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Biomedical subjects

M Isomura

Publications and source records attributed to M Isomura.

At least 55 records · Page 3Linked to original sources
PubMed1993

Studies on RA derivatives. V. Synthesis and antitumor activity of Ala2-modified RA-VII derivatives.

A number of RA-VII derivatives having various amino acids including proline (6), pipecolic acid (11), norvaline (12), ornithine (14), aspartic acid (15) and methionine (20) in place of Ala2 have been synthesized from RA-X methyl ester (3) and evaluated for cytotoxicity to P388 leukemia and KB cells in vitro. Comparison of the cytotoxicity of these compounds suggests that the polarity and the length of the 2nd amino acid residue affect the activity. An NMR study revealed that, in solution, 6 and 11 are locked in one conformational state, corresponding to conformer A of RA-VII.

Animals↗
PubMed1991

Inhibition of the action of the stimulatory GDP/GTP exchange protein for smg p21 by the geranylgeranylated synthetic peptides designed from its C-terminal region.

smg p21B, a member of the ras p21-like small GTP-binding protein superfamily, undergoes post-translational modifications, which are geranylgeranylation of the cysteine residue in the C-terminal region followed by removal of the three C-terminal amino acids (QLL) and the subsequent carboxyl methylation of the exposed prenylated cysteine residue. smg p21B has a polybasic region upstream of the prenylated cysteine residue. We have previously proposed that these C-terminal structures of smg p21B are essential for the action of its stimulatory GDP/GTP exchange protein, named GDP dissociation stimulator (GDS). We studied here which structure of the C-terminal region of smg p21B is important for its interaction with smg p21 GDS. For this purpose, we synthesized a peptide according to the C-terminal structure of smg p21B, which was PGKARKKSSC-geranylgeranyl-carboxyl methyl, and its variously modified peptides and examined their ability to interact with smg p21 GDS and to interfere with the smg p21 GDS action to stimulate the GDP/GTP exchange reaction of smg p21B. The results indicate that the phosphorylated form of PGKARKKSSC-geranylgeranyl stoichiometrically interacts with smg p21 GDS, that the presence of the geranylgeranyl moiety is essential for, but not sufficient for, the smg p21 GDS action, and that the presence of the methyl moiety, removal of the three C-terminal amino acids, and the presence of the polybasic amino acids also affect the smg p21 GDS action. It is likely that all the steps of the post-translational processing and presence of the polybasic region in the C-terminal region of smg p21B are related to its interaction with smg p21 GDS.

Amino Acid Sequence↗
PubMed1991

Regulation of binding of rhoB p20 to membranes by its specific regulatory protein, GDP dissociation inhibitor.

We have previously purified from bovine brain cytosol a novel regulatory protein for the GTP-binding proteins of the rho gene family. This regulatory protein, designated as rho GDP dissociation inhibitor (GDI), makes a complex stoichiometrically with the GDP-bound form of the rho gene products and thereby regulates the GDP/GTP exchange reaction by inhibiting the dissociation of GDP from and the subsequent binding of GTP to them. We show here that rho GDI also regulates the binding of rhoB p20, a member of the rho gene products, to various membranes including rat brain synaptic plasma membranes and human erythrocyte ghosts. Both the GDP- and GTP-bound forms of rhoB p20 bound to the membranes. This binding was not inhibited by prior treatment of the membranes with boiling or tryptic digestion, suggesting that a protein molecule of the membranes is not essential for the binding of rhoB p20 to the membranes. rho GDI inhibited the binding of the GDP-bound form of rhoB p20, but not that of the GTP-bound form, to the membranes. Moreover, rho GDI induced the dissociation of the GDP-bound form, but not that of the GTP-bound form, of rhoB p20 exogenously bound to the membranes from them. These results suggest that the rho gene products bind to the membranes, presumably in a reversible manner and that this binding is regulated by their specific GDI.

Animals↗
PubMed1991

Post-translational modifications of the C-terminal region of the rho protein are important for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins.

We have recently found, by use of the rhoA p21 purified from bovine aortic smooth muscle, that it is similarly post-translationally processed as described for ras p21s: it is first geranylgeranylated at the cysteine residue in the C-terminal region followed by removal of the three C-terminal amino acids and the subsequent carboxyl methylation of the revealed C-terminal cysteine residue. In the present study, we investigated the function(s) of these post-translational modifications of the C-terminal region of rhoA p21 by use of the rhoA p21s purified from bovine aortic smooth muscle and rhoA p21-overexpressing Escherichia coli since the bacterial protein was not modified with a geranylgeranyl moiety. Bovine rhoA p21 bound to plasma membranes and phosphatidylserine-linked Affigel, but bacterial rhoA p21 did not bind to them. The inhibitory GDP/GTP exchange protein for rhoA p21, named GDP dissociation inhibitor (GDI), made a complex with the GDP-bound form of bovine rhoA p21 and thereby inhibited the dissociation of GDP from and the subsequent binding of GTP to it. However, rho GDI neither made a complex with the GDP-bound form of bacterial rhoA p21 nor affected these reactions of the bacterial protein. The stimulatory GDP/GTP exchange protein for rhoA p21, named GDP dissociation stimulator (GDS), stimulated the dissociation of GDP from bovine rhoA p21, but was inactive for the bacterial protein. In contrast, the GTPase activating protein for rhoA p21 is active not only for bovine rhoA p21 but also for the bacterial protein. These results suggest that the post-translational modifications of the C-terminal region of bovine rhoA p21, most presumably the geranylgeranylation, which are absent in bacterial rhoA p21, play important roles in its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins but not with the GAP.

Animals↗
PubMed1990

Regulation of reversible binding of smg p25A, a ras p21-like GTP-binding protein, to synaptic plasma membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor.

We have previously purified from bovine brain cytosol a novel regulatory protein for smg p25A, a ras p21-like GTP-binding protein. This protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP/GTP exchange reaction of smg p25A by inhibiting the dissociation of GDP from and thereby the subsequent binding of GTP to it. We have also previously found that smg p25A is mainly localized in presynaptic plasma membranes and vesicles and moderately in presynaptic cytosol in rat brain synapses. In this paper, we have studied the possible involvement of smg p25A GDI in the localization of smg p25A in the cytosol, plasma membranes, and vesicles in rat brain synapses. Both the GDP- and GTP-bound forms of smg p25A bound to the synaptic membranes and vesicles. smg p25A GDI inhibited the binding of the GDP-bound form of smg p25A, but not that of the GTP-bound form, to the synaptic membranes and vesicles. Moreover, smg p25A GDI induced the dissociation of the GDP-bound form, but not that of the GTP-bound form, of both endogenous and exogenous smg p25As from the synaptic membranes and vesicles. smg p25A GDI made a complex with the GDP-bound form of smg p25A with a molar ratio of 1:1, but not with the GTP-bound or guanine nucleotide-free form. These results suggest that smg p25A reversibly binds to synaptic plasma membranes and vesicles and that this reversible binding is regulated by its specific GDI.

Animals↗
PubMed1990

Partial purification and characterization of GDP dissociation stimulator (GDS) for the rho proteins from bovine brain cytosol.

A novel type of regulatory proteins for the rho proteins (rhoA p21 and rhoB p20), ras p21-like small GTP-binding proteins (G proteins), are partially purified from bovine brain cytosol. These regulatory proteins, named rho GDP dissociation stimulator (GDS) 1 and -2, stimulate the dissociation of GDP from rhoA p21 and rhoB p20. rho GDS1 and -2 are inactive for other ras p21/ras p21-like small G proteins including c-Ha-ras p21, smg p21B, and smg p25A. Since we have previously shown that the rate limiting step for the GDP/GTP exchange reaction of the rho proteins is the dissociation of GDP from these proteins, the present results suggest that rho GDS1 and -2 stimulate the GDP/GTP exchange reaction of the rho proteins. rho GDS1 and -2 are distinct from the GAP- and GDI-types of regulatory proteins for the rho proteins previously purified from bovine brain cytosol. rho GAP stimulates the GTPase activity of the rho proteins and rho GDI inhibits the GDP/GTP exchange reaction of the rho proteins. The present results together with these earlier observations indicate that the rho proteins are regulated by at least three different types of regulatory proteins, GDS, GDI, and GAP.

Animals↗
PubMed1990

Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein.

A novel regulatory protein for smg p25A, a ras p21-like GTP-binding protein, was purified to near homogeneity from bovine brain cytosol. This regulatory protein, designated here as smg p25A GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP, but not of guanosine 5'-(3-O-thio)triphosphate (GTPgamma S), from smg p25A. smg p25A GDI also inhibited the binding of GTPgamma S to the GDP-bound form of smg p25A but not of that to the guanine nucleotide-free form. GDI did not stimulate the GTPase activity of smg p25A and by itself showed neither GTPgammaS-binding nor GTPase activity. GDI was inactive for other ras p21/ras p21-like GTP-binding proteins including c-Ha-ras p21, rhoB p20, and smg p21. The Mr value of GDI was estimated to be about 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, about 65,000 from the S value (4.5 S), and about 82,000 by gel filtration. The isoelectric point of GDI was about pH 5.6. The activities of GDI were killed by tryptic digestion or heat boiling. These results indicate that bovine brain cytosol contains a regulatory protein for smg p25A that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to this protein.

Animals↗
PubMed1989

Release of flagellar filament-hook-rod complex by a Salmonella typhimurium mutant defective in the M ring of the basal body.

A Salmonella typhimurium strain possessing a mutation in the fliF gene (coding for the component protein of the M ring of the flagellar basal body) swarmed poorly on a semisolid plate. However, cells grown in liquid medium swam normally and did not show any differences from wild-type cells in terms of swimming speed or tumbling frequency. When mutant cells were grown in a viscous medium, detached bundles of flagellar filaments as long as 100 microns were formed and the cells had impaired motility. Electron microscopy and immunoelectron microscopy revealed that the filaments released from the cells had the hook and a part of the rod of the flagellar basal body still attached. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis showed that the rod portion of the released structures consisted of the 30-kilodalton FlgG protein. Double mutants containing this fliF mutation and various che mutations were constructed, and their behavior in viscous media was analyzed. When the flagellar rotation of the mutants was strongly biased to either a counterclockwise or a clockwise direction, detached bundles were not formed. The formation of large bundles was most extreme in mutants weakly biased to clockwise rotation.

Bacterial Proteins↗
PubMed1986

Genetic evidence for a switching and energy-transducing complex in the flagellar motor of Salmonella typhimurium.

The flaAII.2, flaQ, and flaN genes of Salmonella typhimurium are important for assembly, rotation, and counterclockwise-clockwise switching of the flagellar motor. Paralyzed and nonchemotactic mutants were subjected to selection pressure for partial acquisition of motility and chemotaxis, and the suppressor mutations of the resulting pseudorevertants were mapped and isolated. Many of the intergenic suppressor mutations were in one of the other two genes. Others were in genes for cytoplasmic components of the chemotaxis system, notably cheY and cheZ; one of the mutations was found in the cheA gene and one in a motility gene, motB. Suppression among the three fla genes was allele specific, and many of the pseudorevertants were either cold sensitive or heat sensitive. We conclude that the FlaAII.2, FlaQ, and FlaN proteins form a complex which determines the rotational sense, either counterclockwise or clockwise, of the motor and also participates in the conversion of proton energy into mechanical work of rotation. This switch complex is probably mounted to the base of the flagellar basal body and, via binding of the CheY and CheZ proteins, receives sensory information and uses it to control flagellar operation.

Chemotaxis↗
PubMed1982

[Enzyme immunoassay of CEA using monoclonal antibody].

Carcinoembryonic antigen (CEA) is important as one of the tumor markers, and enzyme-immunoassay using monoclonal anti-CEA, which is based on the Sandwich method and unnecessary for the treatment of CEA-extract from serum, was tried in this study. The standard curve obtained from this assay showed lineality on low CEA level. Sensitivity could allow to detect CEA below 5 ng/ml of CEA. Correlation between radioimmunoassay and this EIA was not found in especially high CEA-concentration. Reproducibility respect of Intra-assay and Inter-assay had good results. CEA value yielded by dilution of sample serum coincided with nearly expected value; this result indicated that dilution test was effective. CEA value determined by the assay to be necessary for treatment of extract was higher than that by the assay without treatment. Though the number of test samples used for assay of CEA in serum from various diseases were small, we considered it would be appropriate to set up a normal value range in 0.44-3.16 ng/ml (mean +/- 2 SD) and cut off value at 3.9 ng/ml (mean +/- 3 SD). We have been interested in cancer-specificity on monoclonal antibody, but we could not so for evaluate effects of the assay using monoclonal antibody.

Antibodies, Monoclonal↗