Nagao Ogura

Nucleotide Sequence of a cDNA for 1-Aminocyclopropane-1-Carboxylate Synthase from Melon Fruits

Ethylene is a plant hormone that has an essential role in fruit ripening (Yang and Hoffman, 1984; Kende, 1993). ACC synthase (S-adenosyl-L-methionine methylethioadenosine-lyase, EC 4.4.1.14), which is encoded by a multigene family, plays a regulatory role in ethylene production. Severa1 genes for ACC synthase have been isolated from tomato (Rottmann et al., 1991), mung bean (Botella et al., 1992, 19931, winter squash (Nakajima et al., 1990; Nakagawa et al., 1991), and Arabidopsis (Liang et al., 1992; Van Der Straeten et al., 1992). Two ACC synthase genes (LE-ACS2 and LE-ACS4, which are identified as a wounding and a ripening inducing gene, respectively) are expressed during ripening of tomato fruits (Olson et al., 1991; Rottmann et al., 1991). An antisense RNA experiment with LEACS2 reduced the levels of mRNAs for LEACS2 and LEACS4 in tomato fruits and caused retardation of initiation of ripening of tomato fruits (Oeller et al., 1991). These results showed that wound-induced ACC synthase also played an important role in the production of ethylene in tomato fruit during ripening. We isolated a cDNA (pMEACS1,2097 bp) for ACC synthase from wounded mesocarp tissue of melon fruits (Cucumis melo L. cv AMS) (Table I). The polypeptide derived from the cDNA in Escherichiu coli had ACC synthase activity. Sequence analysis of this cDNA revealed the presente of an open reading frame of 493 amino acids. This polypeptide contained seven sequences that were conserved among other ACC synthases. pMEACSl showed high homology at the amino acid and nucleotide levels to wound-induced ACC synthase from squash (Nakajima et al., 1990; Sato et al., 1991). RNA blot analysis showed that the level of mRNA for the gene increased in the mesocarp tissue of melon fruits after wounding and also during ripening. Since we could detect cDNA only for MEACSl ACC synthase in a PCR experiment with the mRNA from mesocarp tissue of ripe melon fruits, MEACSl should be the gene that is preferentially expressed during ripening of

Immunological properties of β-fructofuranosidase from ripening tomato fruit

Abstract The amount of tomato fruit β-fructofuranosidase extractable from the cell walls during ripening parallelled the changes in activity of the enzyme. Using the techniques of radioimmunoassay, double immunodiffusion analysis and immunotitration, no differences in immunological properties of β-fructofuranosidase between the various stages of fruit ripening were detected.

Inactive β-fructofuranosidase molecules in senescent tomato fruit

The present paper deals with the formation of altered molecules of β-fructofuranosidase (β-FFase, EC 3.2.1.26) in the cell wall fraction of tomato fruit in relation to aging. The monospecific antibody prepared from rabbits was used to characterize enzymes at ripened and senescent stages of tomato fruits. Although the activity on a fresh weight basis and the specific activity of the crude extract declined as the fruit aged, no difference was observed in the amount of the enzyme protein on a fr. wt basis between the two stages. With purified enzyme, there was little difference in such properties as Km, heat stability and optimum pH. However, the purified β-FFase from the senescent fruits had a lower specific activity. It is concluded from the results that the decline in the enzyme activity in the senescent fruits is due to the occurrence of immunologically active but catalytically inactive molecules of β-FFase.

A Nitrate Reductase-Inactivator in Spinach Cell Suspension Culture

Summary A nitrate reductase inactivator (NR-I) was detected in crude extracts from spinach ( Spinacia oleracea L. cv. Hoyo) cell suspension cultures. It inhibited not only NADH-NR activity, but also FMNH 2 -NR or MVH-NR activities. In contrast, xanthine oxidase, a flavoprotein with Fe and Mo components, was not affected. NR-I could not be dialyzed and when centrifuged at 105,000 × g for 90 min did not sediment as macromolecule. It was thermo-labile, and was particularly sensitive to metal chelators, EDTA and o-phenanthroline, and to trypsin. However, phenylmethyl sulfonyl fluoride, leupeptin, trypsin inhibitor, N-ethylmaleimide and iodoacet-amide were not inhibitory.

Cell Wall-bound Enzyme Properties of β-Fructofuranosidase Bound to Tomato Cell Wall

Tomato cell wall fraction washed three times with water was used as a preparation of insoluble β-fructofuranosidase (β-FFase). The insoluble β-FFase could not be released from cell wall fraction in the presence of substrate.Comparative studies on kinetics of soluble and insoluble forms of β-FFase were performed.There were no marked differences between the two forms with regard to pH dependency, Michaelis constant and some inhibitors.Considerable difference was observed between the two forms in both temperature sensitivity and dependency. The soluble form was no longer active at 55°C, but the insoluble form still retained 65% of the maximum activity at this temperature. There was a marked break in the Arrhenius plot at 19°C with the insoluble form, but a linear line was obtained at a temperature range of 10°C to 30°C with the soluble form. Activation energies were 10,800 and 17,300 cal/mole in the lower temperature range (below 19°C) for the insoluble and the soluble form, respectively. But they differed w...

Selective degradation of altered tomato β-fructofuranosidase molecules by neutral protease

Abstract Evidence is presented for the selective breakdown of altered tomato β-fructofuranosidase molecules by a neutral protease from Bacillus subtilis .

Reexamination of Methods for Determination of Chlorophyll in Pure Chlorophyll Solution and Mixture of Chlorophyll and Pheophytin.

調製クロロフィル及びフェオフィチンを用いて,クロロフィルの定量法及びクロロフィルとフェオフィチンの混合液中のクロロフィルの定量について再検討を行い,次の結果を得た.(1) 既に報告されている異なったクロロフィルの定量法7法についてクロロフィルを定量した結果,測定溶媒,測定波長が異なるにもかかわらず,それぞれの実験式で求めた値はほぼ一致した.(2) 既知量のクロロフィルとフェオフィチンの混合液についてVERNONの式を用い両色素の定量を行ったところ,クロロフィル量,フェオフィチン量ともに測定値は理論値より高く計算され,混合割合が高くなるに従い,その差は大きくなり,正しい色素量を求めることは困難であった.(3) 既知量のクロロフィルとフェオフィチンの混合液について,DIETRICHの方法に従ってクロロフィルのフェオフィチンへの変化率を求めたところ,両色素の混合率とよく一致した.よって混合液中のクロロフィルの定量は,混合液中の全フェオフィチン量を求め,クロロフィルに換算し,クロロフィルの変化率から求める方法が最も適していると思われた.

Studies on Pectic Enzymes

1. Alkali titration method is inquired and employed in the determination of the pectin-es-terase activity. 2. The PE is extracted from tomato pulp by Na2HPO4 at pH 8. It is more influenced by pH than extractant. 3. The PE is precipitated by (NH4)2SO4 at 11_??_70% saturated. The precipitated enzyme is stable at dry state. 4. Pectin solutin is decomposed by PE and produces acid, but this reaction is inhibited at pH 3.8. 5. The PE has opt. pH 6_??_7.5, and decomposes over 80% methoxyl groups in pectin. 6. The opt. temp. is 60°, and loses the activity at 80° 7. CaCl2 and (NH4)2SO4 accelerate the PE activity. 8. The enzyme does not decrasc the viscosity of pectin or pectic acid solution, and does not produce the reducing sugar. 9. The characters of the PE from tomato differs from the PE in mould or bacteria.