Shoji Fujii

Diacylglycerol acyltransferase in maturing safflower seeds: its influences on the fatty acid composition of triacylglycerol and on the rate of triacylglycerol synthesis

Diacylglycerol acyltransferase in a particulate preparation from maturing safflower seeds showed no strict selectivity for acyl-CoA when acyl-CoA substrates were administered as mixtures. This suggested that the fatty acid composition of position 3 in safflower triacyl-sn-glycerol exclusively depends on the acyl-CoA composition in the cell. The specific activity of the acylation was approximately 3 nmol/min per mg protein of the preparation under the optimum assay conditions. This low activity and other data appeared to indicate that the diacylglycerol acyltransferase reaction may be the rate-limiting step in triacylglycerol synthesis in vivo.

Three cDNA sequences coding for glutamine synthetase polypeptides in Oryza sativa L.

Atsushi Sakamoto i, Masahiro Ogawa 3, Takehiro Masumura l, Daisuke Shibata 1, Go Takeba 2, Kunisuke Tanaka 1 and Shoji Fujii 1 1Laboratory of Biochemistry, Faculty of Agriculture, Kyoto Prefectural University, Shimogamo, Kyoto 606, Japan; 2Laboratory of Applied Biology, Faculty of Living Science, Kyoto Prefectural University, Shimogamo, Kyoto 606, Japan; 3Laboratory of Food Production, The Research Institute for Food Science, Kyoto University, Ufi, Kyoto 611, Japan

cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds

Using a rice maturing seed pUC9 expression library, we isolated a cDNA clone corresponding to 10 kDa sulfurrich prolamin by immunoscreening. A longer cDNA clone was obtained from a λgtll library by plaque hybridization using this 32P-labeled cDNA as a probe. A polypeptide sequence composed of 134 amino acids was deduced from the nucleotide sequence. A 24 amino acid signal peptide was assigned by computer calculation for the membrane spanning region and Edman sequencing of the purified mature polypeptide. Remarkably, 20% of methionine and 10% of cysteine were found in the mature polypeptide as well as high contents of glutamine, and hydrophobic amino acids. Part of the amino acid sequence was homologous with a conserved cysteine-rich region found in other plant prolamins. Two repeats of amino acid sequence were found in the polypeptide.

Cloning and characterization of a cDNA encoding a rice 13 kDa prolamin

SummaryA cDNA library constructed from mRNAs obtained from developing rice endosperm was screened with a cDNA clone (λRM7) of highest frequency of occurrence (1.8%). The translati) product directed by the mRNA which was hybrid-released from λRM7 cDNA in a wheat germ cell-free system showed a molecular size of 13 kDa when coexisting with the protein body fraction of developing maize endosperm. A polypeptide sequence composed of 156 amino acids was deduced from the nucleotide sequence. By comparison with the 19 N-terminal amino acids obtained from Edman degradation of the isolated rice 13 kDa prolamin fraction, the signal sequence was determined as consisting of 19 amino acids. The deduced polypeptide is rich in hydrophobic amino acids such as Leu and Val, and also in Gln, but lacks Lys. Hence, the amino acid composition is consistent with that of rice 13 kDa rolamin. By homology with previously reported cereal prolamins, only a single octapeptide sequence, Gln-Gln-Gln-CysCys-Gln-Gln-Leu, which was observed in 15 kDa and 27 kDa zein, B- and γ-hordein, α/β- and γ-gliadin, and γ-secalin was conserved in the rice 10 kDa and 13 kDa prolamin. No repetitive sequences and/or sequences homologous to other cereal prolamins, except the above octapeptide, were observed for the mature 13 kDa prolamin polypeptide. The signal sequence region of the 13 kDa prolamin, however, shows homology of more than 65% in both the nucleotide sequence and the amino acid sequence with rice 10 kDa prolamin and maize zein.

Intracellular translocation of phosphatidate phosphatase in maturing safflower seeds: a possible mechanism of feedforward control of triacylglycerol synthesis by fatty acids.

Phosphatidate phosphatase activity was found both in the cytosol and in the microsomal membrane of maturing safflower seeds. The combined and relative activities of these two forms varied with seed maturation. During the period of rapid triacylglycerol accumulation in the cell, most of the phosphatidate phosphatase activity was membrane-bound; at the initial and last stages of seed development when triacylglycerol synthesis was at an insignificant level, the majority of the activity was soluble. The potassium salts of palmitic, stearic and oleic acids, which are the fatty acid products of proplastids, caused the translocation of the cytosolic phosphatidate phosphatase to the microsomal membrane, while laurate and linoleate, which are not products of proplastids, showed no effect. Oleoyl-CoA did not convert the soluble form of the enzyme into the membrane-bound form. The translocation induced by oleate was reversible. The cytosolic phosphatidate phosphatase of safflower seeds was not transferred to the microsomal membranes prepared from soybean, a plant species of Leguminosae, and from rapeseed, a species of Cruciferae, but was transferred to that from sunflower, which belongs to the same family as safflower, Compositae. These observations suggest that in maturing oil seeds the rate of fatty acid synthesis in proplastids may regulate the species-specific translocation of phosphatidate phosphatase between the cytosol and the endoplasmic reticulum membrane where triacylglycerol synthesis occurs and that in turn the translocation of this ambiquitous enzyme could control the rate of triacylglycerol synthesis in the cell.

Nucleotide sequence of a cDNA encoding a major rice glutelin.

Glutelin and prolamin are major storage proteins of rice endosperm. Glutelin is synthesized by endoplasmic reticulum-bound polysomes, and then transported and deposited into the vacuoles via Golgi apparatus [5]. It has been shown that preproglutelin (59 kDa) is processed to proglutelin (57 kDa) co-translationally, and proglutelin cleaves to form acidic subunits (37-39 kDa) and basic subunits (22-23 kDa) of mature glutelin polypeptides post-translationally [ 6]. Takaiwa et al. have reported the nucleotide sequences of a glutelin cDNA clone, pREE61 [2, 3], and a glutelin genomic DNA clone, 2INE3 [4]. We constructed a cDNA library for mRNA obtained from ripening rice endosperm, and screened cDNA clones that yielded high populations in the library. We also isolated cDNA clone, 2RG21 encoding a new group of glutelin polypeptide, which was identified by hybridization-released translation method. Figure 1 shows the nucleotide and the deduced amino acid sequence of 2RG21, a total length of 1 644 nucleotides. An open reading frame starts at position 27. The first ATG, AGCTATGGC, is very similar to the consensus sequence surrounding the ATG initiation codon for plants, AACAATGGC [ 1]. No ATG codon is present upstream of position 27, and a stop codon, TAA, is observed at position 9. Thus, the first ATG correspond to the initiation codon. The signal peptide sequence consists of 24 amino acids, and the acidic subunit starting at Gin, position 99-935, consists of 279 amino acids, with a M r of 32 037. The basic subunit starting at Gly, position 936-1526, consists of 197 amino acids, with a M r of 22168. These findings were obtained, by comparing the deduced amino acid sequence with those obtained from pREE61. The consensus polyadenylation signal, AATAAA, is located at position 1622-1627, and the polyadenylic acid chain starts at position 1633. Comparison of the sequence of a new type glutelin cDNA clone 2RG21 with that of a previously reported glutelin cDNA clone pREE61 revealed 67.6?o homology at the nucleotide sequence level and 65.8?/0 at the amino acid sequence level. Analysis of the population of ;tRG21 type clone in cDNA libraries for mRNAs obtained from early, middle and late stage of ripening rice endosperms revealed that 2RG21 and 2RG1 (a clone identical to pREE61) comprised 1.5~ and 0.9~ of total clones, respectively. Therefore, cDNA clone 2RG21 belongs to the major glutelin subfamilies, and differs from the previous reported group of pREE61 type.

2-Amino-2-deoxy-D-erythrose in Agaricus bisporus

2-Amino-2-deoxy-D-erythrose was isolated from the cell wall of the fruit body of Agaricus bisporus. The structure of the amino sugar was determined by mass spectrography and 1H-NMR spectrography of its acetylated derivative and by paper chromatographic comparisons with authentic 2-amino-2-deoxy-D-erythrose. This amino sugar is a component of the glycoprotein fraction from the cell wall. Its content in the glycoprotein increased markedly, especially during the ripening stage of the fruit body.

Rice Storage Proteins: Genetic Analysis of Accumulation Process

Plant seed storage protein is unique as it is expressed at a very specific stage and in very specific tissue. Many legumes usually express their storage proteins, 7S and 11S globulins, in the cotyledons. In contrast, cereal storage proteins which are prolamin, except rice (glutelin) and oat (12S globulin) are strongly expressed in the scutellar and/or endosperm cells. These storage proteins are localized mainly in proteinaceous organelles called the protein bodies to stabilize the protein reserves for the next generation (Pernollet 1978). These storage proteins are also important for animal and human food as an agronomically important material. In rice, the protein of the starchy endosperm consists of about 15% salt-soluble proteins (albumin and globulin), 5% ethanol-soluble proteins (prolamin), and 80% diluted alkali- or acid-soluble proteins (glutelin) (Juliano 1972). Most of these proteins accumulate in concentrated deposits, termed protein body (PB).

Induction of single strand scission in .PHI.X174RFI DNA by D-isoglucosamine.

The mechanism of the DNA breaking activity of 1-amino-1-deoxy-d-fructose (d-iso-glucosamine) in double-stranded replicative form I DNA (RFI DNA) of bacteriophage ϕX174 was investigated by agarose gel electrophoresis under various conditions. Cu2+ was indispensable for DNA strand scission. Induction of scission by d-isoglucosamine was proportional to incubation time and concentration, and was inhibited by the addition of chelating agents (EDTA, DETAPAC), superoxide dismutase (SOD), catalase, and some other free radical scavengers. These results suggested that DNA strad scission by D-isoglucosamine was caused by oxygen radicals generated during the autoxidation of d-isoglucosamine in the presence of Cu2+.