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Hairy roots of medicinal morning glory (Pharbitis nil) showed potent glucosylation activity against umbelliferone and aesculetin, so the glucosylation activity against several phenolic compounds was tested. Some coumarin derivatives and flavone derivatives having phenolic hydroxyl groups were incubated with the hairy roots. The coumarin derivatives and flavone derivatives almost disappeared from the culture medium in half a day. In the case of the coumarin derivatives, a 7-hydroxyl group was easily glucosylated. A methyl group at C-8 somewhat decreased the glucosylation to a hydroxyl group at C-7 of the coumarin skeleton. The 4-hydroxy coumarin derivatives were changed to acetophenone-type glucosides by incubation with the hairy roots through decarboxylation. Several flavonol derivatives were tested for glucosylation by the hairy roots. 3-Hydroxy flavone, 3.6-dihydroxyflavone and 3,7-dihydroxyflavone were glucosylated to give 3-glucosylated derivatives. Of these, 3,6-dihydroxyflavone was highly glucosylated, but not 3-hydroxyflavone or 3,7-dihydroxyflavone to the same degree. In the case of the flavones, a 3-hydroxy group could be predominantly glucosylated, and hydroxyl groups on the A and B ring of the flavones affected glucosylation by the hairy roots.

Hairy root cultures of Pharbitis nil treated with CuSO4 and methyl jasmonate (MeJA) produced umbelliferone (1) and scopoletin (2) in the culture medium, and skimmin (3), a beta-D-glucopyranoside of 1, was isolated from the hairy roots. While 1 in the medium increased and reached a maximal level 16 h after the treatment with CuSO4, the amount of 3 in the hairy roots decreased, reaching a minimal level after 8 h, before recovering to a level higher than the basal level after 24 h and then continuously increasing. These observations suggest that 1 was released by the hydrolysis of 3. Umbelliferone (1) inhibited hairy root growth, while skimmin (3) did not. This result suggests that, after the release of 1 as a phytoalexin, the hairy roots glycosylated 1 for the detoxification and re-use of 3 as a source of phytoalexin.

To clarify the role of L-ascorbic acid (AsA) in the formation of pyridinoline, we examined the effects of AsA in vitro using soluble collagen and partially purified lysyl oxidase from bovine aorta. The concentration of dehydrodihydroxylysinonorleucine decreased when AsA was added in the early stage of pyridinoline formation. However, when AsA was added in a later stage of pyridinoline formation, the concentration of pyridinoline was not affected. These findings indicated that AsA was involved in the initial enzymatic reaction in pyridinoline synthesis. We purified lysyl oxidase to confirm its association of AsA. AsA inhibited the enzyme activity. Erythorbic acid and 3,4-dihydroxybenzoate suppressed the enzyme activity as well as AsA did. The inhibition by AsA of the lysyl oxidase activity arose from characteristics of AsA structure. AsA might be important in the regulation of the oxidative reaction of lysine.

Vacuolar processing enzyme (VPE) is a cysteine proteinase responsible for the maturation of various vacuolar proteins in higher plants. To clarify the mechanism of maturation and activation of VPE, we expressed the precursors of Arabidopsis gamma VPE in insect cells. The cells accumulated a glycosylated proprotein precursor (pVPE) and an unglycosylated preproprotein precursor (ppVPE) which might be unfolded. The N-terminal sequence of pVPE revealed that ppVPE had a 22-amino-acid signal peptide to be removed co-translationally. Under acidic conditions, the 56-kDa pVPE was self-catalytically converted to a 43-kDa intermediate form (iVPE) and then to the 40-kDa mature form (mVPE). N-terminal sequencing of iVPE and mVPE showed that sequential removal of the C-terminal propeptide and N-terminal propeptide produced mVPE. Both iVPE and mVPE exhibited the activity, while pVPE exhibited no activity. These results imply that the removal of the C-terminal propeptide is essential for activating the enzyme. Further removal of the N-terminal propeptide from iVPE is not required to activate the enzyme. To demonstrate that the C-terminal propeptide functions as an inhibitor of VPE, we expressed the C-terminal propeptide and produced specific antibodies against it. We found that the C-terminal propeptide reduced the activity of VPE and that this inhibitory activity was suppressed by specific antibodies against it. Our findings suggest that the C-terminal propeptide functions as an auto-inhibitory domain that masks the catalytic site. Thus, the removal of the C-terminal propeptide of pVPE might expose the catalytic site of the enzyme.

Precursor-accumulating (PAC) vesicles were previously shown to mediate the transport of the precursor of a major storage protein (pro2S albumin) to protein-storage vacuoles in developing pumpkin cotyledons. In this study, we characterized two homologous proteins from PAC vesicles, a 72 kDa protein (PV72) and an 82 kDa protein (PV82). PV72 and PV82 showed an ability to bind to peptides derived from both an internal propeptide and a C-terminal peptide of pro2S albumin. PV72 was predicted to be a type I integral membrane protein with epidermal growth factor (EGF)-like motifs. These results suggest that PV72 and PV82 are potential sorting receptors for 2S albumin to protein-storage vacuoles.

The hypoglycemic effects of Lagerstroemia speciosa L., known by the Tagalog name of banaba in the Phillipines, were studied using hereditary diabetic mice (Type II, KK-AY/Ta Jcl). The mice were fed a test diet containing 5% of the hot-water extract (HWE) from banaba leaves, 3% of the water eluent of the partial fraction unadsorbed onto HP-20 resin of HWE (HPWE), and 2% of the methanol eluent of the partial fraction adsorbed onto HP-20 resin of it (HPME) for a feeding period of 5 weeks. The elevation of blood plasma glucose level in non-insulin dependent diabetic mice fed the cellulose as control (CEL) diet were almost entirely suppressed by addition of either HWE or HPME in place of cellulose in the CEL diet. Water intakes were inclined to increase gradually in the group fed either CEL or HPWE, but lower in the mice fed either HWE or HPME than in the animals given either CEL or HPME. The level of serum insulin and the amount of urinary excreted glucose were also lowered in mice fed HWE. Plasma total cholesterol level was also lowered in mice fed the either HWE or HPME. It is suggested that HWE, especially HPME, obtained from banaba leaves have beneficial effects on control of the level of plasma glucose in non-insulin dependent diabetes mellitus.

Spectra of 125 MHz 13C-nuclear magnetic resonance (NMR) of glycosphingolipids, GlcCer, GalCer, sulfatide, LacCer, and nLc4Cer have been studied, and the following results were obtained. (i) Signals of ring carbons of each sugar component are distributed in a wide field (50-110 ppm) and clearly separated. (ii) Chemical shifts of anomeric carbon (C1) and methylene carbon (C6) of sugars are far from those of other methine carbons of sugars and characteristic of sugar components, which makes it possible to identify each sugar component and its molar raito. (iii) The downfield shifts (about 6-9 ppm) of alpha-carbon signals involved in the glycosidic linkages and upfield shifts (about 1.5-2 ppm) of the neighboring beta-carbons, which are known as glycosylation shifts, could be observed. (iv) Characteristic shifts of aglycon signals caused by the presence of an OH group at the alpha-position of fatty acid were assigned. These observations are useful for the characterization of glycosphingolipid structures.