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.     

Inhibition of glycolipid biosynthesis in chloroplasts by ozone and sulfhydryl reagents

The metabolism of uridine 5′-pyrophosphate-galactose by spinach (Spinacia oleracea) chloroplast preparations was inhibited by ozone. The formation of digalactosyl diglyceride and trigalactosyl diglyceride was inhibited much more than the formation of monogalactosyl diglyceride, steryl glycoside, and acylated steryl glycoside. Essentially identical results were obtained when glycolipid synthesis was inhibited by N-ethyl maleimide, p-hydroxymercuribenzoate, and CdCl₂. Iodoacetate and iodoacetamide affected neither the total incorporation of sugar from uridine 5′-pyrophosphate-galactose nor distribution of the incorporated sugar in the various glycolipids.     

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.     

Partial characterization of steryl ester biosynthesis in spinach leaves

Acetone powders of a 20,000g pellet fraction from spinach leaves (Spinacia oleracea L.) synthesized [4-(14)C]cholesteryl esters when incubated with [4-(14)C]cholesterol. The reaction was inhibited by digitonin. There was a reciprocal relationship between the decline of label in cholesterol and its incorporation into cholesteryl ester, indicating that free cholesterol was the direct precursor for cholesteryl ester biosynthesis. The hydrolysis of cholesteryl [1-(14)C]palmitate into free cholesterol and [1-(14)C]palmitate was not detected in these acetone powder preparations. Exogenous cholesteryl palmitate had no effect on the esterification of [4-(14)C]cholesterol. The data indicate that an esterase-type mechanism was not involved in the biosynthesis of these steryl esters. Label from [1-(14)C]palmitoyl-CoA was incorporated into steryl esters when incubated with spinach leaf acetone powder preparations. The optimal buffer for steryl ester biosynthesis was 2-(N-morpholino)ethanesulfonate and the optimal pH was 6. Iodoacetamide, N-ethylmaleimide, and dithiothreitol had no effect on the esterification reaction. Ethylenediaminetetraacetate, MgCl(2), CaCl(2), MnCl(2), and ZnSO(4) inhibited at concentrations of 10 to 30 mm.     

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.     

Inhibition of steryl glycoside biosynthesis by acyl coenzyme a and by digitonin

ATP, GTP, CoA, Mg²⁺, and Mn²⁺ did not inhibit biosynthesis of steryl glycoside and acylated steryl glycoside when added singly to enzyme preparations from spinach leaves. The combination of ATP (but not GTP), CoA, and Mg²⁺ or Mn²⁺ caused marked inhibition, especially of steryl glycoside biosynthesis, when reaction mixture concentrations of the additions were 0.2 millimolar. Inhibition was attributed to acyl-CoA and could be reproduced by palmitoyl-CoA. The inhibition could be partially prevented by bovine serum albumin. The effects of palmitoyl-CoA were distinct at 10 micromolar, and 50% inhibition of biosynthesis was observed at 40 micromolar.     

Metabolism of conjugated sterols in eggplant. Part 2. Phospholipid : steryl glucoside acyltransferase

A membrane-bound phospholipid : steryl glucoside acyltransferase from Solanum melongena leaves was partially purified and its specificity and molecular as well as kinetic properties were defined. Among the steryl glycosides tested (e.g. typical plant steryl glucosides, steryl galactosides and cholesteryl xyloside) the highest activity was found with cholesteryl glucoside, but some structurally related compounds such as sito- and stigmasteryl glucoside or galactoside as well as cholesteryl galactoside were also acylated, albeit at lower rates. The investigated enzyme was able to use all classes of phosphoglycerolipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol) as an acyl source for biosynthesis of acylated steryl glucoside. Among them 1,2-dimirystoylphosphatidylic acid appeared to be the best acyl donor. Apart from phosphoglycerolipids, 1,2-diacylglycerols were also used as acyl donor for steryl glucoside acylation, although at a distinctly lower rate. The acyl moiety was transferred from the C-1 position of phospholipid molecule. The investigated acyltransferase activity was stimulated by 2-mercaptoethanol, Triton X-100, 1-monoacylglycerols and inhibited in the presence of divalent cations such as Ca(2+), Mn(2+), Zn(2+) or Co(2+), some lipids (MDGD, ceramide), detergents (Tween 20, 40, 60 and 80, Tyloxapol, sodium deoxycholate) and high ionic strength.     

Fractionation and characterization of cellular membranes from root tips of garden cress (Lepidium sativum L.)

The first step in the gravitropic reaction chain, i.e. perception, is known to occur in the statenchyma of the root cap. Because of the importance of the root tip in graviperception, a procedure has been developed to isolate root tips from garden cress (Lepidium sativum L.). The root tip fraction contains the tissues of the root cap plus the lower half of the meristem zone, but is clearly separated from the tissues of the elongation zone, the zone of gravitropic response. Membranes from the root tip and root base fractions have been centrifuged on sucrose density gradients and the marker enzyme profiles analyzed. These results show that the marker enzyme profiles for vacuoles, dictyosomes, mitochondria, and plasma membranes are similar in the root tip or root base fractions. The endoplasmic reticulum (ER) has a shoulder of cytochrome c reductase activity at a density of 1.16 g cm^-3 which is distinct from the other enzyme activities and is only observed in root tip preparations. The specific enzyme activity for ER, cytochrome c reductase, was enriched in root tip membranes 1.7 fold. This latter increase is interpreted as at least in part an increased ER content in the root tip.Abbreviations ASG 6-acyl-steryl glucoside - ER endoplasmic reticulum - IDP inosine-5-diphosphate - INT 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride - PM plasma membrane - SG steryl glucoside     

Acyl carrier protein is present in the mitochondria of plants and eucaryotic micro-organisms

Proteins antigenically similar to the acyl carrier protein (ACP) found in the mitochondria of Neurospora crassa were detected by immunoblotting and radioimmunoassay techniques in mitochondria isolated from yeast, potatoes, and pea leaves. These mitochondrial proteins were similar to Neurospora ACP both in their electrophoretic mobility and in their unusual decrease in mobility upon reduction. Authentic ACP(s) show this type of change upon conversion of the acylated to the unacylated form. Purified ACP from both spinach chloroplasts and Escherichia coli cells cross-reacted with antibodies raised against Neurospora ACP. Purified ACP from Neurospora cross-reacted with antibodies raised against spinach chloroplast ACP and E. coli ACP. Mitochondria isolated from beef heart and rat brain were tested extensively and exhibited no cross-reaction with any of the three anti-ACP preparations. The discovery of ACP in the mitochondria of other organisms raises questions concerning the possible relationship between ACP and beta-oxidation in mitochondria, the involvement of ACP in de novo biosynthesis of some of the acyl chains in mitochondria and the subcellular locations of fatty acid biosynthesis in plants and eucaryotic micro-organisms.     

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     

Comparison of lipids from Spiroplasma citri and corn stunt spiroplasma.

The qualitative lipid composition of Spiroplasma citri and corn stunt spiroplasma is identical. Small amounts of acylated glucose and steryl glucoside were found.     

Lipid Profiles of Leaf Tonoplasts from Plants with Different CO2-Fixation Mechanisms*

Tonoplast preparations were obtained from leaves of Hordeum vulgare (C₃), Kalanchoë daigremontiana (obligate CAM) and Mesembryanthemum crystallinum (C₃ and inducible CAM). Lipid analyses showed reproducible patterns comprising free sterols, glycolipids of plastidic origin, glucose-containing lipids (steryl glucoside, acylated steryl glucoside, cerebroside) and phospholipids. Predominant components were sterols, cerebrosides, phosphatidyl choline and phosphatidyl ethanolamine. Very long chain fatty acids were found in phosphatidyl serine and hydroxy fatty acids in cerebrosides. Isolation of tonoplasts via protoplasts and vacuoles may have resulted in reduced levels of free sterols. The data show a similarity between tonoplasts and plasma membranes with respect to lipid profiles. Lipid composition was neither affected by different CO₂-fixation mechanisms nor by salt-induced changes in Mesembryanthemum crystallinum.     

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.     

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.     

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     

ADP/ATP Translocator from Pea Root Plastids (Comparison with Translocators from Spinach Chloroplasts and Pea Leaf Mitochondria)

The kinetic properties of the adenosine 5[prime]-diphosphate/adenosine 5[prime]-triphosphate (ADP/ATP) translocator from pea (Pisum sativum L.) root plastids were determined by silicone oil filtering centrifugation and compared with those of spinach (Spinacia oleracea L.) chloroplasts and pea leaf mitochondria. In addition, the ADP/ATP transporting activities from the above organelles were reconstituted into liposomes. The Km(ATP) value of the pea root ADP/ATP translocator was 10 [mu]M and that for ADP was 46 [mu]M. Corresponding values of the spinach ADP/ATP translocator were 25 [mu]M and 28 [mu]M, respectively. Comparable results were obtained for the reconstituted ATP transport activities. The transport was highly specific for ATP and ADP. Adenosine 5[prime]-monophosphate (AMP) caused only a slight inhibition and phosphoenolpyruvate and inorganic pyrophosphate caused no inhibition of ATP uptake. With pea root plastids and spinach chloroplasts, Km values >1 mM were obtained for ADP-glucose. Since the concentrations of ATP and ADP-glucose in the cytosolic compartment of spinach leaves have been determined as 2.5 and 0.6 mM, respectively, a transport of ADP-glucose by the ADP/ATP translocator does not appear to have any physiological significance in vivo. Although both the plastidial and the mitochondrial ADP/ATP translocators were inhibited to some extent by carboxyatractyloside, no immunological cross-reactivity was detected between the plastidial and the mitochondrial proteins. It seems probable that these proteins derive from different ancestors.     

Antigenic relationships between chloroplast and cytosolic fructose-1,6-bisphosphatases.

Cytosolic fructose-1,6-biphosphatases (FBPase, EC 3.1.3.11) from pea (Pisum sativum L. cv Lincoln) and spinach (Spinacia oleracea L. cv Winter Giant) did not cross-react by double immunodiffusion and western blotting with either of the antisera raised against the chloroplast enzyme of both species; similarly, pea and spinach chloroplast FBPases did not react with the spinach cytosolic FBPase antiserum. On the other hand, spinach and pea chloroplast FBPases showed strong cross-reactions against the antisera to chloroplast FBPases, in the same way that the pea and spinach cytosolic enzymes displayed good cross-reactions against the antiserum to spinach cytosolic FBPase. Crude extracts from spinach and pea leaves, as well as the corresponding purified chloroplast enzymes, showed by western blotting only one band (44 and 43 kD, respectively) in reaction with either of the antisera against the chloroplast enzymes. A unique fraction of molecular mass 38 kD appeared when either of the crude extracts or the purified spinach cytosolic FBPase were analyzed against the spinach cytosolic FBPase antiserum. These molecular sizes are in accordance with those reported for the subunits of the photosynthetic and gluconeogenic FBPases. Chloroplast and cytosolic FBPases underwent increasing inactivation when increasing concentrations of chloroplast or cytosolic anti-FBPase immunoglobulin G (IgG), respectively, were added to the reaction mixture. However, inactivations were not observed when the photosynthetic enzyme was incubated with the IgG to cytosolic FBPase, or vice versa. Quantitative results obtained by enzyme-linked immunosorbent assays (ELISA) showed 77% common antigenic determinants between the two chloroplast enzymes when tested against the spinach photosynthetic FBPase antiserum, which shifted to 64% when assayed against the pea antiserum. In contrast, common antigenic determinats between the spinach cytosolic FBPase and the two chloroplast enzymes were less than 10% when the ELISA test was carried out with either of the photosynthetic FBPase antisera, and only 5% when the assay was performed with the antiserum to the spinach cytosolic FBPase. These results were supported by sequencing data: the deduced amino acid sequence of a chloroplast FBPase clone isolated from a pea cDNA library indicated a 39,253 molecular weight protein, with a homology of 85% with the spinach chloroplast FBPase but only 48.5% with the cytosolic enzyme from spinach.     

Steryl Glycoside Formation in Seedlings of Nicotiana tabacum L

Particulate enzyme preparations from tobacco seedlings (Nicotiana tabacum L.) were used in the synthesis of steryl glycoside. The data obtained by measuring cholesterol-4-¹⁴C incorporation generally agree with results obtained with UDP-glucose-¹⁴C. The in vitro reaction was linear for the first 10 minutes and had a pH optimum of 7.0 to 7.4. Addition of ATP activated while UDP-glucose inhibited slightly the reaction. In short term experiments, the percentage disappearance of endogenous and added sterol was about the same.     

Studies on uridine diphosphate-galactose pyrophosphatase and uridine diphosphate-galactose: glycoprotein galactosyltransferase activities in microsomal membranes.

Rat liver microsomes showed very active uridine diphosphate-galactose pyrophosphatase activity leading to the hydrolysis of uridine diphosphate-galactose into galactose1-phosphate and finally into galactose. The activity was observed in presence of buffers with wide ranges of pH. Different concentrations of divalent cations, such as Mn²⁺, Mg²⁺, and Ca²⁺ had no significant effect on the enzyme activity. A number of nucleotides and their derivatives inhibited the pyrophosphatase activity. Of these, different concentrations of uridine monophosphate, cytidine 5′-phosphate and cytidine 5′-diphosphate have slight or no effect; cytidine 5′-triphosphate, adenosine 5′-triphosphate, guanosine 5′-triphosphate, cytidine 5′-diphosphate-glucose and guanosine 5′-diphosphate-glucose showed strong inhibitory effect whereas cytidine 5′-diphosphate-choline showed a moderate effect on the pyrophosphatase. All these nucleotides also showed variable stimulatory effects on uridine diphosphate-galactose:glycoprotein galactosyltransferase activity in the microsomes which could be partly related to their inhibitory effects on uridine diphosphate-galactose pyrophosphatase. Among them uridine monophosphate, cytidine 5′-phosphate, and cytidine 5′-diphosphate stimulated galactosyltransferase activity without showing appreciable inhibition of pyrophosphatase, cytidine 5′-diphosphate-choline, although did not inhibit pyrophosphatase as effectively as cytidine 5′-triphosphate, guanosine 5′-triphosphate, adenosine 5′-triphosphate, cytidine 5′-diphosphate-glucose, and guanosine 5′-diphosphate-glucose but stimulated galactosyltransferase activity as well as those. The fact that cytidine 5′-diphosphate-choline stimulated galactosyltransferase more effectively than cytidine 5′-phosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate suggested an additional role of the choline moiety in the system. It has been also shown that cytidine 5′-diphosphate-choline can affect the saturation of galactosyltransferase enzyme at a much lower concentration of uridine diphosphate-galactose. Most of the pyrophosphatase and galactosyltransferase activities were solubilized by deoxycholate and the membrane pellets remaining after solubilization still retained some galactosyltransferase activity which was stimulated by cytidine 5′-diphosphate-choline. In different membrane fractions a concerted effect of both uridine diphosphate-galactose pyrophosphatase and glycoprotein:galactosyltransferase enzymes on the substrate uridine diphosphate-galactose is indicated and their eventual controlling effects on the glycopolymer synthesis in vitro or in vivo need careful evaluation.     

Stimulation of carbon dioxide fixation in isolated pea chloroplasts by catalytic amounts of adenine nucleotides

Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum var. Massey Gem) chloroplasts was increased two to 12 times by the addition of ATP. O(2) evolution was also stimulated by ADP and to a lesser extent by AMP. The ATP effects were not due to broken chloroplasts present in the preparations nor was ATP acting as a phosphate source. We concluded that the adenine nucleotides were acting catalytically. The concentration of ATP required for half-maximum rate of O(2) evolution was 16 to 25 mum. The degree to which ATP stimulated O(2) evolution depended on the age of pea plants from which the chloroplasts were isolated. Spinach (Spinacia oleracea var. True Hybrid 102) chloroplasts did not show a consistent stimulation of O(2) evolution by adenine nucleotides.The adenine nucleotide content of pea chloroplasts was not lower than that of spinach chloroplasts, but pea chloroplasts which showed a large stimulation of O(2) evolution by ATP contained an ATP-hydrolyzing reaction with rates of 10 to 50 mumol ATP hydrolyzed mg chlorophyll(-1) hour(-1). The rate of the ATP-consuming reaction was much lower in spinach chloroplasts and in chloroplasts from older pea plants which did not show large stimulation of O(2) evolution by ATP. We propose that the ATP-consuming reaction, with a high affinity for ATP, decreased the effective size of the ATP pool available for CO(2) fixation. Added adenine nucleotides could be transported into the chloroplasts increasing the concentration of internal nucleotides. Calculations showed that the adenine nucleotide transporter on the outer chloroplast membranes could operate at a sufficient rate to produce such an effect.