164-203) on the other head. This finding suggests that the two HMM heads are in contact with each other at different sites. Ultracentrifugal fractionation revealed that the head-to-head cross-linked gizzard HMM could be reversibly released from F-actin in the presence of Mg-ATP. The yield of the head-to-head cross-linking was not significantly changed with the acto-HMM complex between actin/HMM head molar ratios of 1 and 4, and it was very slightly decreased even at a molar ratio of 8, where HMM molecules were attached sparsely to actin filaments.(ABSTRACT TRUNCATED AT 250 WORDS)

Globin prepared from hemoglobin of the European hedgehog (Erinaceus europaeus) was separated into alpha and beta polypeptide chains by chromatography on a CM 52 column. The S-aminoethylated alpha and beta chains were each digested with trypsin and the resulting peptides were isolated. The sequences of all the tryptic peptides were established. The ordering of these peptides in the alpha and beta chains was deduced from their homology with the primary structures of alpha and beta chains of human adult hemoglobin. Comparing the primary structures of the alpha and beta chains of adult hemoglobin of the European hedgehog thus obtained with those of adult hemoglobin of the tupai (Tupaia glis), 35 amino acids substitutions in the alpha chains and 30 in the beta chains were recognized.

Gene phylogenetic trees were constructed by the maximum parsimony method for various sets of ninety six globin chain amino acid sequences spanning plant and animal kingdoms. The method, executed by several computer programs, constructed ancestor and descendant globin messengers on tree topologies which required the least number of nucleotide replacements to account for the evolution of the globins. The human myoglobin-hemoglobin divergence was traced to a gene duplication which occurred either in the first vertebrates or earlier yet in the common ancestor of chordates and annelids, the alpha-beta divergence to a gene duplication in the common ancestor of teleosts and tetrapods, the gamma divergence from typical beta chains to a gene duplication in basal therian mammals, and the delta separation from beta to a duplication in the basal catarrhine primates. Evidence was provided by the globin phylogenies for the hominoid affinities of the gibbon and the close phyletic relationship of the African apes to man. Over the period of teleos-tetrapod divergence the globin messengers evolved at an average rate of 18.5 nucleotide replacements per 100 codons per 108 years, a faster rate than most previous estimates. Very fast and very slow rates were encountered in different globin lineages and at different stages of descent, reducing the effectiveness of globins as molecular clocks. Rates increased with gene duplication and decreased after selection discovered useful specializations in the products of genes which had previously been freer to accept mutations. The early eutherian radiation was characterized by rapid rates of globin evolution, but the later hominoid radiation by extremely slow rates. This pattern was related to more complicated grades of internal organization evolving in human ancestors. The types of nucleotide replacements in the globin messengers over the long course of globin evolution did not seem indicative of any special mutational mechanisms.