activities and sub cellular localisation of enzymes responsible for lipolysis

Biochemical Systematics and Ecology, Vol. 15, No. 5, pp. 551-558, 1987. 0305-1978/87 $3.00+0.00 Printed in Great Britain. Pergamon Journals Ltd.

Activities and Subcellular Localization of Enzymes Responsible for Lipolysis and Gluconeogenesis during the Germination of Brassica campestris cv. esculenta Seeds

NIEVES VILLALOBOS, FERNANDO SIMON, LUISA MARTIN, MAITE HERRERA and GREGORIO NICOLAS

Department of Plant Biology, Faculty of Biology, University of Salamanca, 37008 Salamanca, Spain

Key Word Index--Brassica campestris cv. escuienta; turnip seed germination; lipolysis; gluconeogenesis.

Abstract--During the growth of turnip seedlings, two new lipases have been demonstrated, one with a maximum activity at pH 4.5 (acid lipase) and the other with a maxima at pH 8.6 (alkaline lipase). Many different enzymes are involved in gluconeogenesis: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase, aconitase, citrate synthetase, fumarase, glycolate oxidase, phosphoenol-pyruvate carboxykinase. All of these show maximum activity coinciding with the stage in which lipid hydrolysis is maximal and when the accumulation of soluble carbohydrates has also reached its peak. The alkaline lipase as found to be located mainly in the spherosomes, whereas the glyoxysomes contained the following main activities: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase and citrate synthetase. Aconitase, together with cytochrome oxidase and fumarase showed their highest activity in the mitochondria, and the presence of malate dehydrogenase, citrate synthetase and glycolate oxidase was also observed in these organelles. In the membrane-bound fraction, the activities of cytochrome reductase, glycolate oxidase and phosphoenol-pyruvate kinase were marked, although the latter enzyme was even more active in the soluble fraction.

Introduction During the germination of oleaginous seeds, fats are rapidly converted into sugars [1]. This conversion involves many enzymes and suggestions have been made concerning the possible existence of a different subcellular compartmentalization for the enzymes involved in both lipolysis and gluconeogenesis. Ultrastructural studies of cells during different stages of the germination process have revealed a decrease in the number of lipid bodies per cell. This fact suggest that some lipid bodies, mainly those adjacent to the glyoxysomes, are degraded before others [2]. Gluconeogenesis is divided mainly into sepa- rate compartments: (a) lipid bodies; (b) glyoxysomes; (c) mitochondria; and (d) the cytosol. The present work describes the results obtained in the study of some of the enzymes involved in the transformation of triglycerides

Abbreviations: IL, isocitrate lyase; MS, maiate synthetase; MDH, malatedehydrogenase: CS, citrate synthetase.

(Received 29 March 1987)

into soluble carbohydrates necessary for the processes of growth and development of the embryonic axis in turnip seeds, An attempt was also made by separating the organelles, to discover the subcellular location of the different enzymes acting during lipolysis and gluconeogenesis in the germination of these seeds.

Results and Discussion Lipolysis during the germination of turnip seeds In ungerminated seeds, approximately 50% of the total dry weight is accounted for by lipids, whereas the soluble carbohydrates only formed 22%. During germination, the lipid content of the seed decreased gradually, coinciding with an increase in the amount of soluble carbohydrates (Fig. la). This is in accordance with findings reported by other authors [3] for jojoba seed cotyledons.

It has been well documented that oleaginous seeds contain lipase(s) that are manifested during germination [4]. Two kinds of lipase have been described in turnip seeds: an acid lipase (Fig. l b) present in dry seeds, whose

551

552 NIEVES VILLALOBOS, FERNANDO SIMON, LUISA MARTIN, MAITE HERRERA AND GREGORIO NICOLAS

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activity increases during the first 24 h of germination and then declines throughout the rest of the germination process, and an alkaline lipase (Fig. ld) which can be detected after the third day of germination; this form is particu- larly active between days 5 and 8 and shows maximum activity on day 6 of the process. These facts are in agreement with the results

found concerning the mobilization of lipids, since their faster hydrolysis coincides with the period of activity of the alkaline lipase which, as shown in Fig. 1 could be considered to be the main catalysing enzyme in the lipolysis of reserve triglycerides.

On studying the optimum pH for activity of these enzymes (Fig. 2), the acid lipase was seen to show maximum activity at a pH of 4.5, whereas for the alkaline form it was 8.6 (Fig. 3). The neutral lipase showed slight activity at pH 6.5 (Fig. 4), though this could be considered negligible compared with the activities of the other two enzyme forms (Fig. lc).

Variation during germination of the enzymes in valved in gluyconeogenesis The process of lipid mobilization involves numerous enzymes. Among them catalase, a glyoxysomal enzyme that participates in the 13-oxidation of fatty acids, has been used as a marker to indicate the development of gluconeogenesis from reserve lipids in different oleaginous species [5, 6]. During the germination of turnip seeds, catalase activity (Fig. 5A), present at very low levels in ungerminated seeds, increases until a maximum was reached during days 3-5; after this it decreased until a very low level was present in the last three days of germination. This finding

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GERMINATION OF BRASS~CA CAMPESTR/S SEEDS 553

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is very similar to what was reported for cotton [7] and castor bean seeds [8].

Isocitrate lyase, malate synthetase and aconitase (Fig. 5) developed their activities during germination in a way similar to that described in ref. [9], since they were undetectable or present at very low levels only when the seeds had not begun the imbibition period. At 24 h of germination, it was already possible to note a

small increase in isocitrate lyase and malate synthetase activities; these continued to rise gradually until maxima were reached during days 3-5, in the case of IL, and days 4-6 in the case of MS (Fig. 5b). Later, the activity of these two enzymes began to fall and continued to do so until the end of the process, although it should be noted that there was still considerable activity up until the eighth day. Similar activity profiles were observed for citrate synthetase and malate dehydrogenase (Fig. 5) which had maximum activities over days 3-5 (CS) and 4-6 (MDH).

Other enzymes participating in gluconeogenesis, such as glycolate oxidase (Fig. 5b), fumarase and phosphoenol pyruvate carboxykinase (Fig. 5c), had maximum activities, like the above-mentioned enzymes, over days 3-6 of germination.

A relevant aspect of all these findings is that all the enzymes participating in the mobilization of reserve lipids show considerable activity during the days when lipid hydrolysis was most pronounced; coinciding with this an accumulation of soluble carbohydrates occurred. These facts demonstrate that in turnip seeds there is active gluconeogenesis, as demonstrated by the presence during germination of all the enzymes participating in the process.

Subcellular localization of the different enzymatic activities The subcellular fractions obtained by centri- fugation were the following: spherosomes, floating on the upper part; a soluble fraction, immediately under the layer of spherosomes; a membrane fraction (using cytochrome reductase as marker) in the interphase between 1.10-1.13 g cm-3; a mitochondrial fraction (using cytochrome oxidase as marker) at a density of 1.19 g cm -3 and the glyoxysome fraction (using catalase as marker) at a density of 1.28 g cm -3.

When the several enzyme activities were assayed using the different cellular fractions (Table 1), most of the alkaline lipase activity was found in the spherosomes (58.61%), whereas only 8.98% was found in the glyoxysomes, 8.07% in the mitochondria; 1.8% in the membrane fraction and 22.22% in the


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GERMINATION OF BRASSICA CAMPESTRIS SEEDS 555

soluble fraction. This high percentage of lipase activity in the soluble fraction may be indica- tive of breakage, during fractionation, of some spherosomes since when their own lipase was used as substrate there was clear autolytic activity. The existence of high activity in lipid bodies has already been reported in jojoba seeds [10] and in ref. [11] working with soybean seeds, who found maximum lipase activity in the soluble fraction; these authors postulated that its origin was mainly from the glyoxysomes.

Catalase was the enzyme showing the highest activity and almost all of it (82%) was present in the glyoxysomes. Similar results concerning glyoxysomal activity were observed for the isocitrate lyase with 73.12% and for the MS, with 82.52%. However, the MDH and CS, typical enzymes of the glyoxylate cycle, showed similar activity in the glyoxysomes (39.39% for the MDH and 39.92% for the CS) and in the mitochondria (40.2% and 27.51% for the MDH and CS respectively). Aconitase, fumarase and cytochrome oxidase had their highest activities in the mitochondria, although a fairly significant activity of all these enzymes was also seen in the soluble fraction. Outstanding too was the level reached by aconitase in the glyoxysomes; this was to be expected in view of the fact that it is one of the enzymes associated with the glyoxylate cycle.

Glycolate oxidase, although apparently pre- senting its highest activity in the membrane fraction (31.21%) was active in the glyoxysomes (14.9%), the mitochondria (20.5% and the soluble fraction (25.16%).

However, cytochrome reductase, used as marker of the membrane fraction--since it is mainly bound to the endoplasmic reticulum-- [12] showed a similar activity in the soluble fraction (40.18%), which seems to suggest that during fractionation the breakage of some microbodies occurs. Such findings coincide with the results of ref. [12] working with jojoba seeds; these authors reported the existence of a certain activity associated with lipid body membranes. Finally, the phosphoenol pyruvate carboxykinase was located mainly in the soluble fraction (42.63%) though its activity was lower in the membrane fraction (31.7%) and in the mitochondria.

Studies with the electron microscope In resting seeds and during the first days of

germination, the cell were almost completely occupied by lipid bodies (Fig. 6). From the day 3 of germination onwards, a pronounced decrease occurred in these lipid bodies accom- panied by the appearance of vacuolated zones. This situation persisted during the remaining days of germination studied (Fig. 7). These results seem to confirm the existence of active lipid metabolism during the germination of these seeds. Figure 8, corresponding to sections of cotyledons at 7 days of germination, highlights the appearance of microbodies inti- mately related to lipid metabolism [13, 14]. During the other days of germination, it cannot be said that such microbodies do not appear but rather that they are masked by the large amount of lipids present.

Experimental Plant material. The plant material employed for all the experiments was turnip seeds (Brass/ca campestris cv. esculenta). The seeds were germinated and grown on a glass plate covered with filter paper in the darkness at 25 and 80% RH. All seeding operations were carried out in a sterile chamber after previously sterilizing the materials to be used with hypochlorite and UV light.

Analysis of tota/ /ip/ds and soluble carbohydrates. Lipids were extracted according te the method ef ref. [15]. Total lipids were determined by drying an aliquot of the CHCI 3 extract in a vacuum oven overnight and weighing the lipid residue. Soluble carbohydrates were extracted, after rerneving the lipids, with 80% EtOH by the anthrone method [16]. Proteins were assayed by the method of ref. [17].

Preparation of enzymatic extracts. All stages were performed between O and 5 . To obtain the different enzymatic extracts the method described in ref. [14] was employed, using cotyledons obtained at different germination times.

Preparation oforganelles. This procedure was carried out according te the method of ref. [18].

Enzymatic assays. Two different assays were used for the lipase: the fluorimetric method [19], with the modifications introduced in ref. [20], using N-methyl-indoxylimyristate as substrate and a temperature of 24 and the colorimetric method [21], using tripalmitin, triestearin, triolein, 1,3-dilino- lein and monolinolein as substrate; these were emulsified in 5% gum arabic for 30 sec in a ultrasonic generator. The effect of pH on lipase activity was studied using the following as buffers: succinate hydrochloride (pH 4-6), imidazoI-HCI (pH 6-7), Tris-HCI (pH 7-9) and glycine-NaOH (pH 9-10).

The isocitrate lyase assay was performed by measuring the formation of glyoxylate phenylhydrazone [22]. The molar extinction coefficient of the glyexylate phenylhydrazone at 324 nrn was determined [23].


Malate synthetase activity (EC 4.1.3.2) was measured by the method of ref. [22]. Catalase activity (EC 1.11.1.6) was assayed by the method of ref. [24]. Similarly, the following were assayed by their respective methods: aconitase (EC 4.2.1.3) [25]; malate dehydrogenase (EC 1.1.1.37) [26]; citrate synthetase [27]; fumarase (EC 1.1.3.1) [15] and phosphoenol pyruvate carboxykinase [28]. Cytochrome oxidase was determined photometrically [29] and cytochrome reductase by the method of ref. [3].

Preparation of tissue for electron microscopy. Both ungerminated cotyledons and cotyledons germinated for 10 days were used. The organs were cut into small blocks and fixed: the pieces were submerged in 3% glutaraldehyde in 0.05 M phosphate buffer, pH 6.8, in a test tube. The tubes were subjected to a vacuum so that after allowing air to enter, thus raising the pressure, penetration of glutaraldehyde would be facilitated and the substance would replace the air remaining between the cells. This operation was repeated several times until the sample remained stationary at the bottom of the tubes, showing that they were totally impregnated with glutaraldehyde. Following this, the samples were washed 3 0.05 M phosphate buffer pH 6.8. Fixing was performed in 1% osmium tetroxide in phosphate buffer for 2 h. The samples were then washed again with phosphate buffer for 10 min and finally with H20 for 20 min each wash.

All the samples were placed in a 1.5% agar solution, dehydrated in a graded acetone series and embedded in Spurr resin [30]. Polymerization was performed at 60 overnight. Thin sections for electron microscopy were cut using a LKB ultramicrotome. After being sectioned the samples were mounted on formvar-coated slot grids. The grids con- taining the thin sections of the samples were first stained with 2% uranyl acetate for 20 min at 20; they were then washed with H20 and stained again with lead citrate. Observations were made with a Philips EM-300 electron microscope.

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FIG. 6. MICROPHOTOGRAPH OF A SECTION FROM A COTYLEDON OF A TURNIP SEED GERMINATED FOR ONE DAY. LB--lipid body; GIN--cell wall.

FIG. 7. MICROPHOTOGRAPH OF A SECTION FROM A COTYLEDON OF A TURNIP TOP SEED GERMINATED FOR SIX DAYS. LS--lipid body; CW--cell wall; V--vacuole.

558

FIG. 8. MICROPHOTOGRAPH OF A SECTION FROM A COTYLEDON OF A TURNIP SEED GERMINATED FOR SEVEN DAYS. LB--lipid body; CW-cell wall; MB-microbody; PL--plastid.

activities and sub cellular localisation of enzymes responsible for lipolysis

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