Characterization of dolichol and dolichyl phosphate phosphatase from soya beans (Glycine max)

Abstract

A series of polyprenols, ranging in length from 15 to 22 isoprene units, has been isolated from soya beans (Glycine max) and purified by high-pressure liquid chromatography. N.m.r., i.r. and mass spectra of the compounds indicated that they are alpha-saturated polyprenols of the dolichol type. The amount present in dry seeds was about 9 mg/100 g, whereas dolichyl phosphate (Dol-P) was present only in trace amounts. Dol-P phosphatase activity was detected in the microsomal fraction of 5-day-old germinating soya-bean cotyledons. The Dol-P phosphatase activity was linear with respect to time and protein concentration and exhibited a broad pH optimum (pH 7-9). Triton X-100 was necessary for significant enzyme activity. Enzyme activity was slightly enhanced by EDTA, whereas dithiothreitol was without effect. An apparent Km of 5 microM was determined for Dol-P. Bivalent metal ions were not required for enzyme activity. A number of phosphorylated compounds tested as enzyme substrates (including a number of nucleoside phosphates, glucose 6-phosphate, sodium beta-glycerophosphate and Na4P2O7) did not compete with [1-3H]Dol-P as substrate. A number of phospholipids were also tested for their ability to act as Dol-P phosphatase substrates. At 1 mM concentration, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and lysophosphatidic acid each inhibited enzymic activity. However, at 0.1 mM concentration, phosphatidylcholine and phosphatidylethanolamine were slightly stimulatory, whereas phosphatidic acid and lysophosphatidic acid were still inhibitory. Phosphatidic acid showed competitive inhibition.

Divergent pattern of polyisoprenoid alcohols in the tissues of Coluria geoides: A new electrospray lonization MS approach

“…A more detailed examination, together with progress in the development of a new chromatographic 2-D TLC technique (22), showed that this simplified polyprenol vs. dolichol dichotomy is, in fact, far more complicated. Dolichols were found as the predominant form in shoots (23) and seeds (24) of dicotyledonous plants. Small amounts of dolichols were also observed in young roots of Hevea brasiliensis.…”

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life

“…In various model plant systems, including Arabidopsis thaliana, Glycine max (soybean), and Coluria geoides , as well as common plants such as magnolia, spinach, parsley, and perilla, the length of most dolichol molecules falls within the range of 14-18 isoprene units [125-128]. Interestingly, dolichols found in soybean seeds contain one additional isoprene unit compared to those in the roots, shoots, and leaves, which typically have 16 – 17 isoprene units [127].…”

Biosynthesis of glycosylated dolichol intermediates and asparagine-linked glycoproteins during the germination of soybean seed embryos

“…followed according to Rip et al (1981) and Ravi et al (1983). The assay contained a final concentration of 100 mM Tris-maleate, pH 7.4, 0.05% (w/v) Triton X-100, 10 mM dithiothreitol, 10 mM EDTA, 10 pM [I-'~c]DO~-P (lo6 dpm/mL), and 75 pg/mL microsomal protein.…”