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K Sutoh

Publications and source records attributed to K Sutoh.

87 records · Page 5Linked to original sources
PubMed1984

Electron microscopic visualization of the SH1 thiol of myosin by the use of an avidin-biotin system.

One of the reactive thiols in the myosin head, SH1, was covalently labeled with a biotin derivative, N-iodoacetyl-N'-biotinylhexylenediamine. When 50% of the SH1 thiol was modified with the biotin reagent as judged from measurements of ATPase activities, the biotinylated myosin bound one mole of avidin per mole of myosin at the saturating level. The avidin-myosin complex was readily formed in the presence of MgADP or MgATP. Peptide maps of the biotinylated myosin revealed that SH1 is actually the site of biotinylation with N-iodoacetyl-N'-biotinylhexylenediamine. Electron microscopic examination of the avidin-myosin complex showed that the attachment site of avidin on the myosin head is 130 A from the head-rod junction, indicating that the SH1 thiol is located there.

Amino Acid Sequence↗
PubMed1984

Actin-actin and actin-deoxyribonuclease I contact sites in the actin sequence.

Actin subunits in F-actin were cross-linked with m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS). Peptide maps of the cross-linked actin dimer have revealed that the attachment sites of the MBS cross-link in actin are Cys-373 and a lysine residue in the CB-17 segment (Lys-191, Lys-213, or Lys-215). Since MBS spans approximately 8 A, the result indicates that Cys-373 in an actin subunit is within the distance of approximately 8 A from the lysine residue in the neighboring actin subunit. Therefore, it seems that Cys-373 and the lysine residue in the CB-17 segment are close to the regions of the actin-actin contact sites. The actin-DNase I complex was cross-linked with 1,5-difluoro-2,4-dinitrobenzene ( FFD ). Peptide maps of the actin-DNase I cross-linked complex have shown that the attachment site of the FFD cross-link in actin is in its CB-10 segment. The CB-10 segment of actin contains Lys-50, Lys-61, Lys-68, Tyr-53, and Tyr-69 as candidates for the attachment site. FFD can span only 3 A, and therefore it is most likely that one of these residues is in the region of the binding site of DNase I in actin.

Actins↗
PubMed1984

Role of subunit interactions in the self-assembly of oligomeric proteins.

In oligomeric proteins, the native conformation and its functional properties depend on the interactions which exist between the different chains. The role of these subunit interactions can be studied using either the unfolded state or the native state as a starting point. During the folding process, the properties which appear following a bimolecular reaction are related to the formation of an association area. Similarly, the properties which are lost upon partial dissociation of the native state are related to the association area which is disrupted. Four examples are presented in this article: phosphofructokinase and aspartokinase-homoserine dehydrogenase from E. coli are studied through their folding process, and fatty acid synthetase from B. ammoniagenes and reptilian ovomacroglobulin are studied through their dissociated forms. In all cases, the function of the protein is a sensitive index of the formation of the subunit interactions, and can be more conveniently measured than other size/shape parameters. The extrapolation from the folding of small proteins to the assembly of large and complex structures can be reasonably achieved by admitting that subunit interactions are coupled to the subtle adjustments required by the protein to exert its biological function.

Animals↗
PubMed1984

Isolation and characterization of a high molecular weight actin-binding protein from Physarum polycephalum plasmodia.

A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.

Actins↗
PubMed1983

Mapping of actin-binding sites on the heavy chain of myosin subfragment 1.

When the rigor complex of actin and myosin subfragment 1 (S1) was treated with a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, covalently linked complexes of actin and S1 heavy chain with apparent molecular weights of 165,000 and 175,000 were generated. Measurements of the molar ratio of actin to S1 heavy chain in the 165K and 175K products showed that they were 1:1 complexes of actin and S1 heavy chain. Chemical cleavages of the cross-linked products followed by peptide mappings revealed that two distinct segments of S1 heavy chain spanning the 18K-20K region and the 27K-35K region from its C terminus participated in cross-linking with actin. Cross-linking of actin to the former site generated the 165K peptide while the latter site was responsible for generating the 175K peptide.

Actins↗
PubMed1982

An actin-binding site on the 20K fragment of myosin subfragment 1.

Myosin subfragment 1 (S1) was covalently labeled with a fluorescent dye, N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM), and then digested by trypsin to cleave S1 heavy chain into fragments. The DACM-labeled and trypsin-treated S1 was complexed with F-actin and treated with a zero-length cross-linker, 1-ethyl-3[3-(dimethylamino)propyl] carbodiimide (EDC). The cross-linking reaction generated a covalently linked complex of actin and the 20K fragment of S1 heavy chain, which exclusively incorporated the fluorescent dye, to form a fluorescent 65K cross-linked product. The 20K and 65K fluorescent peptides were isolated and purified and then subjected to cyanogen bromide and/or hydroxylamine cleavages. Mapping of fluorescent cleavage products on acrylamide gels revealed that the N-terminal 20 residues of the 20K fragment of S1 heavy chain contained a cross-linking site of actin.

Actins↗
PubMed1982

Identification of myosin-binding sites on the actin sequence.

The rigor complex of actin and trypsin-treated myosin subfragment 1 (S1) whose heavy chain was cleaved into three fragments (20K, 25K, and 50K) was cross-linked with a zero-length cross-linker, 1-ethyl-3-[3-(dimethyl-amino) propyl]carbodiimide. The cross-linking reaction generated three types of cross-linked products with apparent molecular weights of 65K, 68K, and 95K. The 65K, 68K, and 95K products were covalently linked complexes of actin-20K fragment of the S1 heavy chain, actin-alkaline light chain 1, and actin-50K fragment of the S1 heavy chain, respectively. Cross-linking sites of S1 heavy and light chains on the actin sequence have been determined by digesting the cross-linked products with cyanogen bromide or with hydroxylamine and then mapping resulting peptides on sodium dodecyl sulfate gels. The result indicates that some of the N-terminal acidic residues of actin at positions 1, 2, 3, 4, and 11 are cross-linking sites of the 20K and 50K fragments of the S1 heavy chain while some of its C-terminal acidic residues at positions 360, 362, and 363 are cross-linking sites of the alkaline light chain 1.

Actins↗
PubMed1981

Location of SH1 and SH2 along a heavy chain of myosin subfragment 1.

Two reactive SH groups (SH1 and SH2) of myosin subfragment 1 (S-1) were selectively labeled with a fluorescent dye, N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM). When the DACM-S-1 was digested with trypsin, 95K heavy chain was cleaved into 50K, 25K, and 20K fragments, and the fluorescent labels incorporated into SH1 and SH2 were exclusively found in the 20K fragment, indicating that these SH groups were located in the fragment. When the trypsin-treated DACM-S-1 was subsequently fragmented with hydroxylamine in 6 M guanidine hydrochloride at pH 9.0, the fluorescent 20K fragment was cleaved into two fluorescent segments, the 13K segment containing SH1 and the 5K segment containing SH2. Since it is known that SH1 and SH2 are only 10 residues apart in the sequence and that SH1 is nearer the COOH terminus than SH2, the results show that these reactive SH groups are separated from the COOH terminus of S-1 heavy chain by a 13K-dalton stretch of polypeptide chain.

Animals↗
PubMed1980

Millisecond photo-cross-linking of protein components in vertebrate striated muscle thin filaments.

Troponin I (TnI) was reacted with a photosensitive heterobifunctional reagent, methyl 4-azidobenzimidate (ABI), and then troponin was reconstituted with the ABI-modified TnI. Flash irradiation of the reconstituted troponin resulted in the formation of cross-links between TnI and other components of troponin, troponin C (TnC) and troponin T (TnT), suggesting that TnI is in contact with TnC and TnT when troponin is free in solution. No effect of calcium on the cross-linking could be detected. When the reconstituted troponin was complexed with F-actin-tropomyosin, flash irradiation of the reconstituted then filament yielded the cross-linked products of TnC-TnI, TnI-TnT, and TnI-actin in the presence ans absence of calcium, indicating that TnI is in contact with TnC, TnT, and actin in the thin filament complex irrespective of calcium concentration. No cross-linking could be detected between TnI and tropomyosin. Calcium was found to affect the cross-linking of TnC-TnI and TnI-actin; when TnC was saturated with calcium, the extent of the TnC-TnI cross-linking increased, while that of the TnI-actin cross-linking decreased. Calcium did not affect the TnI-TnT cross-linking.

Actins↗
PubMed1980

Direct evidence for the calcium-induced change in the quaternary structure of troponin in situ. Millisecond cross-linking of troponin components by a photosensitive heterobifunctional reagent.

Flash irradiation of the reconstituted troponin of thin filament complex in which one of their components, troponin C, was modified with a heterobifunctional photosensitive reagent before reconstitution of the troponin complex resulted in the formation of cross-links between troponin C and other components in contact with it. Quantitative analysis of the cross-linked products by gel electrophoresis has revealed interesting features of the quaternary structure of troponin. When the reconstituted troponin was photo-cross-linked with a xenon flash, an appreciable amount of cross-linking was detected between troponin C and troponin I and also between troponin C and troponin T. No effect of calcium on the cross-linking could be detected. This arrangement of components was found to change when troponin was complexed with F-actin-tropomyosin. The arrangement of troponin components in the thin filament complex was sensitive to calcium and magnesium; maximum cross-linking of troponin C and troponin I was observed when the thin filament was cross-linked in the presence of calcium and magnesium, while an appreciable decrease in the extent of the cross-linking was detected when calcium alone or calcium and magnesium were removed from the cross-linking medium. The cross-linking of troponin C and troponin T remained marginal irrespective of the concentration of calcium and magnesium.

Calcium↗
PubMed1978

Effect of pH on the cross-bridge arrangement in synthetic myosin filaments.

Synthetic thick filaments were cross-linked with dimethyl suberimidate at various pH values over the range pH 6.8---8.3. The rate of cross-linking myosin heads to the thick filament surface decreases significantly over a narrow pH range (7.4--8.0) despite the fact that the rate of the chemical reaction (amidination of lysine side chains) shows a positive pH dependence. The fall in rate cannot be ascribed to dissociation of the filament during the cross-linking reaction since the sedimentation boundary of the cross-linked filament (pH 8.3) remains unaltered in the presence of high salt (0.5 M). The decreased rate of cross-linking is also not caused by a shift in reactivity of a small number of highly reactive lysine groups, since the time course of cross-linking (pH 7.2) is unaffected by preincubation with a monofunctional imidate ester. Our results suggest that the heads of the myosin molecules move away from the thick filament surface at alkaline pH but are held close to the surface at neutral pH.

Dimethyl Suberimidate↗
PubMedDate not supplied

Location of the ATPase site of myosin determined by three-dimensional electron microscopy.

Both ATP hydrolysis by myosin and the accompanying cyclic association-dissociation of actin and myosin are essential for muscle contraction. It is important for understanding the molecular mechanism of contraction to know the three-dimensional locations of the two major functional sites of myosin: the ATPase site and the actin-binding site. We have determined the position of the ATPase site of myosin using three-dimensional image reconstruction from electron micrographs and site-specific labelling with the avidin-biotin system. The ATPase site is about 5 nm from the tip of the myosin head and is about 4 nm away from the actin-binding site of myosin. This is the first report of the three-dimensional location of an enzyme active site by electron microscopy.

Actins↗