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S Aimoto

Publications and source records attributed to S Aimoto.

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

Determination of heteronuclear three-bond J-coupling constants in peptides by a simple heteronuclear relayed E.COSY experiment.

A simple heteronuclear relayed E.COSY pulse sequence with a minimum number of pulses is proposed for the quantitative determination of heteronuclear three-bond J-coupling constants in uniformly 13C-enriched polypeptide samples. Numerous heteronuclear three-bond coupling constants, including 3JHNC, 3JHNC beta, 3JH beta C, and 3JH alpha C gamma, can be determined for each residue from a single heteronuclear relayed E.COSY spectrum. Couplings relevant for stereospecific assignments as well as for the determination of dihedral angles in the amino acid backbone and in side chains are obtained. The method is demonstrated on the uniformly 13C-enriched decapeptide antamanide (-Val1-Pro2-Pro3-Ala4-Phe5-Phe6-Pro7-Pro8-Phe9-Phe1 0-).

Amino Acid Sequence↗
PubMed1995

Comparison of the free and DNA-complexed forms of the DNA-binding domain from c-Myb.

The DNA-binding domain of c-Myb consists of three imperfect tandem repeats (R1, R2 and R3). The three repeats have similar overall architectures, each containing a helix-turn-helix variation motif. The three conserved tryptophans in each repeat participate in forming a hydrophobic core. Comparison of the three repeat structures indicated that cavities are found in the hydrophobic core of R2, which is thermally unstable. On complexation with DNA, the orientations of R2 and R3 are fixed by tight binding and their conformations are slightly changed. No significant changes occur in the chemical shifts of R1 consistent with its loose interaction with DNA.

Amino Acid Sequence↗
PubMed1995

Expression and activity-dependent changes of a novel limbic-serine protease gene in the hippocampus.

A novel murine cDNA which encodes a protein designated neuropsin was cloned. Northern and in situ hybridization analyses demonstrated that neuropsin mRNA is expressed specifically in the limbic system of mouse brain and is localized at highest concentration in pyramidal neurons of the hippocampal CA1-3 subfields. Direct hippocampal stimulation and kindling induced by amygdaloid stimulation caused a significant bilateral change in neuropsin mRNA level in the hippocampal pyramidal neurons. The activity-dependent changes and the specific localization indicate that neuropsin is involved in hippocampal plasticity.

Amino Acid Sequence↗
PubMed1995

Structure and action of MIP (Mytilus inhibitory peptide)-related tetrapeptides synthesized with a multipeptide synthesizer.

Using a multipeptide synthesizer we synthesized 19 peptide libraries, each of which consisted of 19 MIP-related tetrapeptides, and isolated a number of peptides, which have an inhibitory effect on phasic contraction of the ABRM of Mytilus, from the libraries. To the present, the structures of about 30 species of the peptides were determined, and the peptides with the determined structures were synthesized. The structure and action of each synthetic peptide was compared with those of others to explain structure-activity relationship of MIPs.

Amino Acid Sequence↗
PubMed1994

Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II.

IP4BP/Synaptotagmin II is an inositol-1,3,4,5-tetrakisphosphate (IP4) or inositol polyphosphate-binding protein, which is accumulated at nerve terminals. Here we report a novel function of the C2B domain, which was originally thought to be responsible for Ca(2+)-dependent binding to phospholipid membranes. A study of deletion mutants showed that about 30 amino acids of the central region of the C2B domain of mouse IP4BP/synaptotagmin II (315 IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF 346) are essential for inositol polyphosphate binding. This binding domain includes a sequence corresponding to the squid Pep20 peptide, which is also known to be essential for neurotransmitter release (Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163-165), suggesting that inositol polyphosphate has some effect on neurotransmitter release. Rabphilin 3A, another neuronal protein containing C2 domains, cannot bind IP4, indicating that the IP4 binding property is specific to the C2B domain of synaptotagmin. Phospholipid and IP4 binding experiments clearly indicated that the C2A and C2B domains have different functions. The C2A domain binds phospholipid in a Ca(2+)-dependent manner, but the C2B domain binds inositol polyphosphate and phospholipid irrespective of the presence of Ca2+. Our data suggest that the C2B domain of synaptotogamin is the inositol polyphosphate sensor at the synaptic vesicle and may be involved in synaptic function.

Amino Acid Sequence↗
PubMed1994

Sustained tyrosine phosphorylation of p140trkA in PC12h-R cells responding rapidly to NGF.

The PC12h cell, a subclone of PC12 cells, has considerable activities of tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) and shows an NGF-induced increase in both enzyme activities. The TH activity and its inducibility by NGF in PC12h cells were stably maintained in the passage of > 200 generations whereas the ChAT activity was not. We isolated a new cell line, PC12h-R (originally clone 8), from a long-term culture of PC12h cells. PC12h-R cells still showed the considerable TH activity, but not the ChAT activity, and maintained the inducibility of TH activity by NGF. Thus, the responses of PC12h-R cells to NGF were similar to those of chromaffin cells and sympathetic neurons. PC12h-R cells were found to extend neurites and differentiate into sympathetic neuron-like cells in response to NGF much more rapidly than PC12h cells. In addition, PC12h-R cells showed sustained NGF-induced tyrosine phosphorylation of p140trkA and several cellular proteins, including 42-, 44- and 54-kDa proteins, in comparison with PC12h cells. We suggest that the NGF-induced sustained tyrosine phosphorylation signal in PC12h-R cells may be correlated closely with their rapid NGF-induced differentiation into neuron-like cells.

Animals↗
PubMed1994

13C-NMR relaxation study on mobility of the DNA-binding arm of HU.

The mobility of the DNA-binding arm of HU protein was studied by 13C-NMR spectroscopy. The correlation times tau c of Phe47C alpha in the body and Gly60C alpha in the arm of HU were determined for HU and HU-DNA complex. The value of tau c of Phe47C alpha is 2-4 times larger than that of Gly60C alpha irrespective of the presence or absence of DNA. These results show that Gly60C alpha undergoes more rapid motion than Phe47C alpha. The increase in correlation time on addition of DNA is greater for Gly60C alpha than for Phe47C alpha. This suggests that the addition of DNA influences more significantly the motion of Gly60C alpha than that of Phe47C alpha. These results are in accord with the X-ray result, in which the top part of the arm is not visible. Gly60C alpha in the arm is thus more mobile than Phe47C alpha in the body, and the mobility of Gly60C alpha is reduced by the DNA binding.

Bacterial Proteins↗
PubMed1994

Solution structure of the DNA binding octapeptide repeat of the K10 gene product.

A putative transcription factor, the Drosophila K10 gene product, contains eight repeats of the octapeptide sequence SPNQQQHP or close variants. The solution structure of the K10 repeat was studied by NMR using a peptide composed of two SPNQQQHP units (referred to here as HP2). To overcome problems caused by degeneracy of backbone amide signals of Gln residues, a series of synthetic peptides containing an 15N-labelled main chain amide at different positions in HP2 were synthesized. In aqueous trifluoroethanol solution, HP2 folds into two structural units; the SPNQ part of each unit folds into a turn structure, while the C-terminal part shows some helical characteristics but is less structured. The N-terminal turn is likely to provide a core that produces a more stable helical structure upon binding to DNA and probably 'caps' the segmented helical unit at its N-terminus. This model is supported by a DNA footprinting study which shows that one SPNQQQHP unit spans four base pairs upon binding to A/T-rich sequences of DNA.

Amino Acid Sequence↗
PubMed1993

Mg2+ inhibits formation of 4Ca(2+)-calmodulin-enzyme complex at lower Ca2+ concentration. 1H and 113Cd NMR studies.

Our previous 1H NMR studies indicated that when mastoparan (MP) is added to Ca(2+)-half-saturated calmodulin (2Ca(2+)-CaM) in the absence of Mg2+ ions, Ca2+ ions transfer from the C-terminal-half domain of CaM not interacting with MP to the N-terminal-half domain of CaM interacting with MP at lower MP concentrations (Ohki, S., Yazawa, M., Yagi, K., and Hikichi, K. (1991b) J. Biochem. (Tokyo) 110, 737-742). As a consequence, the active form of 4Ca(2+)-CaM.MP complex is formed. In the present study, we studied the effect of Mg2+ ions on Ca2+ transfer. In the presence of Mg2+ ions, such Ca2+ transfer does not occur. The effects of Mg2+ ions are also studied by observing 113Cd NMR in the presence of M13, the 26-residue peptide of the CaM-binding region of myosin light chain kinase. The 113Cd NMR results show that Mg2+ ions prevent to form the active complex. Mg2+ plays an important role as an inactivating factor to CaM.

Amino Acid Sequence↗
PubMed1993

Guanidine hydrochloride-induced folding of proteins.

Guanidine hydrochloride (Gdn-HCl) is the most commonly used denaturant for proteins. Contrary to expectation, we found that Gdn-HCl at low concentrations refolds acid-unfolded apomyoglobin and cytochrome c, stabilizing the molten globule state, i.e. a compact denatured state with a significant amount of secondary structure, but substantially disordered tertiary structure. A further increase in Gdn-HCl concentration, above 1 M, caused co-operative unfolding of the molten globule state. Similar sequential folding and unfolding transitions were also observed at neutral pH with a synthetic amphiphilic peptide consisting of Lys and Leu residues, indicating the generality of the phenomenon. Although the Gdn-HCl-induced refolding and unfolding transitions were puzzling at first glance, we show that they are readily interpreted in terms of the differential action of Gdn-HCl. We also show that the comparison of the unfolding curves for the molten globule and native states provides a measure of the buried surface area upon formation of the molten globule state.

Amino Acid Sequence↗
PubMed1993

Acceleration of bovine neurofilament L assembly by deprivation of acidic tail domain.

Functions of the tail region of neurofilament L have, to date, not been clearly elucidated. Bovine neurofilament L was cleaved into tail-less neurofilament L (50 kDa) and a tail fragment (19 kDa), by thrombin. Tail-less neurofilament L was deficit of the highly acidic domain of the tail region (approximately 77% of the entire region). Assembly of tail-less neurofilament L. was observed to be accelerated by both fluorometric and centrifugal measurements, compared with intact neurofilament L. The critical concentration of tail-less neurofilament L, which constitutes the constant unassembled pool, was approximately 0.25-times lower than that of neurofilament L. Under physiological conditions, tail-less neurofilament L formed a ribbon-like structure, whereas tail-less neurofilament L could form 10-nm filaments in an extremely low ionic-strength buffer in the presence of 1 mM MgCl2. An affinity-purified antibody directed against the tail fragment also accelerated neurofilament L assembly. The tail fragment neither coassembled with neurofilament L nor affect neurofilament L assembly. The acidic domain of the tail region may regulate neurofilament assembly and may be involved in 10-nm filament formation under physiological conditions.

Amino Acid Sequence↗
PubMed1993

Permethylation alters the conformational transitions and the complexing ability of melittin: a model for methylated proteins.

Melittin exhibits a transition from random-coil monomer to helical tetramer as a function of peptide concentration [J. Bello, H. R. Bello, and E. Granados (1982) Biochemistry, Vol. 21, pp. 461-465]. When permethylated on each of the four amino groups (Gly-1 N alpha and Lys-7, 21, and 23 N epsilon) to yield trimethylammonium groups, melittin exists as a random coil and does not show any concentration-dependent conformational transition (up to 290 microM). Acylation of the amino groups of melittin with glycine or 5-aminopentanoic acid followed by permethylation increases helix formation, but to a lesser extent than for the unmethylated aminoacylmelittin derivatives. The results are discussed in relation to hydrophobicity, charge repulsions, and ion binding. Melittin, and more weakly, permethylated melittin (MLT-Me) form helical hybrids with an anionic random-coil melittin analogue (E-MLT), in which all the lysine and arginine residues of melittin were replaced by glutamate residues. The hybrid between MLT-Me and E-MLT shows a concentration-dependent increase in helicity. E-MLT, when succinylated at the N-terminal glycine (E-MLT-suc), forms a stronger hybrid with MLT-Me, possibly as a result of increased electrostatic interaction between equal but opposite charges in E-MLT-suc (net charge -6) and MLT-Me (net charge +6). The hybrids exhibit both cold- and heat-induced denaturation, similar to the phenomenon exhibited by proteins. The hybrids also exhibit significant residual structures in the temperature range of 80-100 degrees C, which may be similar to the molten globular states that have been suggested for proteins.

Amino Acid Sequence↗
PubMed1993

Molecular conformation of porcine amelogenin in solution: three folding units at the N-terminal, central, and C-terminal regions.

Circular dichroism (CD) studies were conducted to gain a better insight into the conformation of amelogenins, which were isolated from developing enamel of piglets. The intact porcine amelogenin and its degraded products were purified chromatographically. The 25-residue peptide corresponding to the segment at the C-terminus was synthesized. CD spectra of these samples were measured at pH 5.0-5.3 in the temperature range between 4 and 90 degrees C. The most remarkable finding was that the CD spectrum of the intact amelogenin was accounted for by the sum of the spectra of the three fragments at the N-terminal, central, and C-terminal regions, supporting the hypothesis that the structure of the whole protein consists of discrete folding units. Furthermore, low-angle laser light scattering analysis provided evidence that the 20 kDa amelogenin, the most abundant extracellular matrix protein in forming enamel tissue, exists in a monomeric form at pH 5.3 and 25 degrees C. It was tentatively concluded that the N-terminal region contains beta-sheet structures, while the spectral characteristics of the C-terminal region are similar to those of a random coil conformation. The conformation of the central region was characterized by a strong negative ellipticity at 203 nm, although its nature remains to be defined.

Amelogenin↗
PubMed1993

Transitional expression of neural cell adhesion molecule isoforms during chicken embryonic myogenesis.

The neural cell adhesion molecule, NCAM, is known to be expressed in chicken muscle as at least three principal molecular forms (molecular masses of 155 kDa, 145 kDa, and 120 kDa). They are generated from a single gene by alternative splicing. To distinguish these molecular species and to investigate their expressions in muscle differentiation during chicken embryonic development, we prepared antipeptide antibodies against three different domains of the NCAM. The antibody named MSD<+> was designed to detect muscle-specific domain (MSD) which was inserted into a muscle-specific NCAM by alternative splicing. The locus encompassing the MSD insertion site was detected with the antibody named MSD<->, and cytoplasmic sites near the transmembrane region were detected with the antibody named CYT. Immunoblot analysis utilizing the peptide antibodies prepared here revealed that, of three NCAMs, two isoforms, 155 kDa and 120 kDa, were recognized with the antibody MSD<+> and the same 155-kDa and the other 145-kDa molecule were recognized with the antibody CYT. The antibody MSD<-> was capable of detecting all three isoforms of the NCAM. Expression of the 120-kDa form with MSD but lacking a cytoplasmic tail increased but that of the 145-kDa form with cytoplasmic tail but lacking MSD declined during embryonic day 5 to day 18. The 155-kDa form NCAM with both MSD and cytoplasmic tail was expressed specifically and transiently during embryonic day 11 to day 14 in chick muscle; this period coincides with the period of extensive myotube formation. Thus, this largest isotype seems to play an important role in muscle differentiation.

Amino Acid Sequence↗
PubMed1992

Charge repulsion in the conformational stability of melittin.

Electrostatic repulsion between positively charged groups has been suggested to be critical in determining the conformation of melittin. To clarify the role of repulsive forces, we prepared a series of succinylated melittins, an acetylated melittin, and a synthetic melittin mutant, with various degrees of charge repulsion. The conformation of the melittin derivatives was examined by far-UV circular dichroism under various conditions of pH and salt at 20 degrees C. The stability of the tetrameric helical state was found to be dependent on the net charge of the peptides. The charge repulsive forces destabilized the helical state of intact melittin by 600 cal/(charge.mol of tetramer). This value was close to the corresponding one (450 cal/(charge.mol)) obtained for the acidic molten globule of horse cytochrome c [Goto, Y., & Nishikiori, S. (1991) J. Mol. Biol. 222, 679-686], which has a molecular weight and a net charge comparable to those of the tetrameric melittin. Small-angle X-ray scattering of the tetrameric melittin and the molten globule of cytochrome c showed that the two states are also comparable to each other in the radius of gyration. These results suggest that the contribution of electrostatic repulsion to the conformational stability of melittin is similar to that of the molten globule.

Amino Acid Sequence↗
PubMed1992

Assembly regulatory domain of glial fibrillary acidic protein. A single phosphorylation diminishes its assembly-accelerating property.

Phosphorylation of glial fibrillary acidic protein (GFAP) induces disassembly of the filaments. An amino-terminal fragment of bovine GFAP (G-Hf) was produced by lysylendopeptidase digestion. G-Hf formed ribbon-like filaments in the presence of GFAP even in low ionic strength, whereas the fragment itself did not form any structures. Only one (PK3) of the five V8 protease fragments of G-Hf accelerated GFAP assembly to the same degree as G-Hf did, whereas the other fragments did not. When PK3 was cleaved into two fragments, it lost the assembly-accelerating property. The sequence of PK3 was determined as RRRVTSATRRSYVSSSE, which corresponded to residues 3-19 of porcine GFAP. It was concluded that PK3 contains a sequence indispensable for GFAP assembly and that neither PK1 (RRRVTS) nor PK2 (ATRRSYVSSSE) included all of the sequence. A single phosphorylation of PK3 by cyclic AMP-dependent protein kinase diminished its assembly-accelerating property. The phosphorylation site was determined as Ser-12 of porcine GFAP. It was shown that single phosphorylation of the amino-terminal head domain, which contains an indispensable sequence for GFAP assembly, might be sufficient for GFAP disassembly.

Amino Acid Sequence↗
PubMed1992

Biological role of an arginine residue present in a histidine-rich peptide which inhibits hemagglutination of Porphyromonas gingivalis.

Inhibitory effects of synthetic fragments in histatin 8, having the sequence Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, on hemagglutination by Porphyromonas gingivalis 381 were examined. The hemagglutinating activity was reduced much more by the peptide Lys-His-His-Ser-His-Arg-Gly-Tyr than by the peptides Lys-His-His-Ser-His and/or Lys-Phe-His-Glu-Lys. These results suggest that the arginine residue may have an important role in the inhibition of hemagglutination by P. gingivalis.

Amino Acid Sequence↗