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K Sutoh

Publications and source records attributed to K Sutoh.

At least 73 records · Page 4Linked to original sources
PubMed1989

End-label fingerprintings show that an N-terminal segment of depactin participates in interaction with actin.

A 1:1 complex of actin and depactin, an actin-depolymerizing protein isolated from starfish oocytes [Mabuchi, I. (1983) J. Cell Biol. 97, 1612-1621], was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) to introduce covalent bonds at their contact site. Locations of cross-linking sites were identified along the depactin sequence by the end-label fingerprinting, which employed site-directed antibodies against the N- and C-termini of depactin as end labels. Mappings with these end labels have revealed that the N-terminal segment of depactin (residues 1-20) contains sites in contact with the N- and C-terminal segments of actin, both of which participate in interaction with depactin [Sutoh, K., & Mabuchi, I. (1986) Biochemistry 25, 6186-6192].

Actin Depolymerizing Factors↗
PubMed1988

Spatial proximity of the glycine-rich loop and the SH2 thiol in myosin subfragment 1.

Subfragment 1 (S1) prepared from rabbit skeletal muscle myosin was digested with trypsin to cleave the 95K heavy chain into three pieces, i.e., the 23K, 50K, and 20K fragments. The trypsin-treated S1 was then cross-linked with p-nitrophenyl iodoacetate. The cross-linker bridged one of the reactive thiols (SH2) in the 20K fragment and a lysine residue in the 23K fragment [Hiratsuka, T. (1987) Biochemistry 26, 3168-3173]. Location of the lysine residue was mapped along the 23K fragment by "end-label fingerprinting", which employed site-directed antibodies against the N-terminus of the 23K fragment and against the C-terminus of the 24K fragment (the 23K fragment plus nine extra residues at its C-terminus). The mapping revealed that Lys-184 or Lys-189 was the residue cross-linked with SH2. Since the cross-linker used here spans only several angstroms, the result indicates that Lys-184 or Lys-189 is very close to SH2 in the three-dimensional structure of myosin head. Examination of the primary structure of the 23K fragment has revealed that these lysine residues are in and very close to the so-called "glycine-rich loop", whose sequence is highly homologous to those of nucleotide-binding sites of various nucleotide-binding proteins.

Animals↗
PubMed1988

The sites on calmodulin cross-linked to myosin light chain kinase and troponin I with water-soluble carbodiimide.

To elucidate the interaction of calmodulin with calmodulin binding proteins, we studied the location of the interaction sites on calmodulin by using a chemical cross-linking reagent. Calmodulin prepared from wheat germ was cross-linked to myosin light chain kinase and troponin-I with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked products were cleaved partially with cyanogen bromide and cross-linked sites were determined by peptide mapping analysis using SDS-urea polyacrylamide gel electrophoresis. Peptides which contain the cross-linked site were displaced from their position because of the attached fragments of myosin light chain kinase or troponin I. The peptide of calmodulin from the N-terminal to Met-73 in the cross-linked product with myosin light chain kinase had the same mobility as that of uncross-linked calmodulin on the map though the amount of the peptide was decreased in the cross-linked product. The peptide from the N-terminal to Met-110 in the cross-linked product was displaced from its position. Similar change in the mobility of the calmodulin peptides was also observed in the cross-linked products with troponin I. It was concluded, therefore, that at least one cross-linked site for myosin light chain kinase and one for troponin I were located between Met-73 and Met-110 of the wheat germ calmodulin.

Binding Sites↗
PubMed1988

[An autopsy case of parathyroid carcinoma with primary hyperparathyroidism].

An autopsy case of a 72-year-old woman who was determined as having had a parathyroid carcinoma with primary hyperparathyroidism is reported. On autopsy, a tumor, measuring 6 X 3 X 2 cm, was found beneath the left lobe of the thyroid gland, adherent to the left recurrent nerve and the trachea, though there was no metastasis evident. The tumor was found to be lobulated on cross section inspection. Histologically, the tumor cells were arranged in trabecular and in solid patterns with fibrous bands. A few tumor cells showed mitosis. Invasions to the capsules and the blood vessels were found present. Immunohistochemically, the parathyroid hormone was found positive in the cytoplasm of the tumor cells. Otitis fibrosa generalisata, metastatic calcification, acute pancreatitis, and core pulmonale also were observed.

Aged↗
PubMed1988

Temperature-induced change of thick filament and location of the functional sites of myosin.

The combination of the small-angle X-ray camera to use the synchrotron radiation (National Lab. of High Energy Physics, Tsukuba) and a sensitive X-ray detecting system (FCR System, Fuji Medical System Inc.) using the imaging plate enabled us to record a small angle X-ray diagram of liver rabbit muscle within few minutes. Live relaxed rabbit muscle kept at 25 degrees C gave clear relaxed pattern, in which myosin layer line can be observed up to 13th order. When it was cooled down to 5 degrees C, it gave the small-angle X-ray pattern which looks like that obtained from contracting frog muscle, whereas cooled muscle produced no tension. The 8th meridional reflection almost vanished. The pattern is different from that obtained from rigor muscle: actin layer line at 72 A spacing can not be observed. The "stiffness" and (1,1) reflection on the equator increased. This indicates that more crossbridges are at the vicinity of the thin filaments without developing tension at low temperature. This might be related to the marked decrease in the rate of the step from M*ADP to M.ADP at low temperature. The position of the ATP binding site, SH1, actin binding site(s) was determined by three-dimensional image reconstruction method. Actin-binding site was determined by comparing the three-dimensional image of actin-tropomyosin-S1 and that of actin-tropomyosin-troponin-Ca. The position of ATP binding site and SH1 was determined by three-dimensional reconstruction of the complexes of actin-tropomyosin and S1 of which the ATP binding site or SH1 was labelled with avidin. It was found that SH1 locates near the actin-binding site at the same side of S1. The distance from head-rod junction to ATP binding site and SH1 was similar. But they locate at the different side of S1 and the distance between two sites is about 5 nm, which is consistent with that obtained by energy transfer method.

Animals↗
PubMed1987

A short hydrophobic segment next to tryptophan-130 in myosin heavy chain is close to the ribose ring of ADP bound in the adenosinetriphosphatase site.

The ATPase site of rabbit skeletal myosin was covalently labeled by an ADP analogue that carried a biotin moiety on its adenine ring and a photoreactive phenyl azide group on its ribose ring [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 212-216]. The ADP analogue was tightly trapped into the ATPase site in the presence of vanadate ions and then covalently cross-linked to the site by UV irradiation. The N-terminal 23,000-dalton tryptic fragment of the heavy chain was selectively labeled with the analogue. Further mapping of the labeled segment along the 23-kDa fragment was carried out by "end-label fingerprinting" which employed site-directed antibodies against both ends of the N-terminal heavy chain fragment. The mapping revealed that a hydrophobic segment of approximately 10 residues next to Trp-130, which was reported to be in proximity to the adenine ring of ADP bound to the ATPase site [Okamoto, Y., & Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1579], was the site of covalent labeling with the ADP analogue. The result indicates that the hydrophobic segment is close to the ribose ring of ADP bound to the ATPase site.

Adenosine Triphosphatases↗
PubMed1987

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.

Animals↗
PubMed1987

Electron microscopic visualization of the N terminus of the myosin heavy chain using a site-directed antibody.

We determined the spatial location of the N terminus of the heavy chain of rabbit skeletal muscle myosin by electron microscopy, using a site-directed antibody raised against its N-terminal eight residues as an electron microscopic probe. By examining rotary-shadowed images of the heavy meromyosin-antibody complex, we measured distances between the head-rod junction and the attachment site of the antibody bound on the head. The average distance was estimated to be about 12 nm. The result indicates that the N terminus of the heavy chain is located at the middle region of the head.

Amino Acid Sequence↗
PubMed1986

Improved method for mapping the binding site of an actin-binding protein in the actin sequence. Use of a site-directed antibody against the N-terminal region of actin as a probe of its N-terminus.

An antibody was raised against the N-terminal 18 residues of rabbit skeletal muscle actin. By the use of this antibody as the N-terminal probe of actin and the fluorescent label at Cys-374 as its C-terminal probe, binding sites of depactin (an actin-depolymerizing protein from starfish oocytes) were identified in the actin sequence according to the method of Sutoh [Sutoh, K. (1982) Biochemistry 21, 3654-3661]. Cross-linking of the one-to-one complex of actin and depactin with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC) generated two types of cross-linked products with slightly different apparent molecular weights, denoted as 60KU and 60KL. By the use of the N-terminal probe, it was unequivocally revealed that the C-terminal actin segment of residues 357-375 participated in cross-linking with depactin to form 60KL. On the other hand, by the use of the C-terminal probe it was revealed that the N-terminal actin segment of residues 1-12 participated in cross-linking with depactin to form 60KU. Since EDC cross-links Lys residue with Asp or Glu residue only when they are in direct contact, the result indicates that some of the N-terminal residues 1-12 and the C-terminal residues 357-375 of actin participate in binding depactin. The introduction of the N-terminal probe (the antibody recognizing the actin N-terminus) has increased the flexibility of the mapping method for locating binding sites of actin-binding proteins in the actin sequence.

Actin Depolymerizing Factors↗
PubMed1986

Actin-fragmin interactions as revealed by chemical cross-linking.

A one to one complex of actin and fragmin (a capping protein from Physarum polycephalum plasmodia) was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linking reaction generated two cross-linked products with slightly different molecular weights (88 000 and 90 000) as major species. They were cross-linked products of one actin and one fragmin. The cross-linking site of fragmin in the actin sequence was determined by peptide mappings [Sutoh, K. (1982) Biochemistry 21, 3654-3661] after partial chemical cleavages of cross-linked products with hydroxylamine. The results indicated that the N-terminal segment of actin spanning residues 1-12 participated in cross-linking with fragmin. The cross-linker used in this study covalently bridges lysine side chains and side chains of acidic residues when they are in direct contact. Therefore, it seems that acidic residues in the N-terminal segment of actin (Asp-1, Glu-2, Asp-3, Glu-4, and Asp-11), at least some of them, are in the binding site of fragmin. It has already been shown that the same acidic segment of actin is in the binding site of myosin or depactin (an actin-depolymerizing protein isolated from starfish oocytes). We suggest that the unusual amino acid sequence of the N-terminal segment of actin makes its N-terminal region a favorable anchoring site for various types of actin-binding proteins.

Actin Depolymerizing Factors↗
PubMed1986

Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog.

An ADP analog carrying a biotin moiety and a photoreactive group was synthesized. In the presence of vanadate ion (Vi), the analog was tightly trapped into the ATPase site of heavy meromyosin (HMM) or myosin subfragment 1 (S1) in an ADP analog/ATPase site molar ratio of 1:1. UV illumination on the HMM (or S1)-Vi-ADP analog complex resulted in covalent incorporation of the analog into the ATPase site. About 15% of the trapped analog was crosslinked to HMM or S1. Mapping of the crosslinking site of the analog showed that the N-terminal Mr 25,000 segment of the heavy chain participated in binding the ADP analog. The biotin moiety of the analog covalently incorporated into the ATPase site was visualized in electron microscopy by attaching an avidin oligomer. Rotary-shadowed images of the HMM-avidin complex revealed that the crosslinked ADP analog was located about 140 A from the head-rod junction on the head. The result indicates that the ATPase site of myosin is about 140 A apart from the head-rod junction along the head.

Adenosine Diphosphate↗
PubMed1985

A new multimeric hemagglutinin from the coelomic fluid of the sea urchin Anthocidaris crassispina.

A hemagglutinin was purified from the coelomic fluid of the sea urchin Anthocidaris crassispina by ion-exchange chromatography on DEAE-cellulose and affinity adsorption to glutaraldehyde-fixed ghosts of human erythrocytes, followed by elution with 10 mM EDTA. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it showed a single protein band with a molecular weight of 13 000 and 26 000 in the presence and absence of 2-mercaptoethanol, respectively. The molecular weight of the native protein with a hemagglutinating activity was determined to be 300 000 by sedimentation equilibrium analysis. Its sedimentation coefficient, S0(20),w, and Stokes radius were 13.7 S and 5.5 nm, respectively. The hemagglutinating activity of this protein required calcium ions. When calcium ions were depleted, no activity was observed and its sedimentation coefficient, S0(20),w, decreased to 11.4 S while its Stokes radius increased to 6.7 nm without a change in its molecular weight. The purified hemagglutinin agglutinated human erythrocytes regardless of their ABO and MN blood types. The hemagglutination reaction was not affected appreciably by various simple sugars but was inhibited by tryptic fragments released from human erythrocyte membranes. The results of alkaline borohydride treatment of the inhibitory tryptic fragments showed that the receptor sites for this hemagglutinin were mainly composed of alkali-labile carbohydrate chains with the structure AcNeu alpha 2----3Gal beta 1----3(AcNeu alpha 2----6)GalNAc----serine (or threonine).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy.

The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 M-NH4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(+/- 3) nm (mean value +/- standard deviation: n = 105).

Animals↗
PubMed1985

Purification of cofilin, a 21,000 molecular weight actin-binding protein, from porcine kidney and identification of the cofilin-binding site in the actin sequence.

Cofilin, a 21,000 molecular weight protein originally purified from porcine brain that is capable of binding to actin filaments in a molar ratio of the protein to actin monomer of 1:1 in the filament (Nishida et al. (1984) Biochemistry 23, 5307-5313), was purified from porcine kidney in the present study. The two cofilins from brain and kidney were indistinguishable from each other with respect to the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the one-dimensional peptide map, and the mode of interaction with actin. Treatment of the actin-cofilin complex with a zero-length cross-linker, 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC), generated a cross-linked product with an apparent molecular weight of 63,000. Analysis of this product by peptide mapping (Sutoh (1982) Biochemistry 21, 3654-3661) showed that cofilin was cross-linked with the N-terminal segment of actin containing residues 1-12.

Actin Depolymerizing Factors↗
PubMed1985

Structural unit of the erythrocyte cytoskeleton. Isolation and electron microscopic examination.

We isolated a protein complex containing major cytoskeletal components from the Triton shell of bovine erythrocytes. This protein complex, which we called the 26-S complex, consisted of three major components, spectrin, band-4.1 protein and actin, and one minor component, band-4.9 protein. The molar ratio of spectrin heterodimer:band 4.1:actin was determined by sodium dodecyl sulfate (SDS) gel electrophoresis to be about 1:2:2, approximately the same as that for the Triton shell. By electron microscopic examinations of rotary-shadowed specimens, it was revealed that the 26-S complex had a "spider-like" morphology with a central core and several spectrin heterodimers radiating from it. The number of spectrin arms in the complex was not constant but was in the range between 3 and 6. The complexes with five spectrin heterodimers were the most numerous. The results showed that the 26-S complex contained on the average five spectrin heterodimers, ten band-4.1 polypeptides and ten actin monomers. As judged from the formation of oligomeric 26-S complexes through spectrin arms, the central core of the complex presumably contains band 4.1 and actin. Supporting this conclusion, the central core acted as a nucleus for actin polymerization when the 26-S complex was mixed with G-actin under an actin-polymerizing condition. The 26-S complex could form large aggregates under a certain condition that spectrin was promoted to associate from dimer to tetramer. We conclude that the 26-S complex is the structural unit of the erythrocyte cytoskeleton.

Actins↗
PubMed1984

Spatial relationship between SH1 and the actin binding site on myosin subfragment-1 surface.

To examine the spatial relationship between SH1 thiol and actin binding site on subfragment-1 surface, we studied the interaction with actin of subfragment-1 whose SH1 was labeled with an iodoacetate derivative of biotin and covered with avidin. Subfragment-1--avidin complex bound F-actin and its Mg2+ ATPase activity was activated by actin. Considering the size and the location of biotin binding site on avidin, our results suggest that SH1 is separated from the actin binding site on subfragment-1 surface by at least 17-20 A.

Actins↗