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Biomedical subjects

K Sutoh

Publications and source records attributed to K Sutoh.

At least 55 records · Page 3Linked to original sources
PubMed1995

Structural studies of myosin:nucleotide complexes: a revised model for the molecular basis of muscle contraction.

The structures of the MgADP-beryllium fluoride and MgADP-aluminum fluoride complexes of the truncated myosin head from Dictyostelium myosin II are reported. These reveal the location of the nucleotide complex and define the amino acid residues that form the active site. The tertiary structure of the beryllium fluoride complex is essentially identical to that seen previously in the three-dimensional structure of chicken skeletal muscle myosin. By contrast, significant domain movements are observed in the aluminum fluoride complex. These structural findings form the basis of a revised model for the structural basis of the contractile cycle. It is now suggested that the narrow cleft that splits the central 50-kDa segment of the heavy chain provides not only the communication route between the nucleotide-binding pocket and actin but also transmits the conformational change necessary for movement.

Adenosine Diphosphate↗
PubMed1994

Isolation of Dictyostelium discoideum cytokinesis mutants by restriction enzyme-mediated integration of the blasticidin S resistance marker.

We have developed an improved REMI (restriction enzyme-mediated integration) system for generating mutant Dictyostelium cells quickly and efficiently for systematic screening of cytokinesis mutants. By means of this system, three cytokinesis mutants that grow as giant and multinucleate cells were isolated from 2,000 Dictyostelium transformants. Southern blot analysis of these mutants revealed that a single copy of the tag DNA was integrated into each genome. The tag with flanking genomic DNA at both ends was rescued from one of the mutants and reintroduced into the parental Ax2 strain. Homologous recombination of the rescued gene and the Dictyostelium genome led to the phenotypical changes expected for cytokinesis mutants.

Animals↗
PubMed1993

Force-generating domain of myosin motor.

To understand the underlying mechanism of force generation by myosin motor, it is crucial to know which part of the molecule is essential for the process. Recent structure determination of myosin motor domain at atomic resolution has revealed that the domain comprises two smaller domains, the "ATPase domain" consisting of only an N-terminal segment of the heavy chain and the "neck domain" consisting of a long alpha-helix of the heavy chain and two light chains. This atomic structure begs the question of whether both domains are required for force generation. To answer it, we genetically truncated the head to generate a recombinant fragment composed of the "ATPase domain" alone. The truncated head drove sliding movement of actin filaments and generated force in a novel in vitro assay system, which allows us to hold a specific site of the head on a glass surface. These results indicate that the compact ATPase domain functions as a force-generating machinery of the myosin motor.

Actins↗
PubMed1993

Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion.

Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin filament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/D363/E364 are not essential for ATP-driven sliding and force generation.

Actins↗
PubMed1993

A transformation vector for dictyostelium discoideum with a new selectable marker bsr.

A new selectable marker for transformation of Dictyostelium discoideum cells was constructed by using the bsr gene from Bacillus cereus, which confers resistance to Blasticidin S. The bsr gene was driven by Dictyostelium actin 15 promoter and Dictyostelium actin 8 terminator for expression in Dictyostelium cells. To demonstrate the feasibility of using the bsr marker, we constructed an extrachromosomal replication vector by replacing the Neor gene of pnDeI (B. Leiting and A. Noegel (1988) Plasmid 20, 241-248) with the bsr gene cassette. A mutant Dictyostelium actin 15 gene was constructed and inserted into the vector. Dictyostelium cells were transformed with the resulting vector and then transformants were selected with Blasticidin S. The selected cells showed high level expression of the mutant actin, indicating an efficient selection of transformed cells with the bsr marker.

Actins↗
PubMed1993

Identification of actin surface interacting with myosin during the actin-myosin sliding.

We constructed several mutant actin genes from the Dictyostelium actin 15 gene by the site-directed mutagenesis. Mutations were designed to change acidic residues in actin subdomain 1 to histidine residues. Amino acid replacements were: D1H (single replacement of Asp1 to His), D4H, D1H/D4H (double replacements of Asp1 and Asp4 to histidine), D1H/E3H/D4H (triple replacements of Asp1, Glu3 and Asp4 to histidine), D24H/D25H, E99H/E100H, E360H/E361H, and D363H/E364H. Mutant genes were then expressed in Dictyostelium cells. In vitro motility assays were carried out for purified actins to see whether the mutations affect sliding motion of actin filaments driven by HMM. The assays showed that replacement of N-terminal acidic residues inhibited the sliding. Replacement of D24/D25 and E99/E100 also resulted in inhibition of the sliding motion. However, replacement of acidic residues at the C-terminal cluster E360/E361/D363/E363 did not resulted in loss of motility.

Actins↗
PubMed1993

Reduction of leaky lymphocyte clones producing immunoglobulins and thymic lymphocytic leukemia by selective inbreeding of SCID (severe combined immunodeficiency) mice.

Selective inbreeding of C.B17-scid/scid mouse pairs showing undetectable IgG and IgM has been carried out in order to reduce the mortality of mice by early occurrence of thymic lymphocytic leukemia and abnormal lymphocyte clones producing immunoglobulins, both of which inhibit the successful heterotransplantation of normal and neoplastic human tissues. Although the majority of C.B17-scid/scid mice showed undetectable (< 1 microgram/ml) or low level (< or = 25 micrograms/ml) of serum IgG and IgM, some produced abnormally high concentrations of IgG and IgM (> 25 micrograms/ml). The incidence of such mice showing higher levels of IgG was very high at F1 and F2 generation (10/55, 18.2%), but significantly low after the F3 generation (18/446, 4.0%, p << 0.001). Although leukemia incidence was very high at F4 to F5 generations (8/40, 20.0%), death from leukemia was not observed early in life (4-6 months after birth) at F7 to F10 generations (0/36, 0%, p < 0.01) and was very low during the age of 6-10 months after the F8 generation (11/66, 16.7% at F4 and F5 vs 4/93, 4.3% at F8-10), p < 0.01). Scid mice improved by the selective inbreeding will provide an invaluable experimental system for the heterotransplantation of normal and neoplastic human tissues.

Animals↗
PubMed1992

Molecular structure of the acyl-enzyme intermediate in beta-lactam hydrolysis at 1.7 A resolution.

The X-ray crystal structure of the molecular complex of penicillin G with a deacylation-defective mutant of the RTEM-1 beta-lactamase from Escherichia coli shows how these antibiotics are recognized and destroyed. Penicillin G is covalently bound to Ser 70 0 gamma as an acyl-enzyme intermediate. The deduced catalytic mechanism uses Ser 70 0 gamma as the attacking nucleophile during acylation. Lys 73 N zeta acts as a general base in abstracting a proton from Ser 70 and transferring it to the thiazolidine ring nitrogen atom via Ser 130 0 gamma. Deacylation is accomplished by nucleophilic attack on the penicilloyl carbonyl carbon by a water molecule assisted by the general base, Glu 166.

Acylation↗
PubMed1992

A chimeric actin carrying N-terminal portion of Tetrahymena actin does not bind to DNase I.

A chimeric actin gene was constructed from Tetrahymena actin sequence corresponding to residues 1-83 and Dictyostelium actin sequence corresponding to residues 84-375, and the gene was expressed in Dictyostelium cells. Using DNase I-affinity column, we revealed that the product of the chimeric actin gene was not retained in the column whereas intrinsic actin was retained. In conjunction with our previous data that Tetrahymena actin does not interact with DNase I [Hirono, M., Kumagai, Y., Numata, O., & Watanabe Y. (1989) Proc. Natl. Acad. Sci. U.S. 86, 75-79], we suggest that the binding site of DNase I in an ubiquitous actin is located in N-terminal region (residues 1-83).

Actins↗
PubMed1991

Site-directed mutations of Dictyostelium actin: disruption of a negative charge cluster at the N terminus.

Aspartic acid residues in the N-terminal negative charge cluster of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis of the actin gene. The mutant actins were expressed in Dictyostelium cells and were purified to homogeneity by HPLC. Functional properties of the mutant actins were compared with those of the wild-type actin. (i) In vitro assays of the sliding movement of actin filaments driven by myosin showed that the movement was slowed by the mutations. (ii) The mutations diminished the actin-activated ATPase activity of myosin in such a way that the maximum turnover rate at infinite actin concentration (Vmax) dropped sharply without an appreciable change in the apparent affinity of actin and myosin (Kapp). These results indicate that the N-terminal negative charge cluster of actin is essential for the ATP-dependent actin-myosin interaction.

Actins↗
PubMed1991

Specific cleavages of arginyl peptide bonds at basic amino acid pairs by a serine proteinase from the microsomal membranes of rat liver.

The specificity of action of a serine proteinase from the microsomal membranes of rat liver was investigated at pH 7.5 and 37 degrees C using various peptides as substrates. HPLC analyses of the peptides produced followed by their amino acid analyses have revealed that the enzyme is a unique endopeptidase specifically cleaving arginyl peptide bonds at paired basic amino acid residues. Thus, the enzyme is suggested to be a kind of processing proteinase involved in the conversion of proproteins to their mature forms. Indeed, the enzyme cleaved specifically the NH2-terminal 20-residue peptide of proalbumin at the Arg-Arg sequence.

Amino Acid Sequence↗
PubMed1991

Structure and structural change of the myosin head.

The ATPase site of myosin was located by three-dimensional electron microscopy using the avidin-biotin system. The site is about 5 nm from the tip of the myosin head, about 4 nm apart from the actin-binding site of myosin. Other functional sites on the myosin head were located by electron microscopy with the avidin-biotin system, monoclonal antibodies and site-directed antibodies. These findings enable us to estimate the domain structure of the head. The shape of the myosin heads was examined by electron microscopy using rotary-shadowing and uni-directional shadowing technique, and two types were seen: a straight one and a bent one. Bending occurs at 12 +/- 2 nm from the head-rod junction. This location corresponds with the images by three-dimensional electron microscopy and electron micrography of the crystal. The bending region locates at the boundary of domains. Bent heads increase in the presence of ADP-Vi. The location and angle of bending were almost the same under all the conditions examined. These findings suggest that the heads of straight and bent shapes are in equilibrium, and that ADP-Vi shifts the equilibrium to the bent shape. The bending of the myosin head may play an important role in the molecular mechanism of muscle contraction.

Animals↗
PubMed1989

End-label fingerprintings show that the N- and C-termini of actin are in the contact site with gelsolin.

Gelsolin was cleaved by chymotrypsin or thermolysin into an N-terminal Mr 45,000 fragment (45N) and a C-terminal Mr 38,000 fragment (38C). The N-terminal half was further cleaved into two fragments with Mr 17,000 (17N) and Mr 28,000 (28N). These fragments were complexed with actin and cross-linked with 1-ethyl-3-[3-(dimethylamino)prophyl]carbodiimide (EDC) to introduce covalent bonds into their contact sites. The location of these bonds was mapped along the actin sequence by end-label fingerprinting with highly sensitive probes for the N- and C-termini of actin. The mapping studies revealed that two gelsolin N-terminal fragments (17N and 28N) were cross-linked with the actin C-terminal segment. The result indicates that the actin N- and C-terminal segments are in the binding site of gelsolin.

Actins↗
PubMed1989

Electron microscopic mappings of myosin head with site-directed antibodies.

Site-directed antibodies were raised against three synthetic peptides whose sequences correspond to a region around the reactive lysine residue and two protease-sensitive regions of subfragment 1 (S1) of skeletal muscle myosin (one at the junction of the 23,000 Mr and 50,000 Mr segments, the J1 junction; and the other at the junction of the 50,000 Mr and 20,000 Mr segments of the heavy chain, the J2 junction). The antisera cross-reacted with intact myosin with titres of 5 x 10(4) (anti-J1 antiserum) and 10(4) (anti-J2 and anti-reactive lysine residue antisera). Site-specific antibodies purified by S1-Sepharose readily bound to myosin. Electron microscopic examinations of antibody-myosin complexes revealed that the J1 and J2 junctions are located 15 nm and 16 nm from the head-rod junction, respectively, while the reactive lysine residue region is 13 nm from the junction.

Animals↗