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%.

Summary. Plasma from a total of 57 patients with adult T-cell leukaemia (ATL) (acute ATL, 39 patients; lymphoma ATL, one patient; chronic ATL, 15 patients; smouldering ATL, two patients) and 20 healthy controls was analysed for the presence of type IV gelatinase activity with clinical features. A signi®cant elevation of plasma matrix metalloproteinase-9 (MMP-9) was observed in some ATL patients, particularly in the patients with malignant cell in®ltration. MMP-9 was found to be secreted into the conditioned medium from all ATL cell lines examined. Moreover, the corresponding mRNA was detectable both in all ATL cell lines examined and in the majority of primary acute ATL cells, indicating that ATL cells are capable of synthesizing and secreting MMP-9. We previously demonstrated that a high incidence of ATL cell in®ltration was closely related to a high plasma level of vascular endothelial growth factor (VEGF) produced by ATL cells themselves. This present study showed that the presence of increased plasma MMP-9 was closely associated with elevated plasma VEGF in ATL patients. Furthermore, we showed that both increased plasma MMP-9 and VEGF were signi®cantly related to high ATL cell in®ltration. All these ®ndings strongly suggest that MMP-9 and VEGF act co-operatively in the process of ATL cell invasion.

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The light chain fraction was separated from chicken gizzard muscle myosin. After S-carboxymethylation or performic acid oxidation, two light chain components (20000-M, and 17 000-M, chains) were isolated by chromatography on a column of DEAE-cellulose in the presence of 4 M urea. Tryptic peptides of the S-carboxymethylated 20000-Mr chain were isolated, and their sequences were determined. The alignment of these tryptic peptides in the chain was deduced from the amino acid compositions and from the partial sequences of peptic peptides of the oxidized protein. The established sequence consists of 171 amino acids and its calculated molecular weight is 19692. Comparing the sequence with those of L-2 chains from chicken and rabbit skeletal muscle myosins, 81 and 78 amino acid substitutions were recognized, respectively, including insertions and/or deletions.Chicken gizzard muscle myosin contains two kinds of light chains. One of them, called the 17 000-molecular-weight light chain or GII, is closely related to the active site of myosin ATPase [l 1. The other, called 20000-molecular-weight light chain, GI or regulatory light chain which has calcium binding ability, is phosphorylated by a specific protein kinase, and is involved in the interaction between actin and myosin [2-41.The physiological roles of the phosphorylation of this chain in the actin-myosin interaction were discussed 15-71. Concerning the primary structure of the light chain, Jakes et al. have reported a sequence of 47 residues including calcium binding regions and a phosphorylated serine [2]. In connection with a relationship between the light chain structure and the mechanisms of the muscle contraction, we have studied the primary structures of the light chains of skeletal, cardiac and gizzard muscle myosins from chicken [8-101. In this paper, we report the whole sequence of the 20000-molecularweight light chain of chicken gizzard muscle myosin. MATERIALS AND METHODS Isolation of the Two Light-Chain ComponentsMyosin was extracted from chicken gizzard muscle according to the method of Ebashi [ll]. The light chain fraction was separated from the heavy chain by the method of Perrie and Perry (121. Oxidation with performic acid and S-carboxymethylation of the light chains were carried out by the methods of Hirs [13] and Crestfield et al. [14], respectively. The two light chain components were separated by DEAEcellulose column chromatography, after the oxidation or S-carboxymethylation. The chromatography was carried out This is a paper VII of a series on Muscular Contractile Proleins. in the same ways as described in the previous paper [9]. Electrophoresis of the light chains on gelatinized cellulose acetate membrane, Cellogel (Chemetron Corp., Italy) was carried out with 20 mM Tris/glycine buffer containing 8 M urea and 2 mM 2-mercaptoethanol, pH 8.3, at 250 V for 30 min.Sodium dodecyl sulfate/polyacrylamide gel (10 %) electrophoresis was performed by the method of Weber and Osborn [15]. Enzymatic Digestion, Isolation and Sequence Analysis of PeptidesPr...

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 (Si) 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: e-N-monomethyflysine at position 35, e-N-trimethyllysine residues at 130 and 550, and 3-N-methylhistidine at 754.The myosin molecule, a major component of the contractile apparatus, consists of two heavy chains and two pairs of light chains. The NH2-terminal portion of each heavy chain and two different light chains form a globular head. The head is generally prepared by limited proteolysis of myosin; it bears both the ATPase and actin-binding functions of myosin (1) and is often called subfragment 1 (S1). Limited tryptic digestion of S1 yields three major fragments of 23, 50, and 20 kDa, which are aligned in that order from the NH2 terminus (2-4). Many studies have been mounted to elucidate the role of myosin in contraction at the molecular level (5), but, to date, only parts of the sequence ofrabbit myosin heavy chain have been published (6, 7). The entire sequence, of course, is necessary for interpretative studies. Here we report the complete sequence ofthe NH2-terminal 808 residues ofheavy chain from the head region of chicken pectoralis myosin. Together with our previously reported light chain sequences (8-10), this work completes the primary structure of the myosin head from chicken-the same myosin head that has been crystallized recently (11,12). MATERIALS AND METHODSMyosin was prepared from adult Hubberd-type chicken pectoralis muscle (13). S1 was prepared by digestion of myosin filaments with a-chymotrypsin (Sigma) as described (14). S1 was digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington) in 0.12 M NaCl/1 mM EDTA/20 mM imidazole hydrochloride, pH 7.0, at 15°C for 30 min. The digest was denatured in 5 M guanidine hydrochloride/0.5 M Tris-HCl, pH 8.1, reduced with 2-mercaptoethanol, and S-carboxymethylated with iodoacetic acid (15). The tryptic fragments were isolated by gel filtration on a Sephadex G-100 column and chromatographed on a QAE-Sephadex A-50 column under conditions as described in Fig. 2. The heavy chain of S1 was separated from the light chain as follows: S1 was denatured, reduced, and Scarboxymethylated, as above, and the rea...

The rigor complexes that formed between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), in which the heavy chains had been cleaved with trypsin into 24K, 50K, and 68K fragments, were examined by using the zero-length chemical cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Two cross-linked products of approximate Mr 115K and 60K were generated. These products were not obtained by EDC treatment of HMM in the absence of F-actin. The HMM fragments that participated in cross-linking were identified by fluorescent labeling and amino acid composition studies. The 115K peptide was determined to be a covalently cross-linked complex that formed between actin and the COOH-terminal 68K fragment of the HMM heavy chain. Our results are in agreement with a previous study which proposed that the site of cross-linking between HMM and F-actin resides within the COOH-terminal 22K fragment of the myosin subfragment 1 heavy chain [Marianne-Pépin, T., Mornet, D., Bertrand, R., Labbé, J.-P., & Kassab, R. (1985) Biochemistry 24, 3024-3029]. The 60K peptide, however, was not a product of cross-linking between HMM and F-actin. On the basis of its amino acid composition, we concluded that this 60K peptide was a cross-linked dimer of the NH2-terminal 24K fragments of the HMM heavy chain. The cross-linking of acto-gizzard HMM significantly increased the Mg-ATPase activity of gizzard HMM without any observable phosphorylation of the regulatory (20K) light chains.(ABSTRACT TRUNCATED AT 250 WORDS)

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Resveratrol, a phytoalexin found in grapes and wine, has been shown to exhibit a wide range of pharmacological properties and is believed to play a role in the chemoprevention of human cancer. Resveratrol has also been shown to induce antiproliferation and apoptosis of several leukemia cell lines. In the present study, we investigated the effect of resveratrol in adult T cell leukemia. Our present observations showed that resveratrol induced growth inhibition in all five human T cell lymphotrophic virus-1-infected cell lines examined, with 50% effective dose of 10.4-85.6 mM. In the resveratrol-treated cells, induction of apoptosis was confirmed by annexin V-based analyses and morphological changes. The most surprising observation was that resveratrol treatment resulted in a gradual decrease in the expression of survivin, an antiapoptotic protein, during cell apoptosis. These findings indicate that resveratrol inhibits the growth of human T cell lymphotrophic virus-1-infected cell lines, at least in part, by inducing apoptosis mediated by downregulation in survivin expression. In view of the accumulating evidence that survivin may be an important determinant of a clinical response in adult T cell leukemia, our present findings have led to the suggestion that resveratrol, a common constituent of the human diet, merits further investigation as a potential therapeutic agent for this incurable disease.