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T Ooi

Publications and source records attributed to T Ooi.

At least 37 records · Page 2Linked to original sources
PubMed1995

Differences in the thermal stability of acclimation temperature-associated types of carp myosin and its rod on differential scanning calorimetry.

Differential scanning calorimetry (DSC) was employed for studying the thermal unfolding of myosin and its rod part prepared from carp acclimated to 10 and 30 degrees C. Differences in the thermal stability reflecting structural properties were clearly demonstrated by the DSC data obtained at pH 8.0 in 0.6 M KCl for the two types of carp myosin and rod. The transition temperatures on myosin and rod given by the major peaks for the 10 degrees C-acclimated carp were 33.9 and 47.4 degrees C and 33.0 and 44.0 degrees C, respectively, assuming two endotherms for this type. Since the shape of the first peaks at 33.9 and 33.0 degrees C was not symmetrical, two peaks having similar transition temperatures overlapped in this temperature range. When the data were analyzed using three endotherms, the three transition temperatures obtained for myosin and rod were 32.8, 34.9, and 47.4 degrees C and 32.9, 33.4, and 44.1 degrees C, respectively. Thus, the position of the first peak for the 10 degrees C-acclimated carp myosin did not change even after removal of the large subfragment-1 part, but the transition of the second peak shifted to a lower temperature by about 3 degrees C. The myosin and rod from carp acclimated to 30 degrees C showed three distinct peaks at 35.9, 39.7, and 49.1 degrees C and 34.5, 39.7, and 46.7 degrees C, respectively. The position of the largest peak for myosin remained unchanged, and the shift of the peak position of the highest temperature was about 3 degrees C, as obtained for the 10 degrees C-acclimated carp.(ABSTRACT TRUNCATED AT 250 WORDS)

Acclimatization↗
PubMed1995

Characterization of vitelline membrane outer layer protein I, VMO-I: amino acid sequence and structural stability.

Vitelline membrane outer layer protein I (VMO-I) tightly bound to ovomucin fibrils of hen's egg yolk membrane was characterized in terms of its amino acid sequence and structural stability. The deduced sequence of VMO-I using the conventional sequencing method is: RTREYTSVITVPNGGHWGKWGIRQFCHSGYANGFALKVEPSQFGRDDTALNGIRLRCLD- GSVIESLVGKWGTWTSFLVCPTGYLVSFSLRSEKSQGGGDDTAANNIQFRCSDEAVLVGD- DLSWGRFGPWSKRCKICGLQTKVESPQGLRDDTALNNVRFFCCK. Thus, VMO-I is composed of 163 amino acid residues with a calculated molecular weight of 17,979. The sequence confirms the cDNA sequence of VMO-I we recently determined and does not show any significant similarity to proteins compiled in the NBRF database. Two of the four disulfide bonds found in VMO-I were estimated to lie between Cys26 and Cys57 and between Cys79 and Cys110. The sequence analyses show that VMO-I contains three 53-residue internal repeats that contain distinctive regions of turns flanked by beta-sheets consistent with the recent finding that the molecule contains a new beta-fold motif, the beta-prism. The molecular characteristics of VMO-I in solution were examined by CD spectroscopy in the far and near ultraviolet regions, NMR spectroscopy, and high sensitive differential scanning calorimetry (DSC). CD spectra in the far UV region at room temperature were similar to that assigned to a random coil, while in the near UV region, small positive peaks were observed. The ellipticity in both regions decreased on raising the temperature. Proton NMR experiments showed the native structure unfolds to unordered conformations at 70 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies of an endoglucanase from Aspergillus aculeatus.

Most fungal cellulases are found in multiple forms varying in size and substrate specificity. Aspergillus aculeatus is known to produce nine cellulolytic enzymes including an endoglucanase (FI CM-cellulase, M(r) = 24,002) as the major component. Single crystals of FI CM-cellulase from Aspergillus aculeatus have been prepared by sitting-drop vapour diffusion using ammonium sulphate as a precipitant. The cellulase crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 52.79(2) A, b = 106.40(4) A and c = 33.15(1) A. The crystals contain one enzyme molecule per asymmetric unit. They diffract to at least 2.0 A resolution and are very stable against X-ray irradiation.

Ammonium Sulfate↗
PubMed1994

High-level expression of porcine muscle adenylate kinase in Escherichia coli: effects of the copy number of the gene and the translational initiation signals.

Porcine muscle adenylate kinase (ADK) was overproduced in Escherichia coli using the expression plasmid with double A-T-G codon at the translational starting site and the Shine-Dalgarno (SD) sequence 10 bp apart from the first A-T-G. We used the expression vectors pKK223-3 and pMK2. pMK2 is about 10-20 times larger in copy number than pK223-3. For both vectors, duplication of A-T-G was effective and the quantity of the expressed ADK from the double A-T-G plasmid was 2 approximately 4-fold more than that achieved when only one A-T-G was present. The amount of the produced ADK was maximum in the case of using pMK2 with double A-T-G. The overproduced ADK formed inclusion bodies in E. coli. It was solubilized in 6 M guanidine hydrochloride and refolded. Through two steps of column chromatography, ADK was purified. It has the same amino acid composition and grossly the same activity as that reported by Schirmer et al. (1970). Its amino acid sequence of the NH2-terminal region was identical with that deduced from the cDNA sequence including the NH2-terminal methionine.

Adenylate Kinase↗
PubMed1994

Thermodynamics of protein folding: effects of hydration and electrostatic interactions.

DNA base sequences contain a variety of information, and this information flows from DNA to a protein through the steps of transcription and translation. The one dimensional information given from DNA is the amino acid sequence of the protein. In an organism, a nascent chain synthesized in vivo from the N-terminus according to the information in DNA folds spontaneously to a native conformation under a given environmental condition, i.e., water is the solvent and concentration of salts, pH, temperature, and pressure are appropriate. The polypeptide chain synthesized chemically from the C-terminus also folds to the native conformation, and often exhibits the proper enzymic activity. X-ray crystallography reveals the three dimensional structure of a protein in crystal, indicating that the location of the constituent atoms in the space is specific for that protein, i.e., the information on how to fold is contained in the amino acid sequence. In order to understand the folding, however, other factors such as geometrical information on each amino acid should be taken into account, because a sequence is one dimensional information and a 3D structure is three dimensional. Environmental conditions are also crucial factors in the folding, since a change in conditions can destroy the native structure. The folding and unfolding phenomena are described in terms of thermodynamics when the processes occur reversibly. As experiments show, the stability of a protein conformation can be described by the free energy of folding or unfolding. Folding of a nascent chain to the native conformation, thus, is interpreted as the process towards a minimum of the folding free energy. Thermodynamic quantities of folding or unfolding are expressed as functions of temperature, pressure, and concentration of reagents such as salts, and the quantities are measured as the differences between states, e.g., the N and D states. Since the effects of surrounding media must be included, the phenomenon in aqueous solution at a given condition of pH and salt concentration is divided into the several steps shown in Fig. 12. The chain molecule has two states, N and D, in vacuo; the molecule is then transferred into water, accompanied by the hydration in both states; next, the ionization process produces electrostatic interactions in the molecule; and finally, addition of chemical reagents like denaturants has other effects on the stability.

Animals↗
PubMed1994

Expression of the cellulase (FI-CMCase) gene of Aspergillus aculeatus in Saccharomyces cerevisiae.

As a step to breed a Saccharomyces cerevisiae strain able to produce ethanol directly from cellulose, we combined cDNA for Aspergillus aculeatus FI-CMCase (FI-carboxymethyl cellulase) with the GAP (glyceraldehyde-3-phosphate dehydrogenase) promoter of S. cerevisiae and used the resultant plasmid, pYEC91, to transform S. cerevisiae. The transformed cells produced active FI-CMCase within the cytoplasm. Western-blot analysis following SDS-polyacrylamide gel electrophoresis demonstrated that the cells contained a peptide having the same molecular mass and immunological identity as A. aculeatus FI-CMCase.

Amino Acid Sequence↗
PubMed1993

Hydration and heat stability effects on protein unfolding.

In summary, the thermal denaturation of proteins has been elucidated in terms of the chain free energy and the hydration free energy as follows. (1) Method to calculate the unfolding free energy. The free energy of unfolding consists of two contributions: the hydration around the molecule, and the intramolecular interactions. A method to calculate the free energy of hydration from the accessible surface area (ASA) of the constituent atomic groups in a protein has been developed. This assumes a proportionality between the free energy and the ASA, where the proportional constants were determined by least-squares fitting to the experimentally derived thermodynamic data on small molecules. Similarly, the free energy of unfolding for the chain in vacuo can be also calculated from the ASA, using the unfolding thermodynamics derived from the experimental data of the ten proteins. (2) Thermodynamics of protein unfolding predicted from the three-dimensional structures and from the amino acid content in proteins. First, our method is applied to predict the thermodynamics of protein unfolding from the X-ray structure. The predicted values of four test proteins agree well with the experimentally derived values. It also accounts for the temperature dependence of the free energy and of the enthalpy upon unfolding for 14 proteins. Second, this method is applied to the helix-coil transition of short peptides of poly(L-Ala)20 and Ac-(AAAAK)3A-NH2. The calculated enthalpy change is close to the experimental values for poly-L-Lys and poly-L-Glu. Since delta Hcu at 25 degrees C significantly contributes to delta Gu, the helix formation is enthalpy-driven through interactions in the chain. Third, the method is applied to predict the unfolding thermodynamics of a globular protein from its amino acid content. It also accounts for the temperature dependence of the free energy of unfolding for the 14 proteins. The agreement between the experimental and the calculated values by this method for the 14 proteins is not so different from those obtained with the three-dimensional structures. Fourth, the values of delta Cpu for 14 proteins may be closely approximated to the predicted values of delta Cp,hu. The delta Cp,hu value in a protein consists of the major contribution from the hydrophobic and the aromatic residues, and the minor one from the hydrophilic residues. (3) Dominant free energies in protein folding.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Expression of the cellulase (FI-CMCase) gene of Aspergillus aculeatus in Escherichia coli.

FI-CMCase cDNA of Aspergillus aculeatus was expressed in Escherichia coli by using the tac promoter of E. coli. Transformants of E. coli harboring a plasmid pHEM06 containing mature form FI-CMCase cDNA produced FI-CMCase in the cytoplasm of the cells. The enzyme from E. coli cells was purified to yield 56% and it was immunological identical to that of FI-CMCase purified from A. aculeatus.

Amino Acid Sequence↗
PubMed1991

Prediction of the thermodynamics of protein unfolding: the helix-coil transition of poly(L-alanine).

The method given earlier for predicting the thermodynamics of protein unfolding from the x-ray structure of a protein is applied here to the poly(L-alanine) helix. First, the fitting parameters derived earlier from a data base of 10 proteins were used to predict the unfolding thermodynamics of 4 other proteins. The agreement between the observed and predicted values is comparable to that found for the 10 proteins studied initially. Next, the temperature dependences of the Gibbs energy and enthalpy changes for unfolding of bacteriophage T4 lysozyme were predicted and compared with data in the literature. The predicted and observed temperature dependences are similar and the predicted results indicate that cold denaturation should be observed at low temperatures, as observed recently for a T4 lysozyme mutant. The fitting parameters derived from thermodynamic data for protein unfolding and for hydration of model compounds were used to predict the unfolding thermodynamics of the poly(L-alanine) helix. The results predict that helix formation is enthalpy-driven, and the predicted enthalpy change for unfolding (0.86 kcal per mol per residue) is close to the value found in a recent calorimetric study of a 50-residue alanine-rich helix.

Kinetics↗
PubMed1991

[A manual for community mental health activities for a public health center in Japan].

A system for treatment of persons with chronic mental disease such as schizophrenics in the present structure of the Japanese public Health Center is proposed. Presently, while cases are handled by responsible staff members, incorporation into the health center system with full utilization of its personnel and resources is seldom accomplished. There is a necessity for a systems approach in order to organize essential programs that would provide care for the mentally diseased. Health centers would have three main roles in this system: as a coordinator of mental health resources in the community, as a specialized agency for case management, and responsibility for public relations in its service area. For proper administration of its system, health centers should follow a team concept. The team would consist of a director of health centers, physicians, part-time psychiatrists, public health nurses, psychiatric social workers and clerical staff. At case conferences discussion of whether a prospective case should be registered, assessment of the needs, and development of management plans for all of the registered cases would be conducted. The files and documents of each case would be deleted or re-registered 5 years after initial registration. Cases would have a case-manager to coordinate public health care, including home visits, family care, psycho-educational consultation and crisis intervention. Health centers would be responsible for bringing together available social resources such as sheltered workshops and transient residential programs. The goal of health centers' mental health activities would be to facilitate the normalization of the mentally diseased within the community.

Community Health Centers↗
PubMed1990

Distinct character in hydrophobicity of amino acid compositions of mitochondrial proteins.

A compact mitochondrial gene contains all essential information about the synthesis of mitochondrial proteins which play their roles in a small compartment of the mitochondrium. Almost no noncoding regions have been found through the gene, but a necessary set of tRNAs for the 20 amino acids is provided for biosynthesis, some of them coding different amino acids from those in a usual cell. Since the gene is so compact that the produced proteins would have some characteristic aspects for the mitochondrium, amino acid compositions of mitochondrial proteins (mt-proteins) were examined in the 20-dimensional composition space. The results show that compositions of proteins translated from the mitochondrial genes have a distinct character having more hydrophobic content than others, which is illustrated by a clustered distribution in the multidimensional composition space. The cluster is located at the tail edge of the global distribution pattern of a Gaussian shape for other various kinds of proteins in the space. The mt-proteins are rich in hydrophobic amino acids as is a membrane protein, but are different from other membrane proteins in a lesser content of Val. A good correlation found between the base and amino acid compositions for the mitochondria was examined in comparison to those of organisms such as thermophilic bacterium having an extreme G-C-rich base composition.

Amino Acids↗
PubMed1990

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus.

We have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonucleotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.

Amino Acid Sequence↗
PubMed1989

Comparison of alpha-helix stability in peptides having a negatively or positively charged residue block attached either to the N- or C-terminus of an alpha-helix: the electrostatic contribution and anisotropic stability of the alpha-helix.

An estimation of the thermodynamic effects of a charged random coil, which is attached either to the N- or C-terminus of polyalanine, upon alpha-helix stability is attempted. A temperature-induced helix-coil transition of Ala20Lys20Phe and Lys20Ala20Phe was studied under various conditions of salt concentration and pH. By combining the results with previous ones for Ala20Glu20Phe and Glu20Ala20Phe, which have opposite electric charges to the present system [S. Ihara et al. (1982) Biopolymers 21, 131-145], the free energy of the coil to helix transition of the polyalanine block could be separated into two terms--one term for the electrostatic interaction of electric charges in the random-coil block with the alpha-helix dipole, and a second term for the intrinsic stability of the helix. The first term indicates the significance of the helix dipole-charge interactions, which affects the helix stability depending on the attaching side of the charged block and on the sign of the charges. This clearly shows the anisotropic stability of the alpha-helix. Furthermore, analysis of the dependence of these thermodynamic quantities on salt concentrations showed, assuming that the effect of the attached electric charges was symmetric (in other words, the absolute values of the electrostatic interaction terms were independent of the sign of electric charges), that the intrinsic stability of the alpha-helix was dependent on which side of the helix was attached to the random coil: a random coil attached to the N-terminus of the alpha-helix had little effect while that attached to a C-terminal significantly destabilized the helix.

Circular Dichroism↗
PubMed1989

Examination of protein sequence homologies: V. New perspectives on evolution between bacterial and chloroplast-type ferredoxins inferred from sequence evidence.

Sequence homologies among 34 chloroplast-type ferredoxins were examined using a computer program that quantitatively evaluates the extent of sequence similarity as a correlation coefficient. The resultant alignment contains six gaps representing insertions or deletions of some residues, all of which are located such that they precisely preserve the domains of structural fragments as determined by crystallographic data on Spirulina platensis ferredoxin. In the search for any total correlation between the chloroplast-type and 27 bacterial ferredoxins, 1891 comparison matrices prepared for possible combinations indicated that the bacterial basal sequence of 55 residues has been conserved evolutionarily in the chloroplast-type sequences corresponding to residue positions 36-90 of Spirulina platensis ferredoxin. In addition, the bacterial "connector sequence" region was found to be conserved. These findings strongly suggest that the bacterial and chloroplast-type ferredoxins descended from a common ancestor, and branched off after the bacterial gene duplication, whereas the chloroplast-type ferredoxins originally were generated by duplicating the already duplicated bacterial gene, i.e., by "double-duplication."

Amino Acid Sequence↗
PubMed1989

Examination of protein sequence homologies. VI. The evolution of Escherichia coli L7/L12 equivalent ribosomal proteins ('A' proteins), and the tertiary structure.

Sequence homologies among 23 complete and two partial sequences of ribosomal 'A' proteins from eukaryotes, metabacteria, eubacteria and chloroplasts, equivalent to Escherichia coli L7/L12, were examined using a correlation method that evaluates sequence similarity quantitatively. Examination of 325 comparison matrices prepared for possible combinations of the sequences indicates that 'A' protein sequences can be classified into two types: one is the "prototype" from eubacteria and chloroplasts, and the other is the "transposition type" from eukaryotes and metabacteria, which must have resulted from the internal transposition of the prototype sequence. The transposition type of eukaryotes can further be classified into P1 and P2 lines. Sequences of the P1 line are closer to those of metabacteria than to those of the P2 line. Eleven gaps, as deletion or insertion sites of amino acid residues, are necessary for an alignment of all the sequences. According to the crystallographic data for the C-terminal fragment (CTF) from E. coli L7, all the gaps involved in the CTF are located between segments that correspond to structural and functional elements such as alpha helix, beta strand, turning loop or hinge part. The existence of specific "preservation units" in these molecules is suggested. In contrast, the transposition site is located at the center of an alpha helix element that is involved in a folding domain, indicating that the transposition event was extremely drastic.

Amino Acid Sequence↗
PubMed1988

Analysis of structure-function relationship of pig calpastatin by expression of mutated cDNAs in Escherichia coli.

Structure-function relationships in pig calpastatin were investigated. Calpastatin is an endogenous inhibitor protein specifically acting on calpains (Ca2+-dependent cysteine endopeptidases). We recently cloned and sequenced the cDNA for pig heart calpastatin and determined the amino acid sequence of the molecule from the nucleotide sequence. Various deletion mutants in one of the four internally repetitive domains (Domain 3, approximately 140 amino acid residues) were created by in vitro site-directed mutagenesis of a cloned cDNA fragment and expressed in Escherichia coli. Deletion of a conserved region on either the amino-terminal or carboxyl-terminal side caused a drastic loss of inhibitory activity against calpain I (low Ca2+-requiring form) and, to a lesser degree, against calpain II (high Ca2+-requiring form). Inhibitory activities were below the detectable level in mutants deleted further toward the central region. Substitution of two amino acids in the latter region of the wild-type Domain 3 protein caused a drastic loss of activity against both calpains. The creation of lowered affinity inhibitors enabled us to perform a conventional kinetic analysis which showed the mode of inhibition to be competitive. Prediction of the secondary structure of Domain 3 suggests that both the amino- and carboxyl-terminal conserved regions form alpha-helical structures, which are largely located in the interior of the calpastatin molecule, whereas the central region does not form alpha-helix or beta-structure. The central region contains a 12-residue consensus sequence common to Domains 1, 2, and 4, and this portion is predicted to be located on the surface of the calpastatin molecule. These results suggest that the central conserved region of each domain of calpastatin is an area for direct interaction either with the active center of calpain or a region in close proximity, and the rest of the domain is a region stabilizing the functionally important tertiary structure of the domain.

Amino Acid Sequence↗