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K Sutoh

Publications and source records attributed to K Sutoh.

At least 19 recordsLinked to original sources
PubMed2001

Stability of capsinoid in various solvents.

To investigate the stability of capsinoid in solvents, the quantitative change of vanillyl nonanoate, a synthetic model capsinoid, in various solvents was measured by HPLC. Vanillyl nonanoate was stable in nonpolar solvents, whereas it was labile in polar solvents. In particular, vanillyl nonanoate tended to decompose in protic solvents such as alcohol and water. Structures of the decomposition products from vanillyl nonanoate in methanol and ethanol were determined to be methyl and ethyl vanillyl ethers, respectively. To clarify the decomposition mechanism of capsinoid, six analogues of vanillyl nonanoate were tested. The stability of the analogues in organic solvents suggested that the hydroxyl group in the para-position of the benzene ring largely contributes to the decomposition of capsinoid.

Capsicum↗
PubMed2001

Mutational analyses of Dictyostelium IQGAP-related protein GApa: possible interaction with small GTPases in cytokinesis.

GAPA is an IQGAP-related protein and is involved in Dictyostelium cytokinesis. Since mammalian IQ-GAPs are effectors for Rac/Cdc42, GAPA is also predicted to bind to small GTPases, which are to be identified. In this study, mutant GAPAs were examined for functions in cytokinesis by genetic complementation of gapA- cells. Positively charged side chains of Arg442 and Lys474 of GAPA, predicted to be present on the surface of interaction with small GTPases, were found to be essential, suggesting an interaction between GAPA and putative small GTPase in cytokinesis. Also, results from truncated GAPAs indicated that almost the entire region of GAPA homologous to IQGAP is required for cytokinesis in Dictyostelium.

Amino Acid Sequence↗
PubMed2000

Insertion or deletion of a single residue in the strut sequence of Dictyostelium myosin II abolishes strong binding to actin.

The strut loop, one of the three loops that connects the upper and lower 50K subdomains of myosin, plays a role as a "strut" to keep the relative disposition of the two subdomains. A single residue was either inserted into or deleted from this loop. The insertion or deletion mutation abolished the in vivo motor functions of myosin, as revealed by the fact that the mutant myosins did not complement the phenotypic defects of the myosin-null cells. In vitro studies of purified full-length myosins and their subfragment-1s (S1s) revealed that the insertion mutants virtually lost the strong binding to actin although their motor functions in the absence of actin remained almost normal, showing that only the hydrophobic actin-myosin association was selectively affected by the insertion mutations. Unlike the insertion mutants, the deletion mutant showed defects both in the strong-binding state and the rate-limiting step of ATPase cycle. These results indicate the functional importance of the strut loop in establishing the strong-binding state of myosin and thereby achieving successful power strokes.

Actins↗
PubMed2000

Characterization of a C-terminal-type kinesin-related protein from Dictyostelium discoideum.

We have determined the full sequence of K2, a kinesin-related protein (KRP) in Dictyostelium discoideum. Sequence homology and domain organization placed K2 in the ncd/Kar3 subfamily of the C-terminal-type KRPs. Bacterially expressed, truncated K2 showed ATP-dependent binding to microtubules and microtubule-stimulated ATPase activity. K2-null cells grew and developed normally, suggesting overlapping functions of K2 with other microtubule motor(s). Overexpression of K2 caused partial mitotic arrest. Green fluorescent protein-tagged full-length K2 localized in the nucleus at the interphase and on the mitotic spindle during mitosis. These results suggest that K2 is a microtubule-dependent motor which may play some roles in mitotic spindles.

Amino Acid Sequence↗
PubMed2000

Novel Dictyostelium unconventional myosin, MyoM, has a putative RhoGEF domain.

We have cloned a novel unconventional myosin gene myoM in Dictyostelium. Phylogenetic analysis of the motor domain indicated that MyoM does not belong to any known subclass of the myosin superfamily. Following the motor domain, two calmodulin-binding IQ motifs, a putative coiled-coil region, and a Pro, Ser and Thr-rich domain, lies a combination of dbl homology and pleckstrin homology domains. These are conserved in Rho GDP/GTP exchange factors (RhoGEFs). We have identified for the first time the RhoGEF domain in the myosin sequences. The growth and terminal developmental phenotype of Dictyostelium cells were not affected by the myoM(-) mutation. Green fluorescent protein-tagged MyoM, however, accumulated at crown-shaped projections and membranes of phase lucent vesicles in growing cells, suggesting its possible roles in macropinocytosis.

Amino Acid Sequence↗
PubMed2000

Structure-function relationships of the two surface loops of myosin heavy chain isoforms from thermally acclimated carp.

The structure-function relationships of fast skeletal myosin isoforms remain poorly understood. To shed some light, we constructed chimeric myosins comprised of Dictyostelium myosin heavy chain backbone with carp loop sequences and analyzed their functional properties. A loop 2-10 chimeric myosin having the loop 2 sequence of the fast skeletal isoform predominantly expressed in carp acclimated to 10 degrees C showed V(max) in actin-activated Mg(2+)-ATPase activity 1.4-fold higher than a loop 2-30 chimera constructed from the loop 2 sequence of the dominant isoform in carp acclimated to 30 degrees C. These two chimera exhibited no significant differences in sliding velocity of actin filaments in in vitro motility assay. Contrastingly, both loop 1-associated chimeras, loop 1-10 and loop 1-30, did not differ in both ATPase activity and in sliding velocity of actin filaments.

Acclimatization↗
PubMed2000

ATP-induced transconformation of myosin revealed by determining three-dimensional positions of fluorophores from fluorescence energy transfer measurements.

The method of fluorescence resonance energy transfer (FRET) is one of the most important techniques for measuring the distance between two fluorophores and for detecting the changes in protein structure under physiological conditions. The use of green fluorescent protein is also a powerful technology that has been used to elucidate dynamic molecular events. From these we have developed a novel method to determine the three-dimensional positions of fluorophores by combining the FRET data and other structural information available. Using this method, we could determine the ATP-induced changes of three-dimensional structure of truncated Dictyostelium myosin in solution. The myosin structure with ADP in solution was found to be similar to that of the crystal structure of MgADPBeFx-bound truncated Dictyostelium myosin (type I structure), whereas myosin with ATP in solution was similar to the crystal structure of MgAdPVi-bound one (type II structure). However, the crystal structure of MgADP-bound scallop myosin (type III structure) could not be explained by any of our FRET data under various conditions. This indicates that the type III crystal structure might represent a transient intermediate conformation that could not be detected using fluorescence energy transfer.

Adenosine Triphosphate↗
PubMed2000

amiB, a novel gene required for the growth/differentiation transition in Dictyostelium.

BACKGROUND: The differentiation programme of Dictyostelium discoideum is initiated by starvation. Nutrient depletion triggers the differentiation of Dictyostelium cells through the transcriptional inactivation of some growth-phase genes, as well as through the transcriptional activation of essential genes required for the aggregation of the cells. The adenylyl cyclase (ACA) gene, acaA, is one of the earliest genes expressed following starvation. ACA produces intracellular and extracellular cAMP that drives further differentiation by inducing chemotaxis, developmental gene expression and morphogenesis of Dictyostelium cells. Although several genes have been identified as being essential for the initiation of differentiation process, such as the transcriptional activation of ACA expression, the molecular mechanisms of the growth/differentiation transition remain to be explored. RESULTS: Using insertional mutagenesis, we have isolated a mutant that does not aggregate upon starvation. The disrupted gene, amiB (aggregation minus B), is predicted to encode a novel protein of 298.9 kDa. When starved, amiB- cells produced an undetectable level of cAMP. Analyses of gene expression showed that amiB- cells fail to turn off the expression of one of the growth-phase genes, cprD, and to turn on the expression of ACA following starvation. The ectopic expression of ACA from a constitutive promoter rescued the differentiation and morphogenesis of amiB- mutants. Furthermore, the ectopic expression of a putative transcriptional factor DdMyb2 or a catalytic subunit of cAMP-dependent protein kinase (PKA-C), both of which are thought to be involved in ACA expression pathway(s), also rescued the starvation-induced ACA expression and further differentiation of the amiB- mutant. CONCLUSION: These results suggest that AmiB plays a role at the start of Dictyostelium differentiation through induction of the ACA expression which is essential for cAMP signalling.

Adenylyl Cyclases↗
PubMed1999

Deletion of the myopathy loop of Dictyostelium myosin II and its impact on motor functions.

One of the putative actin-binding sites of Dictyostelium myosin II is the beta-strand-turn-beta-strand structure (Ile(398)-Leu-Ala-Gly-Arg-Asp(403)-Leu-Val(405)), the "myopathy loop, " which is located at the distal end of the upper 50-kDa subdomain and next to the conserved arginine (Arg(397)), whose mutation in human cardiac myosin results in familial hypertrophic cardiomyopathy. The myopathy loop contains the TEDS residue (Asp(403)), which is a target of the heavy-chain kinase in myosin I. Moreover, the loop contains a cluster of hydrophobic residues (Ile(398), Leu(399), Leu(404), and Val(405)), whose side chains are fully exposed to the solvent. In our study, the myopathy loop was deleted from Dictyostelium myosin II to investigate its functional roles. The mutation abolished hydrophobic interactions of actin and myosin in the strong binding state during the ATPase cycle. Association of the mutant myosin and actin was maintained only through ionic interactions under these conditions. Without strong hydrophobic interactions, the mutant myosin still exhibited motor functions, although at low levels. It is likely that the observed defects resulted mainly from a loss of the cluster of hydrophobic residues, since replacement of Asp(403) or Arg(402) with alanine generated a mutant with less severe or no defects compared with those of the deletion mutant.

Actins↗
PubMed1999

Elasticity of mutant myosin subfragment-1 arranged on a functional silver surface.

Elasticity of a two-dimensionally arranged myosin subfragment-1 (S1) was measured by using a surface forces apparatus. To prepare a two-dimensionally arranged S1-monolayer on a functionalized silver surface, we used genetically engineered Dictyostelium S1 molecules. A highly reactive cysteine residue was fused to the COOH-terminus using the recombinant DNA method. On the other hand, the maleimide groups was self-assembled onto a silver surface. Then the mutant S1 molecules were chemically bound to the functionalized silver surface at its COOH-terminus. This arrangement technique was necessary in order to create a stable S1-monolayer by chemical bond formation onto the silver surface. The occupied area of the single S1 on the silver surface was about 110 nm(2). In the interaction between the S1-monolayer and mica surfaces in aqueous solution, a long-range attractive force was observed. The elastic constants (stiffness and Young's modulus) of myosin S1 were evaluated from force-distance profiles in aqueous solution, using the Hertz theory. We found that the stiffness (or spring constant) and Young's modulus of S1 in the absence of nucleotide are 4.4 +/- 1.0 pN/nm and 0.71 +/- 0.16 GPa, respectively.

Aluminum Silicates↗
PubMed1999

Novel Dictyostelium unconventional myosin MyoK is a class I myosin with the longest loop-1 insert and the shortest tail.

We have identified and sequenced a novel unconventional myosin termed MyoK in Dictyostelium. Like class XIV myosins, MyoK has a very short and basic tail and lacks light chain-binding IQ motifs. In contrast, a phylogenetic analysis of the motor domain (head) clearly indicated that MyoK belongs to class I myosins. Surprisingly, at the loop-1 site of the head, an insert of 142 amino acid residues was found, the longest in all myosins so far sequenced. The insert was rich in Gly and Pro and could serve as a secondary actin-binding site, as is the case with those present in the tail of some class I myosins. The expression of the MyoK transcript was stimulated at very early stages of Dictyostelium development. The growth and terminal developmental phenotype of the Dictyostelium cell were not affected by the myoK- mutation, suggesting the existence of myosin(s) with functions overlapping those of MyoK.

Actins↗
PubMed1999

Identification and possible roles of three types of endopeptidase from germinated wheat seeds.

Little or no endopeptidase activity was detected in extracts of dry mature wheat seeds, but when they were allowed to imbibe water in darkness, the activity expressed per seedling increased notably after d 1, reached a maximum on d 3 and then decreased. Two major endopeptidases, named WEP-1 and WEP-2, were present in the 50-70% saturated ammonium sulfate fraction of d-3 seedlings, and could be separated by hydrophobic column chromatography. WEP-1 was further purified and identified as a 31-kDa polypeptide that was immunoreactive to antiserum raised against REP-1, a major rice cysteine endopeptidase. Experiments with proteinase inhibitors revealed that WEP-1 and WEP-2 are cysteine and serine endopeptidases, respectively. The two enzymes differed in substrate specificity, pH dependence, and the ability to digest major wheat seed proteins. Determination of its amino-terminal amino acid sequence indicated the similarity of WEP-1 to other cereal cysteine endopeptidases which are involved in the digestion of seed storage proteins. The expression of WEP-1 in de-embryonated seeds was induced in the presence of gibberellic acid and its effect was eliminated by abscisic acid. In addition to WEP-1 and WEP-2, a legumain-like asparaginyl endopeptidase was identified in the extract of seedlings on hydrophobic chromatography. The asparaginyl endopeptidase may function in the early step of mobilization of wheat storage proteins in germinated seeds.

Amino Acid Sequence↗
PubMed1998

Swing of the lever arm of a myosin motor at the isomerization and phosphate-release steps.

In muscle, the myosin head ('crossbridge') performs the 'working stroke', in which ATP is hydrolysed to generate the sliding of actin and myosin filaments. The myosin head consists of a globular motor domain and a long lever-arm domain. The 'lever-arm hypothesis' predicts that during the working stroke, the lever-arm domain tilts against the motor domain, which is bound to actin in a fixed orientation. To detect this working stroke in operation, we constructed fusion proteins by connecting Aequorea victoria green fluorescent protein and blue fluorescent protein to the amino and carboxyl termini of the motor domain of myosin II of Dictyostelium discoideum, a soil amoeba, and measured the fluorescence resonance energy transfer between the two fluorescent proteins. We show here that the carboxy-terminal fluorophore swings at the isomerization step of the ATP hydrolysis cycle, and then swings back at the subsequent step in which inorganic phosphate is released, thereby mimicking the swing of the lever arm. The swing at the phosphate-release step may correspond to the working stroke, and the swing at the isomerization step to the recovery stroke.

Adenosine Triphosphate↗
PubMed1998

Morphology and function of human benign tumors and normal thyroid tissues maintained in severe combined immunodeficient mice.

In the improved SCID (severe combined immunodeficient) mice, various human benign tumors of the head and neck region were well maintained morphologically and functionally for 3 years until the experiments were terminated, e.g. transplanted parathyroid adenoma secreted parathyroid hormone (PTH) in the SCID mice for more than 1 year. Normal human thyroid tissue was also well maintained in the SCID mice for 3 years. Rapid and high uptake of radioiodine into the transplanted human thyroid tissue was observed. Furthermore, transplanted human thyroid tissue secreted thyroid hormone (T3) and T3 secretion was stimulated by the injection of human thyroid stimulating hormone (TSH). These findings suggest that the improved SCID mice will provide an invaluable experimental system for investigating the function of normal human tissues and the influence of endogenous and exogenous factors on human tissues.

Adenoma↗