Sterol conjugate interconversions during germination of white mustard (Sinapis alba)

Changes in the content of free sterols (FS), steryl esters (SE), steryl glucosides (SG) and acylated steryl glucosides (ASG) in germinating seeds of white mustard (Sinapis alba) were studied together with parrallel changes in specific activities of some enzymes involved in sterol conjugate transformation. It has been found that a distinct increase in the net SE content and a similar, but less pronounced, increase in SG content at the beginning of germination can be correlated with a distinctly earlier appearance of SE and SG synthesizing enzymes, i.e. triacylglycerol: sterol acyltransferase and UDPG: sterol glucosyltransferase in comparison with hydrolytic activities, i.e. SE hydrolase and SG hydrolase. Our results suggest that metabolism of SG and ASG takes place mainly in the cotyledons while SE metabolism takes place mainly in the roots.     

Steryl glucoside biosynthesis in the alga Prototheca zopfii

A particulate enzyme fraction from the Chlorophyta Prototheca zopfii catalysed the transfer of glucose-[U-¹⁴C]from UDP-Glc-[U-¹⁴C] to endogenous sterol acceptors and the esterification of steryl glucosides with fatty acids from an endogenous acyl donor. Glucose was the only sugar present, and it appeared to have the β-configuration. In the acylated derivatives the glucose-acyl linkage appeared in the C-6 position of glucose, as indicated by periodate oxidation. UDP-Glc:sterol glucosyltransferase was solubilized with detergent and purified 34-fold. The solubilized enzyme showed no specificity for the sterol but a high affinity for the sugar nucleotide UDP-Glc. Time-course incorporation into steryl glucoside (SG) and the acylderivative (ASG) indicated that SG was the precursor of ASG and that phosphatidyl ethanolamine stimulated the formation of the latter compound, presumably acting as acyl donor. A high sterol glucosylating activity was found in the Golgirich fraction. All this evidence indicates that steryl glucosides and their acylated derivatives were synthesized by algae. The early assumption that these compounds were not present in algae must be revised.     

The incorporation of mevalonate-[2-14C] into the sterol fractions of Calendula officinalis

The incorporation of mevalonate-[2-¹⁴C] into the free sterols, steryl esters, steryl glucosides, acylated steryl glucosides and water-soluble complexes was investigated and the sterols of each fraction were separated into stanols, Δ⁷ sterols, Δ⁵ sterols, stigmasterol, clerosterol and methylene-cholesterol. The stanols and Δ⁷ sterols were more strongly labelled in the steryl esters than in the free sterols. The Δ⁵ sterols and stigmasterol were more intensively labelled in the free sterols than in the steryl esters. All sterol types were more labelled in the steryl glycosides than in the acylated steryl glucosides. Stanols were probably formed from Δ⁷ or Δ⁵ precursors.     

Biosynthesis and metabolism of steryl glycosides in Sinapis alba seedlings

With ¹⁴CO₂, d-glucose-[U-¹⁴C] and dl-mevalonate-[4R-4-³H₁] used as precursors, a study was made of the labelling dynamics of the steryl glucosides (SG) and steryl acylglucosides (ASG) in Sinapis alba seedlings. The radioactivity of the sterol and sugar moieties, as well as of the fatty acid moieties in the case of ASG, was analysed separately. The course of incorporation of ¹⁴C from ¹⁴ CO₂ and glucose-[U-¹⁴C] into the sugar part of SG and ASG indicated that about $\text{2}\text{3}$ of the whole pool of the newly synthesized sterol glycosides of both types underwent rapid deglucosylation. Likewise, fatty acids in the ASG pool were rapidly exchanged. The present results point to a high metabolic activity of the sterol glycoside derivatives in plant cells.     

Free sterols,steryl esters,glucosides, acylated glucosides and water-soluble complexes in Calendula officinalis

In 3- and 14-day-old seedlings and in the leaves of Calendula officinalis the following sterols were identified: cholestanol, campestanol, stigmastanol, cholest-7-en-3-β-ol, 24-methylcholest-7-en-3β-ol, stigmast-7-en-3β-ol, cholesterol, campesterol, sitosterol, 24-methylcholesta-5,22-dien-3β-ol, 24-methylenecholesterol, stigmasterol and clerosterol. Sitosterol was predominant in young and stigmasterol in old tissues. Young tissues contained relatively more campesterol but in old tissues a C₂₈Δ^5,22 diene was present suggesting transformation of campesterol to its Δ^5,22 analog, similar to that of sitosterol to stigmasterol. All the identified sterols were present as free compounds and also in the steryl esters, glucosides, acylated glucosides and water-soluble complexes. The variations in the amounts of these fractions in the embryo axes and cotyledons of 3- and 14-day-old seedlings and the distribution of individual sterols among the fractions are discussed.     

Effects of ethephon and norbornadiene on sterol and glycoalkaloid biosynthesis in potato tuber discs

The accumulation of glycoalkaloids that normally takes place in aerobically incubated potato ( Solanum tuberosum L.) tuber discs has been found to be inhibited by the ethylene-releasing substance ethephon. Using ethephon and the ethylene action inhibitor norborna-2,5-diene, the effect of ethylene on the synthesis of sterols and glycoalkaloids, which partly share their biosynthetic pathway, was investigated.
Control discs showed incorporation of (2-¹⁴C)mevalonic acid into free sterols, steryl esters, steryl glycosides and acylated steryl glycosides at 24 h, thereafter the radioactivity decreased in free sterols and steryl esters concomitant with the appearance of radioactivity in glycoalkaloids. Discs with ethephon additions contained more radioactivity in all sterol classes at all time-points, but no glycoalkaloids were formed.
The enzyme S-adenosyl- l -methionine:sterol C24 methyltransferase (SMT, EC 2. 1. 1. 41), located at one presumed branching point in the sterol and glycoalkaloid pathway, was characterized and found to exhibit similar characteristics as in other plants, but a lower specific activity. The activity of SMT increased in ageing tuber discs and this increase was further stimulated by ethephon, but inhibited by norborna-2,5-diene. The activity of the glycoalkaloid-specific enzyme UDP-glucose:solanidine glucosyltransferase (EC 2. 4. 1) also increased after slicing, but here ethephon additions counteracted the induction. The activity of the sterol-specific UDP-glucose:sterol glucosyltransferase (EC 2. 4. 1) was unaffected by either tuber slicing or ethephon additions.
The results indicate that ethylene stimulates sterol synthesis in wounded potato discs, and that the wound-induction of SMT is regulated by ethylene.     

Acyl composition and biosynthesis of acylated steryl glucosides in Calendula officinalis.

Fatty acids C12-C22 are components of acylated steryl glucosides in Calendula officinalis. Various particulate fractions from 14-day-old seedlings catalyze the esterification of the steryl glucosides with utilization of endogenous acyl donors. The activity seems to be associated mainly with the membranous structures being fragments of Golgi complex, as it has previously been suggested for UDPG: sterol glucosyltransferase. Succesive treatment of the particulate enzyme fraction with Triton X-100 and acetone affords a soluble acyltransferase preparation partly depleted of endogenous lipids. As a source of acyl groups for the synthesis of steryl acylglucosides this preparation utilizes various phospholipids obtained from the same plant in the following sequence: phosphatidylinositol greater than phosphatidylethanolamine greater than phosphatidylcholine. It does not utilize triacylglycerols and monogalactosyldiacylglycerols.     

Occurrence of steryl glycosides and acylated steryl glycosides in some marine algae

There is some controversy concerning the presence of steryl glycosides and acylated steryl glycosides in eucaryotic algae. These two classes of sterol compounds were investigated in species belonging to the three major groups of eucaryotic algae: green algae (Ulva gigantea, Cladophora rupestris), brown algae (Fucus vesiculosus, Ascophyllum nodosum), and red algae (Rhodymenia palmata, Porphyridium sp.). All these algae contain both steryl glycosides and acylated steryl glycosides. The sterol components of these compounds vary according to the alga but they are always the same as the free sterols of the alga in question. The most common sugar moiety is glucose. In the acylated steryl glycosides, the fatty acid is mainly palmitic acid. The percentage of these compounds (as a percentage of the total sterol content) is often low.     

Occurrence of UDPG: Sterol glucosyltransferase activity in some lower plants

A study was made of the sterol glucosylating ability of cell-free homogenates obtained from 16 species of photosynthesizing and nonphotosynthesizing lower plants (2 species of Chlorophyceae, 2 species of Cyanophyceae, 1 species of Phycomycetes, 3 species of Ascomycetes, 3 species of Basidomycetes, 1 species of Myxomycetes, 3 species of Musci and 1 species of Sphenopsida). Except for the blue-green and green algae, all the remaining species showed distinct in vitro synthesis of steryl monoglucosides from UDPG and cholesterol or sitosterol. Preliminary studies on the specificity of the relevant enzymes pointed to a correlation between the sterol composition of the plant and the specificity of its glucosylating enzyme. The enzyme from Ascomycetes and Basidomycetes utilized ergosterol better than cholesterol or sitosterol. Enzymic preparations from mosses utilized sitosterol the best.     

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

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.     

UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli/

Steryl glucosides are characteristic lipids of plant membranes. The biosynthesis of these lipids is catalyzed by the membrane-bound UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173). The purified enzyme (Warnecke and Heinz, Plant Physiol 105 (1994): 1067–1073) has been used for the cloning of a corresponding cDNA from oat (Avena sativa L.). Amino acid sequences derived from the amino terminus of the purified protein and from peptides of a trypsin digestion were used to construct oligonucleotide primers for polymerase chain reaction experiments. Screening of oat and Arabidopsis cDNA libraries with amplified labeled DNA fragments resulted in the isolation of sterol glucosyltransferase-specific cDNAs with insert lengths of ca. 2.3 kb for both plants. These cDNAs encode polypeptides of 608 (oat) and 637 (Arabidopsis) amino acid residues with molecular masses of 66 kDa and 69 kDa, respectively. The first amino acid of the purified oat protein corresponds to the amino acid 133 of the deduced polypeptide. The absence of these N-terminal amino acids reduces the molecular mass to 52 kDa, which is similar to the apparent molecular mass of 56 kDa determined for the purified protein. Different fragments of these cDNAs were expressed in Escherichia coli. Enzyme assays with homogenates of the transformed cells exhibited sterol glucosyltransferase activity.     

Phytosterols in tobacco leaves at various stages of physiological maturity

Leaves of varying maturity from 84-day-old tobacco plants were harvested and analyzed for total sterol and their individual sterol components. The mature leaves had a significant higher sterol content than the immature leaves. Separation into free sterols, steryl esters, steryl glycosides, and acylated steryl glycosides showed that the free sterols accounted for most of the sterol increase, and stimgasterol was principally responsible for this increase.     

Sterols in relation to ageing of seeds of Helianthus annuus and Cicer arietinum

Under accelerated ageing at high relative humidity and high temperature for 4 days germination and membrane permeability remained unaffected both in sunflower and chick pea seeds. However, the steryl glycoside concentration in the pooled leachate increased progressively with ageing. Total sterols, as well as steryl glycosides and free sterols of the seeds, increased with a concomitant decline in steryl esters under accelerated ageing. Pretreatment with the sterol biosynthesis inhibitor SK & F 7997A₃ prevented the increase of total sterols under accelerated ageing conditions but there were increases in the amounts of steryl glycosides and free sterols and a decrease in steryl ester after such treatment, therefore, indicating interconversions of the various sterol types. Accelerated ageing also caused increases in free amino acids and soluble carbohydrate. Low relative humidity-high temperature and high relative humidity-low temperature failed to produce such effects.     

Structural analysis of novel bioactive acylated steryl glucosides in pre-germinated brown rice bran

Previous studies from our laboratory indicated that pre-germinated brown rice (PR) contained certain unknown bioactive lipids that activated two enzymes related to diabetes: Na+/K+ATPase and homocysteine-thiolactonase. In this paper, we report on the isolation and structural characterization of the activator lipids from PR bran as acylated steryl glucosides (ASGs). The activator lipid was isolated by silica gel column chromatography, and its chemical structure was determined by NMR, GC-MS, and tandem mass spectrometry. We demonstrated that the bioactive component consists of a mixture of acylated steryl beta-glucosides. Delta8-cholesterol and 2-hydroxyl stearic acid were identified as constituents of ASGs. The steryl glucosides (SGs) subsequent to alkaline hydrolysis lost this enzyme activator activity. Soybean-derived ASGs were not active. This activity may be quite peculiar to PR-derived ASGs. Our findings suggest that the molecular species of ASG may play an important contributing role in the anti-diabetic properties of a PR diet.     

Sterol glucosylation by the plasma membrane from cotyledons of Phaseolus aureus

Membrane fractions were isolated from dark grown cotyledons of Phaseolus auneus by differential and sucrose density gradient centrifugation. Endoplasmic reticulum-, Golgi apparatus- and plasma membrane-rich fractions were identified by their respective enzymic activities and tested for their ability to transfer glucose from UDP-glucose to endogenous sterols to form steryl glucosides. The glucosyltransferase activity was shown to be located mainly at the plasma membrane.ABBREVIATIONS SG steryl glucoside - ASG acylated steryl glucoside - UDP-glc Uridine diphosphoglucose     

Steryl glucoside and acyl glucoside biosynthesis in maturing pea seeds

Sterol glucosyltransferase activity was found in a particulate fraction of pea seeds. The activity was stimulated by Ca²⁺ and Mg²⁺ and inhibited by Zn²⁺, Cu²⁺, Hg²⁺, EDTA and EGTA. Iodoacetamide was without effect but p-chloromercuribenzoate completely inhibited the enzyme. N -Ethylmaleimide gave 60–70 % inhibition over a wide range of concentrations. The activity was stimulated by ATP in the presence of Mg²⁺. Under such conditions, steryl acyl glucoside was formed. The acyl derivative was barely detectable in the presence of Ca²⁺ either with or without ATP. Both oleyl CoA and palmityl CoA stimulated acyl glucoside synthesis. Of the four nucleoside triphosphates, ATP, GTP, UTP and CTP both ATP and CTP stimulated acylation in the presence of Mg²⁺. The observations suggest that acyl donors other than digalactosyl diglyceride and phospholipids may function in steryl acyl glucoside synthesis in plants.     

Sterols in hardening winter wheat

The main sterols in winter wheat crowns and roots were sitosterol and campesterol, with significant amounts of stigmasterol and traces of cholesterol. The main groups of sterol-containing lipids were free sterols, steryl glucosides, steryl esters and esterified steryl glucosides. Sterol analysis within each group showed little difference between them. Steryl esters were relatively rich in cholesterol and poor in stigmasterol. Free sterols were rich in stigmasterol. Low temperature caused an increase in sterol content but had little effect on sterol composition and sterol to lipid P ratio. There was some increase in steryl esters and some decrease in free sterols. Cholesterol and stigmasterol decreased in the steryl ester and free sterol fractions, respectively. There was little evidence for involvement of sterols in winter wheat frost hardening.     

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

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.     

Distribution of different forms of sterols in three cellular subfractions of Calendula officinalis leaves

From Calendula officinalis leaves three cellular subtractions (mitochondrial, Golgi membranes and microsomal) were obtained and enzymatically characterized. The contents of Δ⁰, Δ⁵, Δ⁷, Δ^{5, 22} sterols, as well as those of 24-methylenecholesterol and clerosterol, in the free and bound in the form of esters, glucosides and acylated glucosides were determined in these fractions. The results revealed the predominance of free sterols in the microsomal fraction, of esters in the mitochondrial fraction and of steryl glucosides and acylated glucosides in the Golgi fraction.     

UDP-Glucose: Sterol Glucosyltransferase and a Steryl Glucoside Acyltransferase Activity in Amyloplast Membranes from Potato Tubers

Envelope membranes were isolated from potato tuber amyloplastby a discontinuous sucrose density gradient and high speed centrifugation.These membranes catalyzed the transfer of [¹⁴C]glucose fromUDP-[¹⁴C]glucose to endogenous sterol acceptors and, in turn,catalyzed the esterification of steryl glucosides with fattyacids from an endogenous acyl donor. The synthesis of sterylglucosides was stimulated in the presence of Triton. X-100,while formation of acyl steryl glucosides was inhibited by thedetergent. However, in the presence of an added sterol acceptorand Triton X-100, the inhibition of acyl steryl glucoside synthesiswas overcome by the addition of phosphatidylethanolamine. Theenzyme involved in steryl glucoside formation was solubilizedby treatment of the envelope membranes with 0.3% Triton X-100.The solubilized enzyme had an almost absolute requirement forsterol acceptors. Key words: Solanum tuberosum, Sterol glucosylation, Steryl glucoside acylation, Amyloplast membrane