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C Fukazawa

Publications and source records attributed to C Fukazawa.

At least 19 recordsLinked to original sources
PubMed1998

A class III acidic endochitinase is specifically expressed in the developing seeds of soybean (Glycine max [L.] Merr.).

A soybean chitinase which has an apparent molecular mass of 28 kDa by SDS-PAGE, and has chitinase specific activity of 133 units per mg protein at pH 5.2 and an apparent pI of 5.7, was purified from mature dry seeds. Based upon the selected part (the residue positions 10-17) of the determined N-terminal 38 amino acid sequence, a 23-mer degenerate oligonucleotide was synthesized and used for the PCR cloning of the chitinase cDNA. The resulting 1340 bp cDNA was comprised of a 5'-untranslated region of 39 bases, a coding region corresponding to a 25 amino acid signal sequence, followed by a mature 308 amino acid sequence (calculated molecular mass 34,269, calculated pI 4.7), and a 235 nucleotide 3'-terminal untranslated region including 24 bases of the poly(A) tail. By comparing the deduced primary sequence with those of plant chitinases known to date, this enzyme was more than 50% identical to every class III acidic chitinase, but has no significant similarity to other families of chitinases. The comparison also showed that the C-terminal region of this chitinase is markedly extended, by at least 31 residues. Northern blot analysis demonstrated that this mRNA species is remarkably transcribed from the early stage until the late middle stage of seed development, whilst it is hardly expressed in the leaves and the stems of soybean. Spatial and temporal expression of this single gene imply that this class III chitinase is mainly devoted to the seed defense, not only in development but also in dormancy of soybean seed. This is the first reported isolation and cDNA cloning of a class III acidic endochitinase from seeds. According to the chitinase nomenclature we propose that this enzyme would be classified into a new class of chitinase PR-8 family, together with a Sesbania homologue.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and expression patterns of Cu/Zn-superoxide dismutases in developing soybean seeds.

Assuming that the amount of superoxide radicals generated in vivo correlates with the production of ergastic substances such as storage proteins, the coordinated response of detoxication enzymes such as superoxide dismutases is largely exploited to understand the self-defense systems of plant. Here we examined expression of the genes for superoxide dismutases during seed development of soybean. The cDNAs encoding a cytosolic copper/zinc form and an iron form of the above enzyme have been cloned and then employed as probes, separately. Northern blotting results suggested that both superoxide dismutase mRNAs are expressed at the maximum level, preceding a developmental stage when mRNA encoding glycinin, soybean 11S-storage protein, at the maximum.

Amino Acid Sequence↗
PubMed1996

Functional analysis of Glu380 and Leu383 of soybean beta-amylase. A proposed action mechanism.

Soybean beta-amylase, comprising a (beta/alpha)8-barrel core with a mobile loop, similar to that of triose phosphate isomerase, was mutated by site-directed mutagenesis at residues Glu380 and Leu383. X-ray crystallographic findings suggest that Glu380 is the counterpart of the catalytic site (Glu186) and that Leu383, located near the active-site cavity, forms an inclusion complex with cyclomaltohexaose. Separate substitutions of Glu380 by Gln and Asp completely eliminated the activity without inducing any significant changes in the circular dichroic spectra nor in the binding affinity for cyclomaltohexaose. Glu380, in cooperation with Glu186, therefore, is clearly indispensable for the liberation of beta-maltose from starch. Substitutions of Leu383 by Ile and Gln, in contrast, led to remarkable increases in the Km values of both mutants when compared to that of the non-mutant enzyme. The mutants also showed marked reductions in their binding affinities to cyclomaltohexaose. Overall, it would appear that the kcat/Km of soybean beta-amylase increases in proportion to the length of the substrate molecule, and depends also on the characteristics of the side chain of the residue at position 383. Leu383, therefore, may be important for both substrate penetration and subsequent retention at the active site. Based on the foregoing, we propose an action mechanism of soybean beta-amylase involving the interactions of three essential amino acid residues (Asp101, Glu186 and Glu380) in concert with Leu383, and assumed an indispensable role for Asp101.

Amino Acid Sequence↗
PubMed1996

A rapid purification method for soybean Bowman-Birk protease inhibitor using hydrophobic-interaction chromatography.

A protein fraction was isolated from defatted soybean flour by extraction at acid pH, 40% ammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration chromatography. SDS-PAGE, under reducing conditions, confirmed it as a homogeneous preparation. This conclusion was consistent with N-terminal amino acid sequence data (20 cycles) which showed a major sequence identical to those reported for soybean Bowman-Birk-type protease inhibitor (BBI), and also indicated a minimum 95% purity based on recoveries of PTH-amino acid residues. The purified fraction inhibited both trypsin and chymotrypsin with average specific activities of 350 and 672 units mg(-1), respectively. Compared with classical BBI purification, this procedure is very rapid requiring only 72-96 h to achieve a yield of 37 mg purified BBI per 200 g starting material.

Amino Acid Sequence↗
PubMed1994

The glycinin box: a soybean embryo factor binding motif within the quantitative regulatory region of the 11S seed storage globulin promoter.

The soybean embryo factor binding sequence in the glycinin A2B1a gene promoter was delimited to an A/T-rich 9 bp sequence, 5'-TAATAATTT-3', designated as the glycinin box, by DNA footprinting and gel mobility shift assay using synthetic oligonucleotides. It was shown that the interaction with the factor takes place at a defined DNA sequence rather than at random A/T-rich sequence blocks in the glycinin 5' flanking region. There are four glycinin boxes in the quantitative regulatory region between positions -545 and -378 of the glycinin A2B1a promoter. Multiple nonamer motifs similar to the glycinin box were also found in the equivalent regions of other glycinin and legumin promoters, suggesting that they must be conserved as a binding site for the embryo factor that activates the differential and stage-specific expression of seed 11S globulin genes in leguminous plants.

Base Sequence↗
PubMed1994

Residues essential for catalytic activity of soybean beta-amylase.

To determine which amino acid residues are essential for the catalytic activity of soybean beta-amylase, deoxyoligonucleotide site-directed mutagenesis was employed against aspartyl, glutamyl, and cysteinyl residues located in highly conserved regions found in beta-amylase family to date. Both substitution of aspartic acid at position 101 and that of glutamic acid at position 186 of the enzyme by neutral and acidic amino acids, respectively, led to the complete elimination of activity, but did not induce any significant changes in circular dichroic spectra or the binding affinity for cyclomaltohexaose, a substrate analogue. Taking account of the results obtained here, the above two amino acid residues are involved in the catalytic site of soybean beta-amylase. The replacement of glutamic acid at position 345 decreased activity to below 6% of the non-mutant level, implying that this residue may also play a crucial role in beta-amylase activity, although it may not be involved at the catalytic site itself. In contrast, substitution of cysteinyl residue at position 95 by a serinyl residue led to a drastic reducing of the optimal temperature (from 50 degrees C to 30 degrees C), suggesting that this cysteinyl residue is responsible for the thermal stability of the enzyme.

Amino Acid Sequence↗
PubMed1994

Ginkgo 11S seed storage protein family mRNA: unusual Asn-Asn linkage as post-translational cleavage site.

By reducing the amount of ginkgo water-soluble polysaccharides, which occupy about 35% of the wet seed mass and interfere with the extraction of RNA, cDNA-quality mRNA was obtained from developing seeds of Ginkgo biloba. Based on the NH2-terminal 17-amino acid sequence and an internal 12-amino acid sequence derived from the basic subunit of ginnacin, 11S-seed storage protein family of ginkgo, two degenerate oligonucleotide primers were synthesized and used for polymerase chain reaction (PCR). The resulting PCR product was used for screening the above endosperm cDNA library, and a plaque carrying the 1614 bp cDNA insert, which contained the entire coding region for a precursor of ginnacin was isolated. This is the first reported cloning of cDNA from ginkgo seeds. The deduced primary sequence is composed of a signal peptide segment (25 amino acid residues) and an acidic subunit (248 residues) followed by a basic subunit (187 residues). It was also found that the post-translational cleavage site in the ginnacin precursor is the Asn-Asn rather than the Asn-Gly bond found in a variety of the major subunit precursors in 11S seed protein family known to date. We showed that a purified soybean extract and an extract of ginkgo seeds can specifically hydrolyze -Asn248-Asn249- but not -Asn249-Val250-, in the heptapeptide Gly-Asn248-Asn-Val-Glu-Glu-Leu that corresponds to the ginnacin cleavage region.

Amino Acid Sequence↗
PubMed1993

cDNA nucleotide sequence and primary structure of mouse uterine peptidylarginine deiminase. Detection of a 3'-untranslated nucleotide sequence common to the mRNA of transiently expressed genes and rapid turnover of this enzyme's mRNA in the estrous cycle.

Peptidylarginine deiminase is a protein-modulating enzyme which converts the arginine residues in proteins to citrulline residues. This study describes the complete primary structure of mouse peptidylarginine deiminase, which was deduced from nucleotide sequence analysis of cDNA clones plus proteochemical analysis of the purified enzyme. The composite cDNA sequence contained a 5' untranslated region of 7 bases, an open reading frame of 2019 bases that encoded 673 amino acids, a 3' untranslated region of 2662 bases, and part of a poly(A) tail. The N-terminal and C-terminal sequences of the enzyme matched the sequences deduced from nucleotide analysis. Furthermore, we determined that the N-terminal sequence was N alpha-acetyl-Met-Gln-, a sequence which has never previously been reported among N alpha-acetyl-Met proteins. The Arg 352 of the enzyme was converted to a citrulline residue and the potential Asn-linked glycosylation site (Asn542-Glu543-Ser544) had no carbohydrate moiety. Thus, mouse peptidylarginine deiminase consists of 673 amino acids with a molecular mass of 76,260. Mouse peptidylarginine deiminase mRNA has two AU-rich structures in the 3' untranslated region which exhibit a high degree of similarity to those in lymphokine, cytokine and proto-oncogene mRNA species. Since the rat enzyme (previously reported) does not possess these characteristic structures, we compared the levels of enzyme activity and mRNA in the mouse and rat uterus at four defined phases of the estrous cycle. The degradation of peptidylarginine deiminase and its mRNA proceeded significantly faster in the mouse than in the rat. We speculate that the unusual structure of the mouse enzyme and its mRNA be involved in this species-specific rapid degradation.

Amino Acid Sequence↗
PubMed1993

A high-order structure of plant storage proprotein allows its second conversion by an asparagine-specific cysteine protease, a novel proteolytic enzyme.

During seed embryogenesis, glycinin, the 11-S seed storage protein found in soybeans, undergoes post-translational proteolytic processing, in which a proprotein molecule is cleaved into an acidic and a basic subunit by a one-point cleavage that occurs at the carboxyl side of the asparaginyl residue located at the junction of the subunits. To elucidate the mechanism of this very limited proteolysis, we purified the cysteine endoprotease and used purified proglycinin produced by Escherichia coli as a substrate. This enzyme was separated by isoelectric focusing into three isomeric forms: two had a molecular mass of 33 kDa and the third, 33.8 kDa. The cysteine protease was found both in the proteinaceous vacuoles of cotyledonary tissue of immature seeds and in mature seeds, and is the first proteolytic enzyme to be classified as an asparagine-specific endoprotease. The results also indicate that the above proteolysis is largely attributable to the conformational accessibility of the enzyme to the asparaginyl residue in the cleavage site of proglycinin. The conformation of this single enzyme-accessible region on the proglycinin molecule is relatively flexible and becomes unstable under low salt conditions, or when heat is applied, causing the enzyme to lose its specificity.

Amino Acid Sequence↗
PubMed1993

Expression and mutation of soybean beta-amylase in Escherichia coli.

The cDNA clones corresponding to soybean beta-amylase mRNA were isolated and sequenced. The cDNA contained an open-reading frame composed of 496 amino acids. The comparison of the amino acid sequence deduced from the cDNA with the N-terminal peptide sequence from mature enzyme proved that beta-amylase had no leader sequence. Employing the cDNA, the beta-amylase was directly synthesized in Escherichia coli by the expression vector pKK233-2 controlled by the tac promoter. The enzyme activity detected in E. coli lysate drastically increased with a lower cultivation temperature, and the total activity and specific activity of the enzyme in E. coli lysate cultured at 13 degrees C was 130-fold and 280-fold, respectively, the value at 37 degrees C. The enzyme produced in E. coli was purified by the affinity column chromatography of cyclomaltohexaose-immobilized Sepharose 6B. Employing the established expression and purification system of the enzyme, the functional ionizable groups in the active site were searched. His93, involving an imidazole, and Asp348, involving a carboxylate, in the highly conserved regions within the beta-amylases were replaced by Arg (H93R) and Ash (D348N) by site-directed mutagenesis, respectively. All beta-amylases, including the non-mutant and mutant beta-amylases, produced in E. coli exhibited lower Vmax values than that of beta-amylase isolated conventionally from soybean seeds. Especially the Vmax value of [H93R]beta-amylase was reduced drastically compared to that of the non-mutant; however, none of them lost their enzyme activities completely. Therefore, neither His93 nor Asp348 may participate in the catalytic reaction directly.

Amino Acid Sequence↗
PubMed1993

Affinity purification of beta-amylases originating from plant using cyclomaltohexaose-immobilized Sepharose 6B in the presence of ammonium sulfate.

This paper reports a novel method of affinity purification of soybean and barley beta-amylase on cyclomaltohexaose-immobilized Sepharose. Until now, it has been shown that sweet potato beta-amylase can be purified using the above absorbent but beta-amylases from soybean and barley seeds cannot. We found that soybean and barley beta-amylase becomes adsorbed specifically on the above absorbent if it is in solution with 1 to 2 M ammonium sulfate, and the adsorbed enzyme can be easily eluted with a buffer containing no ammonium sulfate. Employing this procedure, soybean beta-amylase was demonstrated to be purified about 10-fold to homogeneity as judged from analysis of both a sodium dodecyl sulfate-gel electrophoresis and the specific activity, using a crude enzyme preparation (sp act 95 U/mg) as a starting material. The specific activity of this highly purified enzyme (950 U/mg) was almost the same as that of crystallized soybean beta-amylase at 37 degrees C.

Ammonium Sulfate↗
PubMed1993

cis-acting regulatory regions of the soybean seed storage 11S globulin gene and their interactions with seed embryo factors.

A 2.2 kb fragment containing the 5'-flanking region of the soybean glycinin A2B1a gene and its successive deletions with a shorter 5'-flanking sequence were fused, in frame, to the beta-glucuronidase (GUS) reporter gene. The resultant fusions were introduced into tobacco plants via Agrobacterium tumefaciens. Assays of the GUS activity in seeds of transgenic tobacco showed that the upstream region, -657 to -327 (relative to the transcription initiation site [+1]), of the glycinin gene is required for optimal expression of the transformed gene. Interactions between embryo nuclear factors and DNA fragments covering the downstream region of -326, in which are included the TATA box and legumin boxes, were not apparent. The embryo factors capable of binding specifically to three subregions, -653 to -527, -526 to -422, and -427 to -321, of the upstream regulatory region were detected. Such factors appeared to be organ-specific and could be found solely in developing seeds at the early middle stage of embryogenesis (around 24 days after flowering). Evidence obtained by characterizing the nature of the binding proteins and by gel mobility shift assays established that the same factor does interact with a consensus motif 5'-ATA/TATTTCN-/CTA-3' which occurs four times in the cis-acting regulatory region between -657 and -327. Moreover, this conserved motif could also be found in the 5' regulatory region of another glycinin A1aB1b gene. Thus it is likely that the observed interaction between the nuclear factor and the conserved motifs would lead to activation of transcription from the glycinin genes in maturing soybean seeds.

Base Sequence↗
PubMed1992

Overexpression of apolipoprotein E in transgenic mice: marked reduction in plasma lipoproteins except high density lipoprotein and resistance against diet-induced hypercholesterolemia.

Apolipoprotein E (apoE) has a high affinity to cell-surface low density lipoprotein (LDL) receptor. To determine the role of apoE in plasma lipoprotein metabolism, transgenic mouse lines with integrated rat apoE gene under the control of the metallothionein promoter were established. We found that a high expressor line produced rat apoE mainly in the liver, and the gene product was almost entirely associated with plasma lipoproteins. The plasma level of rat apoE in homozygotes for the transgene was 17.4 mg/dl after zinc induction (vs. 4.56 mg/dl of mouse apoE in controls). In this group, plasma cholesterol and triglyceride levels were 43% and 68% reduced as compared with controls, respectively. Heterozygotes showed decreases in both lipids to a lesser extent. Gel filtration chromatography showed that lipid reduction was mainly due to decreased very low density lipoproteins (VLDL) and LDL. Especially in zinc-treated homozygotes, VLDL had almost disappeared, and a remarkable decrease in LDL and a slight decrease in high density lipoprotein were also observed. Consistently, the plasma level of apoB, a structural protein of VLDL and LDL, was 78% lower than that of controls, indicating a marked reduction in lipoproteins containing apoB. Furthermore, the transgenic mice, in contrast to controls, did not develop hypercholesterolemia when fed a high cholesterol diet. These results demonstrated that overexpression of apoE reduces plasma cholesterol and triglyceride levels and prevents diet-induced hypercholesterolemia. From dramatic and dose-related decreases in plasma lipoproteins in transgenic mice, we conclude that apoE plays a key role in plasma lipoprotein metabolism.

Animals↗
PubMed1991

The effect of apo E secretion on lipoprotein uptake in transfected cells.

To investigate the role of apolipoprotein E (apo E) secreted by peripheral tissues in local lipoprotein metabolism, we developed a cell strain that constitutively produced and secreted apo E. A fusion plasmid containing rat apo E genomic DNA under control of mouse metallothionein promotor was constructed and transfected into Chinese hamster ovary cells. A stable transformant designated CHO-MAEII constitutively secreted rat apo E mainly in the form of sialylated free protein. The secretion was further enhanced by metal induction up to 1 micrograms apo E/ml per 12 h. When incubated with 125I-labeled very low density lipoprotein (125I-VLDL) at 37 degrees C, CHO-MAEII took up and degraded 125I-VLDL with higher affinity than control cells. Furthermore, considerable amount of methylated 125I-VLDL was degraded by CHO-MAEII, while no methylated 125I-VLDL was degraded by control cells. No significant differences were found in the uptake of 125I-LDL. The data indicated that apo E molecules secreted by CHO-MAEII were transferred to 125-VLDL particles, which caused a higher affinity of these particles for LDL receptors on the cells. It is suggested that apo E secreted from peripheral tissues enhances the uptake of lipoproteins by themselves or by surrounding cells in the local environment which demand cholesterol and express LDL receptors. CHO-MAEII was a good model for these 'auto- or paracrine-like functions' of apo E.

Animals↗