Search PubMed⌕ Search

Biomedical subjects

F Arisaka

Publications and source records attributed to F Arisaka.

At least 19 recordsLinked to original sources
PubMed2001

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures.

Random mutagenesis coupled with screening of the active enzyme at a low temperature was applied to isolate cold-adapted mutants of a thermophilic enzyme. Four mutant enzymes with enhanced specific activities (up to 4.1-fold at 40 degrees C) at a moderate temperature were isolated from randomly mutated Thermus thermophilus 3-isopropylmalate dehydrogenase. Kinetic analysis revealed two types of cold-adapted mutants, i.e. k(cat)-improved and K(m)-improved types. The k(cat)-improved mutants showed less temperature-dependent catalytic properties, resulting in improvement of k(cat) (up to 7.5-fold at 40 degrees C) at lower temperatures with increased K(m) values mainly for NAD. The K(m)-improved enzyme showed higher affinities toward the substrate and the coenzyme without significant change in k(cat) at the temperatures investigated (30-70 degrees C). In k(cat)-improved mutants, replacement of a residue was found near the binding pocket for the adenine portion of NAD. Two of the mutants retained thermal stability indistinguishable from the wild-type enzyme. Extreme thermal stability of the thermophilic enzyme is not necessarily decreased to improve the catalytic function at lower temperatures. The present strategy provides a powerful tool for obtaining active mutant enzymes at lower temperatures. The results also indicate that it is possible to obtain cold-adapted mutant enzymes with high thermal stability.

3-Isopropylmalate Dehydrogenase↗
PubMed2000

Structural/functional assignment of unknown bacteriophage T4 proteins by iterative database searches.

Among the total of 274 orfs within bacteriophage T4, only half have been reasonably well characterized, and the functions of the rest have remained obscure. In order to predict the molecular functions of the orfs, a position-specific iterated (PSI)-BLAST search of bacteriophage T4 against the sequence database of known 3D structures was carried out. PSI-BLAST is one of the most powerful iterative sequence search methods using multiple sequence alignment, with the ability to detect many more proteins with distant homology than standard pairwise methods. The 3D structures of proteins are considered to be better preserved than the sequences, and the detected distantly homologous proteins are likely to possess highly similar 3D structures. Thirteen orfs of phage T4, whose homologues were not detected by standard pairwise methods, were found to have significantly homologous counterparts by this method. The plausibility of the results was confirmed by checking whether important residues at substrate/ligand-binding sites were conserved. Among them, two orfs, vs.1 and e.1, which are similar to Escherichia coli lytic enzyme and MutT protein, respectively, had not been studied previously. Also, gp rIIA, a rapid lysis protein, whose gene structure had been intensively studied during the development of molecular biology in the 1950s and yet whose molecular function remains unknown, has an N-terminal domain that is significantly similar to the N-terminal region of the heat shock protein Hsp90.

Amino Acid Sequence↗
PubMed2000

Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio.

Silk fibroin produced by the silkworm Bombyx mori consists of a heavy chain, a light chain, and a glycoprotein, P25. The heavy and light chains are linked by a disulfide bond, and P25 associates with disulfide-linked heavy and light chains by noncovalent interactions. Quantitative enzyme-linked immunosorbent assay revealed that molar ratios of the heavy chain, light chain, and P25 were 6:6:1, both in cocoons and in fibroin secreted into the lumen of posterior silk gland. Trace amounts of fibroin produced by three "naked pupa" mutants of B. mori lacked the light chain, but the molar ratio of heavy chain and P25 was also 6:1. Gel filtration chromatography and sedimentation equilibrium analysis demonstrated that a large protein complex of approximately 2.3 MDa, designated an elementary unit of fibroin having 6:6:1 molar ratios of the heavy chain, light chain, and P25, existed in posterior silk gland cells. Inaccessibility of biotinylated concanavalin A to the native elementary unit and partial dissociation of the elementary unit after incubation with excess N-glycosidase F or endoglycosidase H suggest that a single molecule of P25 is located internally and plays an important role in maintaining integrity of the complex.

Amino Acid Sequence↗
PubMed2000

Stoichiometry and inter-subunit interaction of the wedge initiation complex, gp10-gp11, of bacteriophage T4.

Association of gp10 and gp11 (gp=gene product) is the first step in the assembly pathway of the wedge part of the baseplate of bacteriophage T4. The gp10-gp11 complex constitutes the six tail pins at the corners of the baseplate hexagon on the distal side. The stoichiometry of the subunits, gp10 and gp11, of this complex was determined in combination with sedimentation equilibrium, Edman degradation of the complex and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the results of Edman degradation and SDS-PAGE, the molar ratio of gp10 and gp11 was approximately 1. On the other hand, the molecular weight of the purified gp10-gp11 complex was determined by sedimentation equilibrium to be 284000+/-7000, which is in good agreement with the expected value of 269840 if the stoichiometry is 3:3. Furthermore, comparison of the results in the presence and in the absence of reducing reagent, 2-mercaptoethanol (2-ME), in SDS-PAGE revealed that two molecules of gp10 in the complex formed a disulfide bond, while the third gp10 molecule does not participate in the disulfide bond formation.

Chromatography, Liquid↗
PubMed1999

Chimeric virus-like particle formation of adeno-associated virus.

Adeno-associated virus (AAV) capsids are composed of three proteins, VP1, VP2, and VP3. These capsid proteins have a common amino acid sequence, being expressed from different initiation codons on the same open reading frame. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but their functions are poorly understood. To investigate the role(s) of the capsid proteins in capsid formation, we used a baculovirus protein expression system to produce virus-like particles (VLPs). We found that varying the ratios of VP2 and VP3 did not affect VLP formation. Further, their physical properties were equivalent to those of empty wild-type particles. The function of VP3 was studied further by fusing a peptide tag, FLAG, to its N-terminus. This chimeric viral protein, in combination with VP2, could assemble into VLPs, indicating that the chimerism of VP3 did not affect VLP formation. Although the monomeric native form of the FLAG-VP3 chimera could react with anti-FLAG antibody, VLP containing the chimeric VP3 could not, suggesting that the N-terminal region of VP3 is located inside the VLP. These observations indicate that it may be possible to utilize AAV VLP as vectors of a broad range of drugs since fusion of the VP3 N-terminus with defined molecules could impose distinct physical properties onto the internal environment of the VLP.

Animals↗
PubMed1999

A newly identified horseshoe crab lectin with specificity for blood group A antigen recognizes specific O-antigens of bacterial lipopolysaccharides.

A 14-kDa lectin, named tachylectin-3, was newly identified from hemocytes of the Japanese horseshoe crab, Tachypleus tridentatus. This lectin exhibited hemagglutinating activity against human A-type erythrocytes, but not against the B- and O-types of erythrocytes and animal erythrocytes, including those of sheep, rabbit, horse, and bovine. The hemagglutinating activity of tachylectin-3 was equivalent to that of a previously identified lectin, named tachylectin-2, with affinity for N-acetyl-D-glucosamine or N-acetyl-D-galactosamine. However, the activity of tachylectin-3 was not inhibited by these two N-acetylhexosamines at 100 mM but was inhibited by a blood group A-pentasaccharide at a minimum inhibitory concentration of 0.16 mM. Furthermore, the hemagglutinating activity was strongly inhibited by bacterial S-type lipopolysaccharides (LPSs) from Gram-negative bacteria but not by R-type LPSs lacking O-antigens. One of the most effective S-type LPSs was from Escherichia coli O111:B4, with a minimum inhibitory concentration of 6 ng/ml. These data suggest that tachylectin-3 specifically recognizes Gram-negative bacteria through the unique structural units of O-antigens. Ultracentrifugation analysis revealed that tachylectin-3 is present in dimer in solution. A cDNA coding for tachylectin-3 was isolated from a hemocyte cDNA library. Tachylectin-3 consisted of two repeating sequences, each with a partial sequence similarity to rinderpest virus neuraminidase. Tachylectin-3 and three previously isolated types of tachylectins were all predominantly expressed in hemocytes and released from hemocytes in response to external stimuli. These lectins present at injured sites suggest that they probably serve synergistically to accomplish an effective host defense against invading microbes.

ABO Blood-Group System↗
PubMed1999

The C-terminal fragment of the precursor tail lysozyme of bacteriophage T4 stays as a structural component of the baseplate after cleavage.

Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in beta-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.

Bacteriophage T4↗
PubMed1999

Nuclear transport of the major capsid protein is essential for adeno-associated virus capsid formation.

Adeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.

Amino Acid Sequence↗
PubMed1998

Discovery of the tail tube gene of bacteriophage Mu and sequence analysis of the sheath and tube genes.

The nucleotide sequence was determined for 2.75 kbp of phage Mu DNA encoding the contractile tail sheath protein L. N-terminal sequence analysis of Mu tail tube and sheath proteins identified the open reading frame just downstream of gene L as the tube gene. This clustering and order of the sheath and tube genes appear to be common among the myoviridae. Database homology searches revealed high similarity between the Mu sheath and tube proteins and two proteins in a Haemophilus influenzae Mu-like prophage, suggesting that they are the sheath and tube proteins of that prophage.

Amino Acid Sequence↗
PubMed1998

Mapping of functional sites on the primary structure of the tail lysozyme of bacteriophage T4 by mutational analysis.

Tail lysozyme of bacteriophage T4, product of gene 5 (gp5), functions upon infection by locally digging a hole in the peptidoglycan layer, so that the tail tube, through which the phage DNA is injected, can penetrate to the inner membrane. It has been inferred from DNA sequence and expression of the tail lysozyme on a plasmid in Escherichia coli that the tail lysozyme is synthesized as a precursor of 62 K and is later cleaved to form a mature tail lysozyme of 42 K. Furthermore, gp5 has a region that is highly homologous to T4 lysozyme, gpe, that is the product of gene e and functions for 'lysis from within'. As an approach to elucidation of structure-function relationship of gp5, we determined mutational sites of gene 5 mutants that have heat sensitive virions, are temperature sensitive for growth, or require an amber suppressor. All the mutational sites were mapped in the region corresponding to the mature tail lysozyme. Among the ts mutants, 5ts1 was a pseudo-revertant of an amber mutant which bypasses gene e. It was mapped in the region which had a high homology to gpe, which is well known as T4 lysozyme. The other mutational sites will be also discussed in relation to the phenotypes of the mutants.

Bacteriophage T4↗
PubMed1997

Identification of kinesin neck region as a stable alpha-helical coiled coil and its thermodynamic characterization.

The kinesin heavy chain consists of an N-terminal globular domain, referred to as the motor domain, a rod-like middle region, and a C-terminal domain. In this study, the human kinesin neck region, the region adjacent to the motor domain which promotes dimerization, has been investigated. First, we predicted coiled-coil regions including the neck region by our newly devised statistical method. The sequence (335-372) was predominated by a unique heptad amphipathy. A comparison of the bacterially expressed human kinesin heavy chain fragments, K349 (1-349), a monomeric motor domain, and K379 (1-379), a dimer, by circular dichroism (CD) spectroscopy showed that K379 had more alpha-helical content. Chemically synthesized peptides, (332-349), (350-379), and (332-369), gave CD spectra with an alpha-helix-rich pattern, but the spectra varied depending on the peptide concentration. Analysis of the molar ellipticity at 222 nm indicated that those peptides were in monomer-dimer equilibria, and the dissociation isotherms established dissociation constants of 9.6 mM. 60 microM, and 62 nM for the above peptides, respectively. Sedimentation equilibrium measurements verified that the peptide (332-369) existed as a dimeric form. These results strongly suggest that the sequence from 332 to 369 of the neck region forms an alpha-helical coiled coil. The differential peptide of K349 and K379, (350-379), did not show sufficient ability to make K379 dimeric. It is likely that the region (350-379) forms a stable alpha-helical coiled coil only together with the (332-349) region. Fluorescence energy transfer studies of [Cys363]-(332-369) labeled with a fluorescence donor and an acceptor revealed that the peptide formed a parallel coiled coil. This coiled coil was thermodynamically stable against urea and thermal denaturation, and peptide exchange of the coiled coil was undetectable, or extremely slow, at neutral pH. The dissociation free energy was estimated to be 57.7 kJ mol-1 at a peptide concentration of 22 microM. These results indicate that the neck region of kinesin forms a stable coiled coil which may be important for the motility of dimeric kinesin.

Amino Acid Sequence↗
PubMed1997

Isolation and characterization of a molecular chaperone, gp57A, of bacteriophage T4.

A molecular chaperone of bacteriophage T4, gp57A, which facilitates the formation of the long and short tail fibers, was isolated and characterized by peptide analysis, sedimentation equilibrium, and circular dichroism (CD). Sequence analysis confirmed the predicted sequence of 79 amino acids from the nucleotide sequence of the gene with the N-terminal methionine removed. The result led to the conclusion that the apparent smaller molecular weight of 6,000 from Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the expected molecular weight of 8,710 was due to its abnormal electrophoretic behavior instead of cleavage or processing of the gene product. Estimation of the secondary structure from far-UV CD indicated a 94% alpha-helix content, which was in accord with the prediction from the primary structure. A sedimentation equilibrium study, on the other hand, revealed that gp57A assumes a tetrameric subunit structure.

Amino Acid Sequence↗