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  • Author or Editor: Yoshinori Kanayama x
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The properties of sucrose synthase (SS) isozymes partially purified from immature fruit (SS I) of Japanese pear (Pyrus serotina Rehder var. culta Rehder) were different than those of mature fruit (SS II). A clear difference in elusion pattern during DEAE-cellulose chromatography was observed, although the apparent molecular weight of the native proteins extracted from both stages was 350 kD. The Km value of SS II for UDP was similar to that for UDP-glucose; while with SS I, the Km for UDP was lower than that for UDP-glucose. This suggests that SS II activity favors sucrose synthesis compared with SS I, which favors sucrose cleavage. The optimum pH for activity toward sucrose synthesis was 8.0 for SS II and 8.5 to 9.5 for SS I. SS II from mature fruit may be an isozyme of SS occurring during periods of rapid sucrose accumulation, while SS I from immature fruit is more similar to the typical SS which functions mainly toward sucrose cleavage in many plants.

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Seasonal changes in the amounts of the NAD-dependent sorbitol dehydrogenase (NAD-SDH) (enzyme code, protein in developing apple (Malus pumila Mill var. domestica Schneid) fruit were determined by immunoblotting analysis. The amounts of the enzyme protein were very low in young fruit and rose as fruit matured. The weak correlation between enzyme protein and NAD-SDH activity and also the changes in NAD-SDH specific activity suggested that there could be posttranslational modification to the pre-existing enzyme or isoenzyme(s) of NAD-SDH. The changes in the amounts of NAD-SDH protein did not show the same pattern as those in relative growth rate, which is used to express sink activity, especially in young fruit. The role of NAD-SDH on sink activity in apple fruit, therefore, could not be explained simply by the amount and activity of the enzyme. In young fruit, it seems that enzymes other than NAD-SDH would be more directly related with fruit growth.

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Sorbitol plays a important role in the translocation of photosynthate in apple. Sorbitol-6-phosphate dehydrogenase (S6PDH, is the key enzyme regulating sorbitol biosynthesis. The cloning of functional gene like S6PDH provides the potential to elucidate the mechanism of production and translocation of sugar in the Rosaceae family and to manipulate endogenous sorbitol production in horticultural crops.

Poly(A)+RNA was prepared from apple seedlings and cDNA library constructed in an expression vector was screened by the loquat-S6PDH antibody prepared by Hirai (Natl. Res. Inst. Veg. Ornam. Plants & Tea, Japan). The cloned cDNA contained an open reading frame of 930 base pairs encoding a sequence of 310 amino acids. Identification of the cDNA was accomplished by expression of active enzyme in Escherichia coli harboring the cDNA and by the presence of a partial amino acid sequence identical to that found in the purified enzyme. Northern blot analysis showed the expression of S6PDH gene in apple seedlings.

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