Zdzisław A. Wojciechowski

The structure of triterpenic glycosides from the flowers of Calendula officinalis L.

Abstract From the flowers of Calendula officinalis L., five glycosides of oleanolic acid were isolated and their structures determined as: 3-glucuronide; 3-(galactosyl-glucuronide) ; 3-(galactosyl-glucuronide); 17-glucoside; 3-(galactosyl-(glucosyl)-glucuronide); and 3-(galactosyl-(glucosyl)-glucuronide); 17-glucoside.

Specificity of sterol-glucosylating enzymes from Sinapis alba and Physarum polycephalum

1. Sinapis alba L. seedlings contain glycosyltransferase catalyzing the synthesis of sterol glucosides in the presence of UDPglucose as sugar donor. The major activity occurs in the membranous fraction sedimenting at 300--9000 x g. Successive treatment of the particulate enzyme fraction with acetone and Triton X-100 affords a soluble glucosyltransferase preparation which can be partly purified by gel filtration on Sephadex G-150. Molecular weight of the glucosyltransferase is 1.4 . 10(5). Apparent Km values for UDPglucose and sitosterol are 8.0 . 10(-5) M and 5.0 . 10(-6) M, respectively. 2. Comparison was made of the S. alba glucosyltransferase with a similar sterol-glucosylating enzyme isolated from non-photosynthesizing organism Physarum polycephalum (Myxomycetes). UDPglucose was the most efficient glucose donor in both cases but the enzyme from Ph. polycephalum can also utilize CDPglucose and TDPglucose. Glucose acceptors are, in case of both enzymes, sterols containing a beta-OH group at C-3 and a planar ring system (5 alpha-H or double bond at C-5). The number and position of double bonds in the ring system and in the side chain, as well as the presence of additional alkyl groups in the side chain at C-24 are of secondary importance. 3. The present results indicate that both enzymes can be regarded as specific UDPglucose:sterol glucosyltransferases. Certain differences in their specificity towards donors and acceptors of the glucosyl moiety suggest, however, a different structure of the active sites in both enzymes.

The Formation of Sugar Chains in Triterpenoid Saponins and Glycoalkaloids

Triterpenoid saponins and structurally related steroidal glycoalkaloids are a large and diverse family of plant glycosides. The importance of these compounds for chemical protection of plants against microbial pathogens and/or herbivores is now well-documented. Moreover, these compounds have a variety of commercial applications, e.g. as drugs or raw materials for pharmaceutical industry. Until recently there were only sparse data on the biosynthesis of saponins and glycoalkaloids, especially at the enzyme level. Substantial progress has recently been made, however, in our understanding of biosynthetic routes leading to the formation of the diverse array of aglycone skeletons found in these compounds as well as mechanisms of synthesis of their sugar moieties. This review highlights some of the advances made over past two decades in our understanding of the formation and modification of sugar moieties in triterpenoid saponins and glycoalkaloids.

Biosynthesis of oleanolic acid glycosides by subcellular fractions of Calendula officinalis seedlings

Abstract The synthesis of oleanolic acid 3β- d -glucuronoside from oleanolic acid and UDPGlcA has been demonstrated in cell-free preparations from C. officinalis seedlings. Moreover, the formation of more complex glycosides by successive additions of galactose and glucose to oleanolic acid glucuronoside was observed when cell-free preparations were incubated with UDPGal or UDPGlc. The consecutive steps of oleanolic acid glycosylation are localized in three different cellular compartments. The biosynthesis of the 3-glucuronoside takes place in the microsomes, the elongation of the sugar chain at C-3 of the aglycone proceeds in heavy membrane structures which are probably fragments of the Golgi complex while a cytosol enzyme(s) is involved in glucosylation of the C-17 carboxyl group of oleanolic acid.

Free and bound sterols in seedlings of Cucumis sativus

Abstract Free sterols, sterol esters, sterol monoglucosides and sterol acylmonoglucosides have been obtained from 10 days old seedlings of Cucumis sativus . Free sterols and sterol esters consist mainly of Δ 7 di- and triunsaturated sterols, whereas Δ 7 mono-unsaturated and Δ 5 mono- and diunsaturated sterols predominate in the glucosides and acylglucosides. Both acetates and derivatives of higher fatty acids, mainly linoleic and linolenic acids, have been found in the sterol esters. Sterol acylglucosides contain mostly saturated fatty acids, palmitic and stearic acids being the main components.

Purification and some properties of steryl β-d-glucoside hydrolase from Sinapis alba seedlings

Abstract Homogenates of 7-day-old S. alba seedlings hydrolysed cholesteryl[4-H14C] β- d -glucoside or sitosteryl β- d -glucoside-[6-3H]. Activity was located predominantly in the cell membrane structures sedimenting at 1000–15 000 g and was solubilized by acetone treatment. Partially purified enzyme preparation, with an about 1500 times higher specific activity with respect to the crude homogenate, was obtained by repeated acetone precipitation and subsequent chromatography on DEAE-Sephadex and Sephadex G-100. During this procedure a considerable separation from other enzymes with β-glucosidase activity was achieved. The enzyme had MW 65 000 daltons, pH optimum at 5.2–5.6. Two observations suggested that the enzyme was a specific steryl β- d -glucoside hydrolase. Firstly, there was no substrate competition between steryl glucosides and several other β- d -glucosides. Secondly, enzyme activity wasstrongly inhibited by low concentrations of various 3β-OH sterols with a planar ring system and an intact side chain.

The structure of glycosides of oleanolic acid isolated from the roots of Calendula officinalis

Abstract A new series of glycosides of oleanolic acid was discovered in the roots of old Calendula officinalis plants. These compounds derivatives of 3-glucoside of oleanolic acid are different, from those previously isolated from the flowers of this plant, derivatives of 3-glucuronoside of oleanolic acid. The sugar components of 8 representatives of the new series are glucose and galactose in following ratios: in glucoside I, 1:0; in II, 1:1; in III, 1:2; in IV, 2:1; in V, 3:1; in VI, 3:2; in VII, 4:1; in VIII, 4:1. The sugars in I–VII were attached only in position 3 of oleanolic acid, but in VIII one glucose molecule was joined to 28-carboxyl of oleanolic acid. I was identified as 3-monoglucoside and II as 3-(4′-galactosyl)-glucoside of oleanolic acid. Besides these compounds, 6′-methyl ester of 3-glucuronoside of oleanolic acid was found in Calendula roots.

Subcellular localization of udpg: Nuatigenin glucosyltransferase in oat leaves

Abstract Cell-free enzyme preparations from oat leaves effectively catalyse the conversion of both phytosterols and nuatigenin (a furostanol sapogenin) to the corresponding 3β- d -glucosides, with UDPG acting as a sugar donor. Subcellular fractionation has shown that UDPG: sterol glucosyltransferase activity is present almost exclusively in the membranous fraction (105 000 g pellet) while a large part ( ca 70 %) of UDPG : nuatigenin glucosyltransferase activity occurs in the cytosol (105 000 g supernatant). The results obtained indicate clearly that oat leaves contain at least two UDPG-dependent glucosyltransferases catalysing glucosylation of 3β-hydroxysteroids which are localized in different cell compartments and exhibit different specifirity patterns.

Modulation of activities of steryl glucoside hydrolase and UDPG: Sterol glucosyltransferase from Sinapis alba by detergents and lipids

Abstract Interactions of detergents and lipid compounds on the activity of delipidated preparations of UDPG: sterol glucosyltransferase and steryl β- d -glucoside hydrolase (SG hydrolase) isolated from white mustard seedlings were studied. It has been found that various lipids exert diverse effects on the activity of SG hydrolase. This activity was distinctly stimulated by several neutral, relatively unpolar compounds such as phytol, tripalmitoylglycerol, methyl stearate or cholesteryl acetate and, to a lesser extent, by free fatty acids. On the other hand a number of phospho- and glycolipids were inhibitory. A particularly strong inhibition was observed with charged, zwitterionic phospholipids such as PC, PE or their 2-lyso derivatives. These results point to the possibility of in vivo regulation of the membrane-bound SG hydrolase by its lipid microenvironment. In contrast to SG hydrolase no evidence was found for a clear-cut effect of lipids on the activity of UDPG: sterol glucosyltransferase even after a pretreatment of the enzyme preparation with phospholipase C.

Acyl donors for sterol esterification by cell-free preparations from Sinapis alba roots

Abstract Homogenates or crude 300-16 000 g membrane fractions from Sinapis alba roots catalysed esterification of [4- 14 C]cholesterol with utilization of endogenous acyl sources. With acetone powder preparations cholesterol esterification was distinctly stimulated by a neutral lipid fraction isolated from S. alba roots. Among neutral lipids triacylglycerols were the most active in this process. Experiments with various acyl-labelled acylglycerols as acyl donors and non-labelled sterols as acceptors confirmed that triacylglycerols are directly utilized as the source of fatty acids for sterol esterification. Di- and mono-acylglycerols were much less effective.