Acylation of steryl glucosides by phospholipids. Solubilization and properties of the acyl transferase.

Particulate enzyme preparations of cotton fibers catalyze the acylation of exogenous steryl glucoside to form acylated steryl glucoside. The acyl transferase involved in this reaction was solubilized by treatment of the membrane fractions with Triton X-100 and was partially purified by chromatography on DEAE-cellulose and gel filtration. This solubilized enzyme had an absolute requirement for Triton X-100 and phospholipid in order to catalyze the acylation of the steryl glucoside. The best phospholipid substrate was phosphatidylethanolamine but egg and soybean phosphatidylcholine were also active. The phospholipid was shown to function as an acyl donor by demonstrating that [¹⁴C]fatty acid from ¹⁴C-labeled phospholipid could be transferred to steryl-[³H]glucoside to form [¹⁴C,³H]acylated steryl glucoside. Saponification of this compound yielded [¹⁴C]fatty acid and steryl-[⁷H]glucoside.     

Acylated Steryl Glycoside Synthesis in Seedlings of Nicotiana tabacum L

In tobacco seedlings (Nicotiana tabacum L.), glucose from supplied uridine diphosphate-[U-¹⁴C]glucose was first incorporated into steryl glycosides and later into acylated steryl glycosides. However, when [¹⁴C]cholesterol was used as substrate, the acylated steryl glycosides became labeled earlier than the steryl glycosides. With [¹⁴C]cholesteryl glucoside as substrate, most of the radioactive label was recovered as free sterol, and the acylated steryl glycosides were not readily labeled; however, palmitoyl [¹⁴C]cholesteryl glucoside was rapidly converted to steryl glycoside. In feeding experiments with free sterol, an unknown, highly radioactive steroid component was isolated. Incorporation of radioactivity into the unknown occurred before the acylated steryl glycosides were labeled.     

Metabolism of conjugated sterols in eggplant. Part 1. UDP-glucose : sterol glucosyltransferase

A membrane-bound UDP-glucose : sterol glucosyltransferase from Solanum melongena (eggplant) leaves was partially purified and its specificity as well as molecular and kinetic properties were defined. Among a wide spectrum of 3-OH steroids (i.e. typical plant sterols, androstane, pregnane and cholestane derivatives, steroidal alkaloids and sapogenins) and triterpenic alcohols, the highest activity was found with 22-oxycholesterol. UDP-glucose appeared to be the best sugar donor. The enzyme preparation was also able to utilize UDP-galactose, TDP-glucose and CDP-glucose as a sugar source for sterol glucosylation, however, at distinctly lower rates. The investigated glucosyltrasferase was stimulated by 2-mercaptoethanol, Triton X-100 and negatively charged phospholipids, and inhibited in the presence of UDP, mono-, di- and triacylglycerols, divalent cations such as Zn(2+), Co(2+), high ionic strength, cholesteryl glucoside, galactoside and xyloside and some amino acid-modifying reagents (SITS, DIDS, PLP, DEPC, pCMBS, NEM, WRK and HNB). Our results suggest that unmodified residues of lysine, tryptophan, cysteine, histidine and dicarboxylic amino acids are essential for full enzymatic activity and indicate that a glutamic (or aspartic) acid residue is necessary for the binding of sugar donor, i.e. UDP-glucose in the active site of the GT-ase while histidine and cysteine residues are both important for the binding of the nucleotide-sugar as well as of the steroidal aglycone.     

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.     

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.     

Uptake and acylation of 2-acyl-lysophospholipids by Escherichia coli.

The efficiency of extracellular 2-acyl-lysophospholipid incorporation into Escherichia coli membranes and the acyl donor utilized to acylate the 2-acyl-lysophospholipid was determined. Exogenous 2-acyl-lysophospholipids were acylated via the acyl-acyl carrier protein synthetase/2-acylglycerophosphoethanolamine acyltransferase pathway. The maximum extent of 2-acyl-lysophospholipid incorporation into the membrane was approximately 2.5% of the normal phospholipid biosynthetic rate.     

Activation of phosphatidylcholine-sterol acyltransferase by human apolipoprotein E isoforms

The reaction catalysed by phosphatidylcholine-sterol acyltransferase (EC 2.3.1.43) is believed to be the major source of cholesteryl ester in human plasma; the enzyme requires a protein activator. Several human apolipoproteins were found to exhibit an activator function, the major one being apolipoprotein A-I. Human apolipoprotein E exists in the population mainly in three different genetic isoforms; apolipoprotein E-2, E-3 and E-4. These isopeptides were isolated from subjects homozygous for one of the isoforms, incorporated into phospholipid/cholesterol/[14C]cholesterol complexes by the cholate dialysis procedure and used to measure capacity to activate phosphatidylcholine-sterol acyltransferase in comparison to apolipoprotein A-I lipid substrate particles prepared by the same procedure. Acyltransferase activity was measured by the formation of [14C]cholesteryl ester from [14C]cholesterol using purified enzyme. With egg yolk phosphatidylcholine as acyl donor, apo E was 15-19% as efficient as apolipoprotein A-I for activation of the acyltransferase. Apo-E-stimulated cholesteryl ester formation by the enzyme was enhanced when 1-oleoyl-2-palmitoyl-glycerophosphocholine was used as a substrate phospholipid (45% of apo A-I/phosphatidylcholine control) and most pronounced with dimyristoylglycerophosphocholine (75% of apo A-I/phosphatidylcholine control). No significant difference in activation was found between apo E isoforms. It is concluded that apolipoprotein E activates phosphatidylcholine-sterol acyltransferase in vitro and that apolipoprotein E isoforms are similarly effective.     

The Biosynthesis of Steryl Glucosides in Plants

Mitochondrial preparations from pea root (Pisum sativum L. var. Alaska) cauliflower inflorescence (Brassica cauliflora Gars.) and avocado inner mesocarp (Persea americana Mill. var. Fuerte), and chloroplast preparations from spinach leaf (Spinacia oleracea L. var. Bloomsdale) incorporate glucose into steryl glucoside and acylated steryl glucoside when either uridine diphosphate-glucose or uridine diphosphate-galactose is supplied as precursor. In the case of pea root mitochondria, galactosyl diglycerides are not formed from either nucleotide sugar. In the case of spinach chloroplasts only 3% of the metabolized uridine diphosphate-galactose is found as steryl glycosides. Time course experiments indicate that the steryl glucoside is the precursor of the acylated steryl glucoside. The effect of pH on the over-all reaction and analysis of the reaction products suggest that the glucosylation of the sterol has a pH optimum of 8 to 9, and the pH optimum for the acylation of the steryl glucoside is 6.5 to 7. The synthesis of steryl glucoside and acylated steryl glucoside, catalyzed by acetone powders of pea root mitochondria, is stimuated by added sitosterol and stigmasterol.     

Separation of Soybean Sterols by Florisil Chromatography and Characterization of Acylated Steryl Glucoside

Florisil column chromatography was demonstrated to be effective in differentiation between different forms of sterols. Sterols of ground soybeans are in four forms, free, ester, and free and acylated glucosides, as analyzed on acetone extracts. In soybean oil foots, steryl ester is present in negligibly small amount. The acylated steryl glucosides were isolated from oil foots in a crystalline state. A chemical structure, steryl 6-acyl d-glucoside, was assigned to the compound, and its probable identity with the glucosides reported by Lepage is discussed. The acylated glucoside preparation was shown to be heterogeneous in composition, carrying palmitic, stearic, oleic, linoleic and linolenic acids as the main acyl moieties and campesterol, stigmasterol and β-sitosterol as steryl moieties. The presence of the three sterols is common to three other forms of sterols.     

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     

Steryl glucoside and acyl steryl glucoside formation in the amyloplast membrane during the development of potato tuber

Steryl glucoside (SG) and acylated steryl glucoside (ASG) synthesis was investigated in amyloplast membranes from young, intermediate and mature potato tubers. The synthesis ratio SG/ASG was lowest in young tubers (3:2) and highest in mature tubers (6:1).About a 3-fold stimulation of [¹⁴C] glucose incorporation into a mixture of SG was observed in amyloplast membranes from mature tubers in the presence of β-sitosterol, while radioactivity incorporation in young tubers was unaffected, thus indicating that different availabilities of endogenous acceptors occur in the membranes.The enzymes involved in sterol modification exhibit a different behavior towards Triton X-100, depending on the developmental stage of the tubers. Low concentrations of the detergent (0.045%) are required to stimulate the enzymes present in young tuber membranes (2-fold). On the other,hand, 0.15% of Triton increased the enzymatic activity in mature tubers 5-fold. These results, together with those obtained after studies of pH dependence, could be related to the lipidic structure of the vesicles formed at different developmental stages of the tubers.It is concluded that the major changes in the enzymatic activities occur as a consequence of the sterol acceptors and acyl donor content during potato tuber growth.     

Turnover of fatty acids in the 1-position of phosphatidylethanolamine in Escherichia coli

Phosphatidylethanolamine is the major membrane phospholipid of Escherichia coli, and two experimental approaches were used to investigate the metabolic activity of the fatty acids occupying the 1-position of this phospholipid. [3H]Acetate pulse-chase experiments with logarithmically growing cells indicated that 3-5% of the acyl groups were removed from the phosphatidylethanolamine pool/generation. The reacylation aspect of the turnover cycle was demonstrated by the incorporation of fatty acids into the 1-position of pre-existing phosphatidylethanolamine when de novo phospholipid biosynthesis was inhibited using the plsB acyltransferase mutant. 2- Acylglycerophosphoethanolamine would be the intermediate in a 1-position turnover cycle, and this lysophospholipid was identified as a membrane component that could re-esterified by a membrane-bound acyltransferase. The acyltransferase either utilized acyl-acyl carrier protein directly as an acyl donor or activated fatty acids for acyl transfer in the presence of ATP and Mg2+. Acyl-acyl carrier protein was also indicated as an intermediate in the latter reacylation reaction by the complete inhibition of phosphatidylethanolamine formation from fatty acids by acyl carrier protein-specific antibodies and by the observation that the inhibition of the acyltransferase by LiCl was reversed by the addition of acyl carrier protein. Coenzyme A thioesters were not substrates for this acyltransferase. These results suggest the existence of a metabolic cycle for the utilization of 1-position acyl moieties of phosphatidylethanolamine followed by the resynthesis of this membrane phospholipid from 2- acylglycerophosphoethanolamine by an acyl carrier protein-dependent 1-position acyltransferase.     

Cholesteryl glucoside in Candida bogoriensis

Extraction of lipids with CHCl3/CH3OH/1 M HCl from fresh and frozen cells of Candida bogoriensis showed the presence of a steryl glucoside. The product was purified and identified as a beta-cholesteryl glucoside. It hydrolyzes in methanolic HCl and therefore it is a steryl glucoside. The quantitation of the extracted cholesteryl glucoside was pursued with high-pressure liquid chromatography. The chemical ionization mass spectra of the isolated and the synthesized beta-cholesteryl glucosides were compared and found identical. The cell wall of Candida bogoriensis was weakened with enzyme (glusulase) and after homogenisation and sucrose density gradient centrifugation, five layers separated. Only the pellets of one layer showed the presence of cholesteryl glucoside in thin-layer chromatography after extraction with solvents. In the same layer, the activity of sterol glucosyl transferase was found.     

Activation of lecithin: cholesterol acyltransferase by human apolipoprotein A-IV

Human plasma apoproteins (apo) A-I and A-IV both activate the enzyme lecithin:cholesterol acyltransferase (EC 2.3.1.43). Lecithin:cholesterol acyltransferase activity was measured by the conversion of [4-14C] cholesterol to [4-14C]cholesteryl ester using artificial phospholipid/cholesterol/[4-14C]cholesterol/apoprotein substrates. The substrate was prepared by the addition of apoprotein to a sonicated aqueous dispersion of phospholipid/cholesterol/[4-14C]cholesterol. The activation of lecithin:cholesterol acyltransferase by apo-A-I and -A-IV differed, depending upon the nature of the hydrocarbon chains of the sn-L-alpha-phosphatidylcholine acyl donor. Apo-A-I was a more potent activator than apo-A-IV with egg yolk lecithin, L-alpha-dioleoylphosphatidylcholine, and L-alpha-phosphatidylcholine substituted with one saturated and one unsaturated fatty acid regardless of the substitution position. When L-alpha-phosphatidylcholine esterified with two saturated fatty acids was used as acyl donor, apo-A-IV was more active than apo-A-I in stimulating the lecithin:cholesterol acyltransferase reaction. Complexes of phosphatidylcholines substituted with two saturated fatty acids served as substrate for lecithin:cholesterol acyltransferase even in the absence of any activator protein. Essentially the same results were obtained when substrate complexes (phospholipid-cholesterol-[4-14C]cholesterol-apoprotein) were prepared by a detergent dialysis procedure. Apo-A-IV-L-alpha-dimyristoylphosphatidylcholine complexes thus prepared were shown to be homogeneous particles by column chromatography and density gradient ultracentrifugation. It is concluded that apo-A-IV is able to facilitate the lecithin:cholesterol acyltransferase reaction in vitro.     

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     

Effect of steryl glycosides on the phase transition of dipalmitoyl lecithin

The phase transition of dipalmitoyl lecithin, measured by thermal analysis, was eliminated by the plant sterol, sitosterol, and by the derivatives steryl glucoside and acylated steryl glucoside, which were isolated from soybean lipids.     

Role of the cell wall in the ability of tobacco protoplasts to form callus

Cellular membranes from dark grown hypocotyls of Phaseolus aureus Roxb. were separated by centrifugation on a continuous sucrose gradient. Each gradient fraction was monitored for activity of inosine diphosphatase (EC 3.6.1.6) and the ability to transfer glucose from UDP-[¹⁴C]glucose to endogenous lipids in vitro. The highest incorporation of radioactivity into lipids occurred in a particulate fraction correlated with the Golgi apparatus, sedimenting at sucrose densities of 31.5–33% w/w. Three endogenous lipids were glucosylated in vitro. The two main lipids were characterized as steryl glucoside and acylated steryl glucoside; data from chromatography and hydrolysis of the third lipid suggests that it is dolichyl-monophosphate-glucoside. Steryl glucoside was found to be the main glucoside synthesized, but the proportion of the acylated form increased with time. The results are discussed in the context of the role of the Golgi apparatus as a centre of membrane modification within the plant cell.Abbreviations DMP-mannose dolichyl monophosphate mannose - ER endoplasmic reticulum - GA Golgi apparatus - ID-Pase inosine diphosphatase     

Expression of cholesteryl glucoside by heat shock in human fibroblasts

We investigated the heat-induced alteration of glycolipids in human cultured cells, TIG-3 fibroblasts, to show the expression of steryl glucoside by heat shock. A glycolipid band was detected on a thin-layer chromatography plate in lipid extracts from TIG-3 cells exposed to high temperature (42 degrees C) for 15 and 30 minutes, while it was hardly detectable without heat shock. Both cholesterol and glucose were almost exclusively detected by gas liquid chromatography as degradation products of the lipid. The structure of the lipid molecule was elucidated by electrospray mass spectrometry to be a cholesteryl glucoside. This is the first report to show the occurrence of a steryl glucoside in mammalian cells, and this substance is considered to have a significant role in heat shock responses in mammalian cells.     

Perinatal hepatocyte/hepatoma hybrids: construction of clones that express the developmentally regulated monoacyglycerol acyltransferase activity

Microsomal monoacyglycerol acyltransferase is a developmentally expressed enzyme that catalyzes the synthesis of sn-1,2-diacylglycerol from sn-2-monoacylglycerol and palmitoyl-CoA. The activity is present in liver from fetal and suckling rats but is absent in the adult. In order to obtain a stable permanent cell line that expresses this activity, Fao rat hepatoma cells and hepatocytes from 8-day-old baby rats were hybridized and clones were selected. Two hybrids (HA1 and HA7) expressed monoacylglycerol acyltransferase activity. Like fetal hepatocytes, but unlike hepatocytes from postnatal rats, the HA cells had high rates of [14C]acetate incorporation into glycerolipids, cholesterol, and cholesteryl esters, and they secreted triacylglycerol into the media. Monoacylglycerol acyltransferase specific activity increased 2.5-fold as the cells divided in culture, suggesting growth-dependent regulation. The specific activities of glycerol-P acyltransferase, the committed step of the microsomal pathway of glycerolipid synthesis, and diacylglycerol acyltransferase, the activity unique to triacylglycerol biosynthesis, were comparable to the levels of the corresponding activities in fetal hepatocytes. Addition of insulin or dexamethasone to the media increased the incorporation of [14C]oleate into triacyglycerol about 1.7-fold within 2 h, but had little effect on [14C]oleate incorporation into phospholipid. These hormonally responsive rat-hepatoma/hepatocyte hybrids reflect the fetal stage of hepatocyte development in five major aspects of lipid metabolism: sterol, fatty acid, and triacylglycerol biosynthesis, glycerolipid secretion, and the presence of the developmentally expressed monoacylglycerol pathway.     

Selective acylation of alkyllysophospholipids by docosahexaenoic acid in Ehrlich ascites cells

Ehrlich ascites cells were cultured with 1-O-[3H]alkylglycero-3-phosphoethanolamine (1-[3H]alkyl-GPE) or 1-O-[3H]alkylglycero-3-phosphocholine (1-[3H]alkyl-GPC) to reveal the selective retention of polyunsaturated fatty acids at second position of ether-containing phospholipids. Although small percentages of the lysophospholipids were degraded into long-chain alcohol, both alkyllyso-GPE and -GPC were acylated at the rate of approximately 2 nmol/30 min per 10(7) cells. Alkylacylacetylglycerols were prepared from the acylated products by phospholipase C treatment, acetylation and TLC, and fractionated according to the degree of unsaturation by AgNO3-TLC. The distribution of the radioactivity among the subfractions indicated that both alkyllysophospholipids were mainly esterified by docosahexaenoic acid and to a somewhat lesser extent by arachidonic acid. The selectivity for docosahexaenoic acid in the esterification of 1-alkyl-GPE was much stronger than in that of 1-alkyl-GPC. Although acyl-CoA: 1-alkyl-glycerophosphoethanolamine acyltransferase activity of Ehrlich cell microsomes with arachidonoyl-CoA and docosahexaenoyl-CoA as acyl donors was negligible compared with the acyl-CoA:1-alkyl-glycerophosphocholine acyltransferase activity, a significant amount of 1-alkyl-GPE was acylated in the microsomes without exogenously added acyl-CoA. HPLC analysis revealed that docosahexaenoic acid and arachidonic acid were mainly esterified by the microsomal transferase. Acylation of 1-alkyl-GPC with docosahexaenoic acid and arachidonic acid was also observed in the absence of added acyl-CoA, but the activity was lower than that for 1-alkyl-GPE. Although the source of the acyl donor in the acylation has not been determined, the acylation is probably due to the direct transfer of acyl groups between intact phospholipids. The above results provided the first evidence that the lysophospholipid acyltransferase system including the transacylase activity participates in the selective retention of docosahexaenoic acid in intact cells and a cell free system.