Acyl composition and biosynthesis of acylated steryl glucosides in Calendula officinalis

111

Biochimico et Biophysics Acta, 398 (1975) 111-117 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 56628

ACYL COMPOSITION AND BIOSYNTHESIS OF ACYLATED GLUCOSIDES IN CA LENDULA OFFZCZNA LZS

STERYL

ZDZISkAW A. WOJCIECHOWSKI and JAN ZIMOWSKI Department (Poland)

of Biochemistry,

(Received December llth,

Warsaw University,

02-089

Warsaw, ALZwirki

i Wigury 93

1974)

Summary Fatty acids C1 2 -C 2 2 are components of acylated steryl glucosides in Calendulu officinalis. Various particulate fractions from 14day-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 > phosphatidylethanolamine > phosphatidylcholine. It does not utilize triacylglycerols and monogalactosyldiacylglycerols.

Introduction Steryl 3/3-D-glucosides and their acylated forms (i.e. steryl6’-O-acyl-3&Dglucosides) seem to be widely distributed in higher plants together with free sterols and steryl esters [l-3 1. However, their functions in plant cells are not clear. Recently it has been suggested [4--51 that the reversible processes of glucosylation of sterols incorporated into membranes as well as subsequent esterification of steryl glucosides may have some regulatory effect on the membrane organization. Enzymatic synthesis of steryl glucosides using UDPglucose as the sugar donor as well as enzymatic transformation of steryl glucosides into monoacyl derivatives using endogenous acyl donors has been demonstrated in plant tissues [6-lo]. However, the data obtained, up to now, on the localization and properties of acyltransferase participating in transformation of steryl glucosides into steryl acylglucosides are rather scarce and in part contradictory. In wheat

112

roots [ 111 the enzyme has been found in the microsomes and its ability to utilize phosphatidyleth~olamine as a source of acyl groups has been proved. Acyltransferase from carrot has been described [ 121 as a cytosol enzyme utilizing in some measure monogalactosyl- and digalactosyldiacylglycerols. Methods Subcellular fractionation 2-week-old C&end&a officinalis cv. Radio plants were homogenized

with 0.1 M Tris-HCl buffer, pH 7.5 (3 ml per 1 g plants). The homogenate was squeezed through a cloth and successively centrifuged at 3000, 15 000 and 105 000 X g. Organelles from 15 000 X g pellet were further fractionated by sucrose density gradient (21-52%) cent~fugation as described elsewhere [ 131. Marker enzymes, chlorophyll and protein were estimated as described previous1YE131. Solubilized enzyme preparation

The 15 000 X g pellet (28.9 mg protein) was suspended in 0.1 M Tris-HCl pH 7.5, containing 0.1% Triton X-100 (100 ml). The suspension was stirred at 0°C for 15 min and then centrifuged at 15 000 X g (40 min). The supernatant (8.7 mg only slightly active protein) was discarded and the pellet was treated as above with buffer containing 0.8% Triton X-100. After cent~fugation at 15 000 X g (40 mm), the supe~a~t obtained was added dropwise to a 5-fold amount of cold (-15°C) acetone. The precipitating protein (8.2 mg) was collected by centrifugation, washed twice with acetone and dried in a vacuum. Acyltransferase assay

The standard incubation mixture contained in a total volume of 0.7 ml: enzyme preparation (0.1-0.5 mg protein); Tris-HCl, pH 7.2 (50 ,umol); steryl [’ 4 C ] glucoside ( lo5 dpm) and 50 Ergof individual lipids, In some experiments, non-labelled steryl glucoside (250 ,ug) and acyl-’ 4 C-labelled lipids (lo5 dpm) were used. After incubation (1 h, 3O”C), radioactive steryl acylglucosides were isolated and counted as described earlier [ 13,141. Steryl [’ ’ C] glucoside, spec. act. 3.0 Ci/mol, was obtained enzymatically [14]. Aeyl-’ 4 C-labelled lipids were prepared biosynthetic~ly: isolated young shoots of 3-month-old C. officina~is plants were suplied with [l-l 4 C] acetate as described previously [15] and, after 3 days, lipids were extracted and separated as below. Non-labelled steryl glucoside and various acyl lipids were obtained from 2-week-old seedlings. Isolation of lipids

Lipids were extracted and preliminarily separated by column chromatography according to Lepage [ 11. Steryl glucosides and steryl acylglucosides were purified by thin-layer chromatography as described earlier [ 161. Other lipid fractions were isolated by thin-layer chromatography using solvent systerns described by Schwertner and Biale [ 171, Steryl acylglucosides were hydrolyzed and their acyl component assayed by gas-liquid chromatography on a 9 ft column filled with 10% polyethyleneglycol adipate on Diatomite CQ, at 200°C as described previously 1161.

TABLE

I

2-week-old seedlings: whole seedIings crude cbIoroI&~&s crude mitochondria crude microsomes cytoaol(lo5ooo x g sugernatant)

roots

fl0weE3

2.8 4.4 4.6 3.2

1.3

10

0.3 0.3

0.4

42 5 16 7

27 15 11

3.9

5.0 5.7 4.8 6.3

2.0 2.4 2.1

c14:

0

c12: 0

5.5

7.0 6.0 5.5 4.4

1.2 1.9 1.3

ClS : 0

~~~~Cr~A~~~

trglg fresh tissue)

IN C,

Acyl composition

ACYLGLUCOSIDES

Steryl acylglucoaide content

OP STERYL

3-month-old plants: leaves

Material assayed

tr. trace,

DISTRIBUTION

29.4

14.9 17.4 28.1 15.1

37.6 30.6 33.6

c16:

AND

0

12.3

10.1 7.9 9.2 8.7

1.5 2.2 1.8

1

ACYL

cl6:

THEIR

6.7

10.3 10.0 9.8 6&O

8.5 7.0 8,Q

ClB:

0

12.6

27.1 20.6 17.0 26.3

4.2 3.9 3.9

cl8:

COMPOSITION

I

17.9

11.2 18.3 13.6 16.6

25.2 24.3 25.9

cl81

2

10.4

9.2 7.5 7.6 9.6

13.2 18.5 14.0

c18:

3

tr.

0.9 2.2 tr. 3.4

2.1 2.5 0.8

c2o:o

tr.

1.6 tr. tr. tr.

1.9

3.1 2.4

C20:

1

ix.

tr. tr. tr.

tr.

3.1 0.5

1.0

C22 : 0

114

Results and Discussion Acyl components of acylated steryl glucosides from C. officinalis We have previously reported the occurrence and sterol composition of steryl monoacylglucosides from C. officinalis leaves [9]. Table I sumarises some observations on the distribution and acyl composition of these compounds in various organs of 3-month-old plants as well as in different subcellular fractions obtained from 2-week-old seedlings. All preparations contained fatty acids. Various organs of mature plants exhibited rather similar C1*-G2 acyl compositions characterized by a high content of palmitic acid (30.638.6%), linoleic acid (24.3-25.9%) and linolenic acid (13.2-18.5%). Steryl acylglucosides of Z-week-old seedlings clearly distin~ished themselves by a high content of monounsaturated C , 8 and C1 6 acids, i.e. of oleic and palmitooleic acids. It is of interest that the acyl composition of steryl acylglucosides of various subcellular fractions obtained by differential centrifugation of seedling homogenate was markedly different. For example, in steryl acylglucosides of the cytosol and crude mitochondria (15 000 X g pellet), palmitic acid predominated, whereas in crude chloroplasts (3000 X g pellet) and crude microsomes (105 000 X g pellet) oleic acid was the main component. The cytosol fraction contained, contrary to the remaining fractions, more Iinoleic than oleic acid. The assumption that the biosynthesis of steryl acylglucosides can take place in different cell comp~tments, with utilization of different acyl donor pools, seems to be the simplest explanation of this fact. Subcellular localizafiw~ of steryl acylglucoside biosynthesis To localize the subcellular structures in which acylation of steryl glucosides takes place, an homogenate of whole 2-week-old seedlings was separated by differential centrifugation, whereupon the ability of the obtained fractions

Fig. 1. Acylation of steryl glucosides by crude subcellular fractions from C. offrcindis seedlings: (*) Per mg protein, left scale; (0) per 1 g fresh plant tissue, right scale. ia) 3000 X g pellet Node cNoro~lasts>: (b) 15 000 X g pellet (crude mitochondria): (c) 105 000 X g pellet (crude microsomes); (d) 105 000 X B supernatant. The reaction mixtures (0.7 ml) contained: a subcellular fraction (0.2 mg protein): lkis-HCI pH 7.2 (50 pmol) and stew1 [ 14cl glucoside (10s dpm). Incubation was carried out for 1 h at 30°C.

115

to synthesize steryl acylglucosides was investing by incubation with steryl [I 4 C!] glucoside. The incubation was performed without addition of any exogenous acyl donors. It should be mentioned that in the above experiments, incubation of the individual subcellular fractions in the presence of an additional source of acyl groups (crude phospholipid fraction or various individual phospholipids, see below) failed to cause pronounced enhancement of the formation of ’ 4 C-labelled steryl acylglucosides indicating preferential utilization of endogenous acyl donors. It is evident (Fig. 1) that the activity is associated with particulate fractions; in the cytosol only traces of activity were found. The fraction sedimenting at 15 000 X g was most active, exhibiting a lo--13~times higher activity (per mg protein) than the rem~ning particulate fractions. Microscopic studies showed that this fraction conned mainly mitochondria with a smaller amount of chloroplast material. Therefore, for more precise localization of the site of steryl acylglucoside formation, the 15 000 X g fraction was further fractionated by sucrose density gradient centrifugation. In the resulting subfractions, the ability to acylate steryl glucoside as well as the activity of some markers of cell organelles were determined. It can be readily observed (Fig. 2) that the distribution of the acyltransferase activity is superposed onto neither that of the succinate dehydrogenase (mitochondrial marker) nor chlorophyll content (chloroplast marker). A similar distribution of the acyltransferase activity to that of the UDPG : sterol glucosyltransferase indicates that acylation of steryl glucosides proceeds in the same structures in which sterol glucosylation takes place. These structures are, most probably, fragments of the Golgi complex, since such localization has recently been suggested for UDPG : sterol glucosyltransferase [5,13]. Acyl donors in steryl acylglucoside biosynthesis

As it has been pointed above, we were unable, using subcellular fractions,

i

bottom

2

3

4

5 Fraction

6 NO

7

8

9 top

Fig. 2. Sucrose density gradient profile of the 15 000 X g fraction. Enzymie activities and chlorophyll content are expressed in relative vahzes taking specific. activities (oi C~O~OP~Y~ content per mg protein) of the crude 15 000 X g fraction as 1.00. (cross hatching) acyltransferase: (0) UDPG : steroi glucosyltransferase; (a) succinate dehydrogenase: (a) chlorophyll. Aeultransferase was assayed as described in Fig. 1. For other details see Methods.

116

to obtain a measurable enhancement of the acylation of steryl glucosides in the presence of any exogenous lipids, The formation of steryl acylglucosides by acetone powder obtained by direct extraction of the 15 000 X g fraction with cold acetone was only slightly (up to 15%) stimulated by addition of crude phospholipid fraction or individual phospholipids. Therefore, we developed a method for obtaining a partly lipid-depleted enzyme preparation by treatment of the 15 000 X g fraction with 0.8% Triton X-100 and subsequent acetone extraction. The resulting preparation failed to sediment upon centrifugation at 20 000 X g (40 min) and about 60% of acyltransferase activity remained in the supernatant also after centrifugation at 105 000 X g (1 h). Although this preparation still exhibited a fairly high ability to synthesize steryl acylglucosides without addition of exogenous lipids the addition of certain lipids greatly enhanced the rate of the synthesis (Table II). The results obtained indicate that all main phospholipid fractions isolated from the same plant were utilized for the synthesis of steryl acylglucosides; phosphatidy~i~osito~ and phosphatidylethanolamine being utilized much more efficiently than phosphatidylcholine. Triacylglycerols and monogalactosyldiacylglycerols somewhat inhibited, in fact, the acylation of steryl glucosides. A slight stimulatory effect of digalactosyldiacylglycerols was observed. Detailed analysis of digalactosyldiacylglycerol prep~ation obtained showed, however, a slight contam~ation (a few percent) with an unidentified phospholipid material which could not be separated by chromatography. Thus, it cannot be ruled out that the small stimulating effect of digalactosyldiacylglycerols results from the presence of this contamination. Some doubts could arise whether the enhancement of steryl acylglucosides synthesis observed in the above experiment was due to real utilization of phospholipids as acyl donors or resulted only from the stimulator effect of these lipids on the utilization of endogenous acyl donors. The stimulatory effect of some phospholipids on various enzymic systems is well known [5,18,19]. Therefore, the above experiment was repeated using acyl-’ 4 C-labelled lipids and nonradioactive steryl glucosides in the incubation mixture. TABLE

Ii

ACYLATION

OF STERYL

[‘“ClGLUCOSIDES

BY

SOLUBILIZED

ENZYME

OF C. QFFICINALIS

Complete assay system: steryl [14Clglucoside traction (lo5 dpm); Tris-HCl (pH 7.2), 50 /.mol; Tween 80 (0.35 mg) and partly solubilized enzyme preparation (0.24 mg protein). Total volume 0.7 ml; incubation 1 h at 30°C (stew1 glueosides and acyl lipids were added as emulsions in 2% Tween 80). Assay system

14C Incorporation (dpm X 103)

Complete Complete, boiled enzyme Complete + lipid (50 pg): f triaeylglycerols + monog~actosyldiaeylglycerols + digalactosyldiacYlglycerOls + phosphatidylethanolamine + phosphatidylcholine + phosphatidyhnositol

3.31. 0.04

* Mean value from 3 independent

2.57 3.19 3.54 4.84 4.26 4.96 experiments.

into stew1 acylglucosides*

117 TABLE Ifi UTILIZATION MATION

OF VARIOUS

14C-ACYL

BY THE SOLUBILIZED

LABELLED

ENZYME

LIPIDS FOR STERYL

ACYLGLUCOSIDE

FOR-

OF C. OFFICINALIS

Complete assay system: steryl glue&de fraction (250 pgg); mk+-HC1 (PH 7.2). 50 pmol; individuaf acyl14C-1abe&d lipids (10’ dam); Tween 80 (0.36 mg) and partly solubilized enzyme preparation (0.44 mg protein). Total volume 0.7 ml: incubation 1 h at 30°C. AcyI-14C-iabelled lipid added

14C incorporation into stery1 acytglucosides (dpm X lo31

Triacylgiycerols Monogaiectosyidiscylglyeerols Di9~actosyfdiacylg~y,gfyeesols Phosphatidylathanotslmine Pho~batidylcho~ne Phosphatidylinositol

0 0 0.10 3.60 0.42 0.96

Radioactive lipids were obtained by incubation of shoots of 3-month-old plants with [l-’ 4 C] acetate (see Methods). The results (Table III) unequivocally confirmed the direct uti~zation of the acyl groups from phospholipids for acyhtion of steryl glucosides in a manner similar to the esterification of cholesterol in blood serum by a lecithin: ~holes~ols acyltr~sfe~e system [20 1. In this case, however, quanti~tive ~~~~tation of the data obtained could arise doubts, since the specific ra~oa~ti~ty of various ~hos~ho~~ids obtained might have been dissimilar, Acknowledgment We thank Miss E. Gasiorowska for her skilful technical help. References 1 2 3 4 6 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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