Masaomi Arahira

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 34269, 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-termininal 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.

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.

Increasing the conformational stability by replacement of heme axial ligand in c‐type cytochrome

To investigate the role of the heme axial ligand in the conformational stability of c‐type cytochrome, we constructed M58C and M58H mutants of the red alga Porphyra yezoensis cytochrome c 6 in which the sixth heme iron ligand (Met58) was replaced with Cys and His residues, respectively. The Gibbs free energy change for unfolding of the M58H mutant in water (ΔG°unf=1.48 kcal/mol) was lower than that of the wild‐type (2.43 kcal/mol), possibly due to the steric effects of the mutation on the apoprotein structure. On the other hand, the M58C mutant exhibited a ΔG°unf of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol compared with that of wild‐type. This increase was possibly responsible for the sixth heme axial bond of M58C mutant being more stable than that of wild‐type according to the heme‐bound denaturation curve. Based on these observations, we propose that the sixth heme axial ligand is an important key to determine the conformational stability of c‐type cytochromes, and the sixth Cys heme ligand will give stabilizing effects.

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 β-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.

Production and some properties of branched cyclo-malto-oligosaccharides

Abstract Cyclomalto-oligosaccharides (cyclodextrins, CDs) with maltosyl and panosyl branches were produced from maltose or panose and CDs by the reverse action of pullulanase. Purification on columns of octadesylated silica gave maltosylcyclo-malto-hexaose and -heptaose and panosylcyclomalto-hexaose and -heptaose. The solubility of maltosylcyclomaltoheptaose in aqueous 80% ethanol was higher than that of cyclomaltoheptaose in water. The relative rates of degradation of maltose, maltosylcyclomaltohexaose, and maltosylcyclomaltoheptaose with glucoamylase were 1:3.6:5.0, and those of panose, panosylcyclomaltohexaose, and panosylcyclomaltoheptaose were 1:3.0:2.2. The rates of degradation of branched and unbranched malto-oligosaccharides were markedly different, as were those of maltosyl- and panosyl-cyclomalto-oligosaccharides.

An Isoelectric Separation of Soybean β-Amylase Isoforms and Their Enzymic Characteristics

Authentic soybean β-amylase preparation, purified to homogeneity as judged by SDS-PAGE by using an affinity purification step, was composed of four pI-differing isoforms. By chromatofocusing, these isoforms were separated into three fractions, designated as fractions 1-3 in the order of elution. Fraction 1 contained two isoforms having the same molecular mass (55,989 Da), as measured by mass spectrometric analysis, with different pIs, 5.32 (Isoform I) and 5.22 (Isoform II). Fraction 2 showed a single isoform having a molecular mass of 55,994 Da and having a pI of 5.09. This component, named Isoform III, existed rather in excess in a mixture of the authentic enzyme isoforms. The remainder (fraction 3) also contained a single component (Isoform IV) which has a molecular mass of 56,310 Da with a pI of 4.97. Chemical analyses indicated that the N-termini and the C-terminal tripeptides of four pI-separated isoforms mentioned are similar to one another, and are blocked and are NH2-Val-Asp-Gly-COOH, respectively. Moreover, enzymic properties involving specific activity and the value of kcat/Km for the above three fractions are almost the same, and also agreed completely with those of an unfractionated authentic β-amylase preparation.