Characterization of galactosyltransferases in spinach chloroplast envelopes

Biochimica Elsevier

et Biophysics

189

Acta 918 (1987) 189-203

BBA 52500

Characterization of galactosyltransferases

in spinach chloroplast envelopes *

Johan W.M. Heemskerk * *, Frans H.H. Jacobs, Martin A.M. Scheijen, Johannes P.F.G. Helsper and Jef F.G.M. Wintermans Department

of Botany, Faculty of Science, University of Nijmegen,

Toernooioeld, Nijmegen

(The Netherlands)

(Received 10 November 1986)

Key words: Galactolipid metabolism; Galactosyltransferase; Chloroplast envelope membrane; (Spinach)

Diacylglycerol;

Two galactosyltransferases involved in the galactolipid metabolism of spinach chloroplast envelopes were studied by specific assays: UDPgalactose: 1,Zdiacylglycerol galactosyltransferase, which synthetizes monogalactosyldiacylglycerol from UDPgalactose plus diacylglycerol; and galactolipid: galactolipid galactosyltransferase (GGGT), which forms di- , tri- and tetragalactosyldiacylglycerol by dismutation of galactosyl groups between two galactosyldiacylglycerols. In the assay developed for UDPgalactose: 1,Zdiacylglycerol galactosyltransferase, chloroplast envelope membranes were mixed with liposomes of phosphatidylcholine, and the mixture was treated with phospholipase C (from Bacillus cereus). The diacylglycerol produced from phosphatidylcholine is used by UDPgalactose: 1,2-diacylglycerol galactosyltransferase for synthesis of monogalactosyldiacylglycerol in the presence of UDPgalactose. Several characteristics of this enzyme were studied, including the effect of substrate concentrations, pH and temperature. UDPgalactose: 1,2-diacylglycerol galactosyltransferase did not require cations for activity, but was stimulated by Mg2+ and Mn2’. Of the mono- and divalent cations tested only Zn2’, Cd2+ and Fe’+ were inhibitory. Specific inhibitors for UDPgalactose: 1,2-diacylglycerol galactosyltransferase were UDP and N-ethylmaleimide. In contrast to UDPgalactose: 1,Zdiacylglycerol galactosyltransferase, galactolipid: galactolipid galactosyltransferase was strongly stimulated by a series of mono- and divalent cations, most stimulatory being Mn2’, Ba2’, Ca2+ and was specifically inhibited by low concentrations of Mg2+. Galactolipid: galactolipid galactosyltransferase Zn” (1 mM) and by chelating anions. UDPgalactose: 1,2-diacylglycerol galactosyltransferase synthetized monogalactosyldiacylglycerol from various molecular species of diacylglycerol; the highest activity was measured with distearoylglycerol and dioleoylglycerol. On the other hand, digalactosyldiacylglycerol synthesis by galactolipid: galactolipid galactosyltransferase proceeded most rapidly by galactosyl transfer to hexaene species of monogalactosyldiacylglycerol.

* This paper is dedicated to Prof. Dr. H.F. Linskens (Nijmegen) on the occasion of his 65th birthday in 1986. * * This paper presents a part of the Ph.D. Thesis of J.W.M. Heemskerk, University of Nijmegen, 1986 - present address: Laboratory of Biochemistry, State University of Limburg, Biomedisch Centrum, P.O. 616, 6200 MD Maastricht (The Netherlands). Abbreviations: Hepes, 4-(2-hydroxyethyl)-l-piperatineethanesulfonic acid; Mes, 4-morpholineethanesulphonic acid; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine; PC, phos0005-2760/87/$03.50

phatidylcholine. Molecular species of phosphatidylcholine and lipids derived from it are abbreviated by the notation sn - 1 fatty acyl/sn-2 fatty acyl-PC (fatty acyl residues are indicated by a number denoting the number of carbon atoms followed by a colon and a number denoting the number of double bonds). Correspondence: J.F.G.M. Wintermans, Department of Botany, University of Nijmegen, Toemooiveld, 6525 ED Nijmegen, The Netherlands.

0 1987 Elsevier Science Publishers B.V. (Bit>medical Division)

190

Introduction All chloroplast membranes, i.e., the outer and inner envelope membrane and the thylakoids, are built from phosphoand glycolipids. Quantitatively, the most important lipids are the two galactolipids monogalactosyldiacylglycerol and digalactosyldiacylglycerol: together they comprise 46-79% of the glycerolipids of the chloroplast membranes [l], and as such the galactolipids can be considered the most abundant lipids on earth [2]. It has been generally accepted now that galactolipid biosynthesis takes place in the chloroplast envelope membranes. However, in spite of their abundance, the exact biochemical way of synthesis in vivo is still a matter of discussion [1,3]. In the last decade, three galactosyltransferases have been described which may be involved in the formation of mono- and digalactosyldiacylglycer01. UDPgalactose: 1,Zdiacylglycerol galactosyltransferase synthetizes monogalactosyldiacylglycerol from UDPgalactose and diacylglycerol [4-81. The enzyme is located in the inner envelope membrane of spinach chloroplasts [9-lo], but has been reported in the outer envelope membrane of pea chloroplasts [ll]. Digalactosyldiacylglycerol is synthetized either by an UPDgalactose: monogalactosyldiacylglycerol galactosyltransferase [12] or by galactolipid: galactolipid galactosyltransferase [13]. Evidence for the first enzymatic activity is still poor [3], e.g., it could not be detected in purified spinach envelope membranes [14]. On the other hand, galactolipid: galactolipid galactosyltransferase is a very active enzyme in spinach chloroplasts. It produces digalactosyldiacylglycer01 by dismutation of two molecules of monogalactosyldiacylglycerol, simultaneously liberating diacylglycerol [13-151. Galactolipid: galactolipid galactosyltransferase is accessible from the cytosolic face of the spinach chloroplast [16], and its localization in the outer envelope membrane has been confirmed recently [15]. However, by similar transgalactosylation reactions it also produces the artificial galactolipids, tri- and tetragalactosyldiacylglycerol [13], which are not natural constituents of the chloroplast membranes. For this reason the physiological significance of galactolipid: galactolipid galactosyltransferase for digalactolipid synthesis has been questioned by some authors

L161.

Measurement of activities of the spinach galactosyltransferases is greatly complicated by a close cooperation of UDPgalactose: 1,2-diacylglycerol galactosyltransferase and galactolipid: galactolipid galactosyltransferase, which occurs both in isolated envelope membranes [13,14] and in intact chloroplasts [17]. The monogalactosyldiacylglycerol, newly produced by UDPgalactose: 1,2-diacylglycerol galactosyltransferase, is consumed rapidly by galactolipid: galactolipid galactosyltransferase and converted into di- , triand tetragalactosyldiacylglycerol. Conversely, the diacylglycerol liberated by galactolipid: galactolipid galactosyltransferase is a substrate for monogalactolipid synthesis by UDPgalactose: 1,2-diacylglycerol galactosyltransferase. Consequently, in most studies on galactolipid synthesis where chloroplast membrane fractions were incubated with galactose-labeled UDPgalactose, synthesis of monogalactolipid and of di- , tri- and tetragalactolipid are measured simultaneously. Another complication is that envelope membranes become artificially enriched with diacylglycerol during their isolation procedure, as a result of the activation of galactolipid: galactolipid galactosyltransferase by breakage of the chloroplasts [15,16]. So two main problems arise for the determination of UDPgalactose: 1,2-diacylglycerol galactosyltransferase activity in envelope membranes: it is difficult to control the concentration of diacylglycer01 substrate, and to measure it apart from galactolipid: galactolipid galactosyltransferase activity. In view of the key function of UDPgalactose: 1,2-diacylglycerol galactosyltransferase in galactolipid metabolism, we developed an assay procedure fairly specific for the enzyme, in which the interference of galactolipid: galactolipid galactosyltransferase activity is fairly reduced, and controlled amounts of diacylglycerol (with a known fatty acid composition) can be furnished to the enzyme. The development of a specific assay for galactolipid: galactolipid galactosyltransferase [14] enabled us earlier to study this enzyme apart from UDPgalactose: 1,2-diacylglycerol galactosyltransferase. Now, the possibility for separate measurement of both galactosyltransferases clears the way for characterization studies with respect to the effects of cations and inhibitors, and to the suita-

191

bility of various molecular species of lipids as substrates for each one, as described in this paper. Experimental

procedures

Lipids. All phosphatidylcholines used were 1,2diacyl-sn-glycero-3-phosphatidylcholines. Egg phosphatidylcholine and molecular species of phosphatidylcholine (Sigma, U.S.A.) were chromatographically pure. Crude phosphatidylcholine from soybean meal (Serva, Heidelberg, F.R.G) was purified by three runs of preparative TLC, with elution by chloroform/ methanol/ water (65 : 25 : 4, v/v). [ g[ycerol-3H]Phosphatidylcholine (175 MBq/mmol) was obtained from germinating pollen of Lilium longijlorum L., as described by Helsper and Pierson [18]. 1,2Di[l-‘4C]oleoyl-sn-3phosphatidylcholine (Amersham International, U.K.) was diluted with unlabeled dioleoylphosphatidylcholine to a specific radioactivity of 63 MBq/mmol. Galactose-labeled [ 14C]monogalactosyldiacylglycerol (28-34 MBq/mmol) and [‘4C[digalactosyldiacylglycerol (16 MBq/mmol) were prepared by incubation of spinach envelope membranes with UDP[U-14C]galactose (71 MBq/mmol), followed by separation of the labeled galactolipids [14]. An aliquot of the purified [‘4C]monogalactosyldiacylglycerol was reduced by hydrogenation over Pd-charcoal. The resulting (partially) saturated [r4C]monogalactosyldiacylglycer01 was repurified by TLC on silica gel (elution with chloroform/ methanol/ water, 65 : 35 : 4, v/v), and had a specific radioactivity of 34 MBq/ mmol. Labeled galactolipids were solubilized by sonication with sodium desoxycholate [14] in 50 mM Tricine (pH 7.2 with Tris), in a concentration ratio of 100 I_IM galactolipid/200 PM desoxycholate. Quantification of lipids by GLC [19], analysis of radioactive lipids [14] and determination of protein [lo] were described before. Argentation TLC of labeled mono-, and digalactosyldiacylglycerols was according to Siebertz et al. [20]. Liposomes of phosphatidylcholine were prepared in a concentration of 7 mg lipid/ml by sonication in 5 mM Tricine (pH 7.2 with NaOH) for 5 min at 15 pm using a Soniprep 150 (M.S.E., Crawley, U.K.) sonicator.

Membranes. Envelope membranes were isolated from spinach chloroplasts according to Deuce and Joyard [21]. The following modifications were employed in order to reduce diacylglycerol production by galactolipid: galactolipid galactosyltransferase activity during the isolation procedure. The intact, purified chloroplasts were burst and thylakoids were collected by centrifugation in a SS 34 rotor (Sorvall, Norwalk, U.S.A.) for 10 min at 12 000 x g,,, (4OC). The supernatant was supplied with 10 mM EDTA and then layered on top of a sucrose gradient [21]. After gradient centrifugation, the envelope membranes were collected, pelleted and stored at - 80 o C [14]. All buffers for centrifugation and pelleting contained 1 mM EDTA. Diacylglycerol in the membranes was quantified for each batch, and was 60-130 nmol/ mg protein. Envelope membranes were sonicated before use in 5 mM Tricine (pH 7.2 with NaOH) for 5 s, in a concentration of 2 mg protein/ml. Sonication did not influence enzymatic activities as was shown for example in a study of galactolipid metabolizing enzymes [15], where envelope membrane fractions resuspended by pottering [9] were compared with fractions resuspended by sonication [lo]. Assay for UPDgaIactose: 1,2-diacylglycerol galactosyltransferase. Envelope membranes (200 pg protein/ 100 ~1) and phosphatidylcholine liposomes (700 pg lipid/100 ~1) were sonicated together in 5 mM Tricine (pH 7.2 with NaOH) for 5 s on ice. The suspension was divided over ten assay tubes. To each of these, 0.2 units of phospholipase C from B. cereus (Sigma, Type V) were added. After 15 min of pre-incubation at 30°C, all phosphatidylcholine was converted into diacylglycerol, as was checked by TLC and HPLC [19]. Then 35 mM Tricine buffer (pH 7.2 with NaOH) and 0.12 mM UDPgalactose were added (final concentrations) to a final vol. of 100 ~1, and incubations were carried out for 15 min at 30°C unless stated otherwise. Radioactive label was either in phosphatidylcholine ([glycerol- ‘H]phosphatidylcholine, 175 MBq/mmol, or di[l-‘4C]oleoylphosphatidylcholine, 63 MBq/mmol) or in UDPgalactose (UDP[U-‘4C]Gal, 71 MBq/mmol, or LJDP[6-3H]galactose, 610 MBq/mmol). Incubations were stopped by the addition of 1.5 ml methanol and 1.5 ml chloroform. When applied,

192

inhibitors and other effecters were added after the pre-incubation period, except for oleic acid and cholesterol, which were included in the phosphatidylcholine liposomes. Alterations from standard conditions are indicated in the text. Data are representative of at least three experiments. Rates were calculated from incubations of standard conditions and duration. Assay for galactolipid: galactolipid galactosyltransferase. Galactolipid: glactolipid galactosyltransferase activity was assayed, basically as described before [14]. Briefly, a suspension of [ “C]monogalactosyldiacylglycerol/ desoxycholate (75 : 150, nmol/nmol), or of [i4C]digalactosyldiacylglycerol/desoxycholate (150 : 300, nmol/ nmol) was sonicated with envelope membranes (about 500 pg protein) for 10 s. The resulting mixture, in 25 mM Tricine (pH 7.2 with Tris), was kept on ice until the reaction was started. Incubations contained envelope membranes (20 pg protein), [ 14C]digalactosyldiacylglycerol (3 or 6 nmol, respectively) in 25 mM Tricine (pH 7.2 with Tris), and 10 mM MgCl, (final concentrations). Total vol. was 100 ~1. Incubations were for 10 min at 30 o C, and were started by the addition of MgCl 2. Inhibitors were added just before the start of the incubation, except that oleic acid and cholesterol were included in the galactolipid/ desoxycholate micelles. Results of galactolipid: galactolipid galactosyltransferase were quantified by taking into account the total amount of mono- or digalactosyldiacylglycerol, i.e., the amount of added labeled galactolipid plus the amount of galactolipid already present in the envelope membranes. Both sources of galactolipid are converted by galactolipid: galactolipid galactosyltransferase at the same rate, as has been established for monogalactosyldiacylglycerol [15], and derived for digalactosyldiacylglycerol [19]. Since it has been shown that galactolipid: galactolipid galactosyltransferase activity is strongly dependent on the total galactolipid content present during the incubation [15], the total amount of mono- or digalactosyldiacylglycerol was quantified for each experiment. Generally, data are given from at least three experiments, which were performed in duplicate. Chemicals. The sources of lipids are mentioned

above. Other chemicals were of analytical grade. Metallic chlorides, 2-iodoacetamide and bovine serum albumin were supplied by Merck (F.R.G.); uridine-containing compounds were from Boehringer (Mannheim, F.R.G.); N-ethylmaleimide was from Serva (F.R.G.); all other potential inhibitors tested were purchased from Sigma (U.S.A.). Results Control of the diacylglycerol content of envelope membranes A reliable assay for the measurement of UDPgalactose: 1,2-diacylglycerol galactosyltransferase in envelope membranes requires control of the amount and fatty acid composition of its substrate diacylglycerol. A preferable manner of operation would be to use membranes low in endogenous diacylglycerol and add the required diacylglycerol externally. Unfortunately, envelope membranes contain, after their isolation, variable and generally high amounts of endogenous diacylglycerol, as a consequence of the activation of galactolipid: galactolipid galactosyltransferase during the isolation procedure [15,22]. Therefore, membranes low in diacylglycerol can be obtained only when galactolipid: galactolipid galactosyltransferase is inactivated, for instance by proteinase treatment of the chloroplasts [15,16]. Since we wanted to isolate envelope membranes in which both UDPgalactose: 1,2-diacylglycerol galactosyltransferase and galactolipid: galactolipid galactosyltransferase could be studied, the use of proteinase was not chosen. We followed an altemative method: the membranes were isolated by a fast isolation procedure which was partially in the presence of EDTA (see Experimental procedures), thus minimizing the cation-induced stimulation of galactolipid: galactolipid galactosyltransferase [14]. By this method, the diacylglycerol content of the membranes was reduced to 60-130 nmol/mg envelope protein. A further depletion of diacylglycerol, e.g., by incubation of the membranes with UDPgalactose, was not possible due to the activity of galactolipid: galactolipid galactosyltransferase during the subsequent pelleting of the membranes. Several ways were studied of incorporating external diacylglycerol into envelope membranes. In

193

a preliminary experiment, aqueous suspensions of diacylglycerol with desoxycholate or n-octylglucoside were sonicated with the membranes, but UDPgalactose: 1,2-diacylglycerol galactosyltransferase activity varied considerably with the concentrations of protein and detergent. When detergent was omitted, even high sonication power resulted in poor incorporation of the added diacylglycerol. A reproducible loading of envelope membranes with external diacylglycerol, however, could be achieved by short sonication of the membranes with phosphatidylcholine liposomes, followed by conversion of the phosphatidylcholine with phospholipase C. Fig. 1 shows the results of such an experiment. Pre-incubation of a mixture of envelopes and [glycerol- 3H]phosphatidylcholine with phospholipase C resulted in the formation of diacyl[ 3H]glycerol. Addition of UDPgalactose then caused a rapid production of labeled monogalactosyldiacylglycerol, followed by a rather slow production of digalactosyldiacylglycerol. Hence, the labeled diacylglycerol was a good substrate for UDPgalactose: 1,2-diacylglycerol galactosyltransnmol I mg protein

1

125

100

: 75

50

ii

\ \

\\ \ \ I

\

25

0

@_ Ae

7 %

\\ \ 0

l ‘+-----.. y /.HAPA --20

I

--+____ LO

60 time (min

1

Fig. 1. UDPgalactose: 1,Zdiacylglycerol galactosyltransferase assay, time course of galactolipid synthesis from diacyl[ 3H] glycerol. Envelope membranes (50 pg protein) were sonicated with liposomes of [ glycerof-3H]phosphatidylcholine (4.8 pg), and 0.2 units of phospholipase C were added. At t = 0, buffer was adjusted to 35 mM Tricine (pH 7.2 with NaOH) and UDPgalactose was added. Further conditions are described in Experimental procedures. Radioactivity is given for phosv), diacylglycerol (Ophatidylcholine (v.)* monogalactosyldiacylglycerol (0 ~ 0), and digalactosyldiacylglycerol (AA).

ferase. Since only a small amount of diacyl[ 3HIglycerol was generated in this experiment, UDPgalactose: 1,2-diacylglycerol galactosyltransferase soon became depleted of its labeled substrate, and the rate of monogalactosyldiacylglycerol synthesis slowed down in time (Fig. 1). Based on this indirect way of diacylglycerol generation, standard conditions were developed for the measurement of this enzyme (see Experimental procedures). Assay for UDPgalactose: syltransferase

1,2-diacylglycerol

galacto-

UDPgalactose: 1,2-diacylglycerol galactosyltransferase activity was measured routinely with an excess of phosphatidylcholine as source for diacylglycerol: we used 3.5 mg soybean phosphatidylcholine (equivalent to 4450 nmol, i.e., 35-75 times the amount of endogenous diacylglycerol)/mg membrane protein, sufficient phospholipase C to convert all phosphatidylcholine within the pre-incubation period, and UDP[‘4C]galactose as label. Under such standard conditions 92% of the incorporated i4C label was found in monogalactosyldiacylglycerol, and the remaining 8% was found in di- , tri- and tetragalactosyldiacylglycerol (Table I, Expt. A). The molecular species composition of the produced [‘4C]monogalactosyldiacylglycerol corresponded quite well with the species composition of the phosphatidylcholine source (see below). It was necessary to check the specificity of the assay for monogalactosyldiacylglycerol synthesis under conditions of optimal galactolipid: galactolipid galactosyltransferase activity. Since galactolipid: galactolipid galactosyltransferase is strongly stimulated by Mg2+ ions [14], the influence of Mg2+ on galactolipid synthesis was tested in the presence and in the absence of external phosphatidylcholine and phospholipase C. In the presence of phosphatidylcholine and phospholipase C, total galactolipid synthesis and galactolipid: galactolipid galactosyltransferasecatalyzed formation of di- , tri- and tetragalactolipid doubled by addition of Mg2+, but the contribution of monogalactosyldiacylglycerol remained constant on about 92% (Table I, Expt. A). Without phosphatidylcholine and phospholipase C, addition of 10 mM MgCl, resulted in a 6.5-fold

194

TABLE

I

SPECIFICITY OF THE UDP-GALACTOSE: ACYLGLYCEROL GALACTOSYLTRANSFERASE SAY FOR MONOGALACTOSYLDIACYLGLYCEROL SYNTHESIS

1,2-DIAS-

Expt. A: Standard assay conditions. Envelopes (19 pg protein) were mixed with 60 pg soybean phosphatidylcholine, pre-incubated with 0.2 units of phospholipase C and incubated with UDP[t4C]galactose for 15 min at 30“ C. Expt. B: Conditions as for Expt. A, except that phosphatidylcholine and phospholipase C were omitted. During incubation with UDP[i4C]galactose 10 mM MgCl, was present where indicated. Total UDPgalactose incorporation is given, i.e., incorporation into mono- , di- , tri- and tetragalactosyldiacylglycerol (MGDG, DGDG, TGDG and TeGDG, respectively); also given is galactolipid: galactolipid galactosyltransferase-dependent incorporation into DGDG + TGDG +TeGDG. Data are from experiments (SD, n = 3) performed with one batch of envelope membranes with a diacylglycerol content of 60 nmol/mg envelope protein. Expt.

MgCla

(nmol/mg

protein

synthesis of MGDG + DGDG + T(e)GDG A

+

135+ 6 292k17

B

+

78& 519*

2 5

tolipid: galactolipid galactosyltransferase-stimulated UDPgalactose: 1,2-diacylglycerol galactosyltransferase activity. Although Table I shows that the assay did not prevent completely the production of di- , tri- and tetragalactolipid, it may be noted that those lipids were mostly fairly constant and minor products, comprising together only 5-9% of the produced galactolipids (see Figs. 2-4). Various characteristics of the UDPgalactose: 1,2-diacylglycerol galactosyltransferase assay are shown in Figs. 2-4. A time course (Fig. 2, curve A) indicates that monogalactosyldiacylglycerol synthesis continued for at least 1 h; continuing synthesis was measured even after 6 h of incubation (not shown). Since the synthesis already decreased slowly after 10 min, the UDPgalactose: 1,2-diacylglycerol galactosyltransferase rates de-

per h)

synthesis of DGDG + T(e)GDG

11.0+0.4 25.9 + 1.6 4.6kO.2 263 &5

& of label in MGDG

nmol / mg protem

92 91 94 49

increase of total galactolipid synthesis and in a 60-fold increase of galactolipid: galactolipid galactosyltransferase products, the contribution of di- and higher galactolipids increasing from 6% to 51% (Table I, Expt. B). So it is clear that the Mg’+-induced activation of galactolipid: galactolipid galactosyltransferase is greatly reduced under assay conditions, and consequently the delivering of diacylglycerol by galactolipid: galactolipid galactosyltransferase to UDPgalactose: 1,2-diacylglycerol galactosyltransferase is largely prevented. Apparently, the presence of phosphatidylcholine and phospholipase C inhibits cooperative action of UDPgalactose: 1,2-diacylglycerol galactosyltransferase and galactolipid: galactolipid galactosyltransferase, and measurement of monogalactosylglycerol synthesis under these conditions reflects real UDPgalactose: 1,2-diacylglycerol galactosyltransferase activity, instead of a galac-

time (tin1 Fig. 2. UDPgalactose: 1,2-diacylglycerol galactosyltransferase assay, time course of galactolipid synthesis from UDP [‘4C]galactose. Galactolipid synthesis in nmol/mg envelope protein is indicated for various experimental conditions. The amount of endogenous diacylglycerol in the membranes was 110 nmol/mg protein. A. Assay conditions: Envelope membranes (21 ng protein) were sonicated with liposomes of soybean phosphatidylcholine (70 pg), pre-incubated with 0.2 units phospholipase C, and incubated for various times with UDP[ i4C]galactose. Synthesis of monogalactosyldiacylglycerol 0), and of di- , tri- and tetragalactosyldiacylglycerol (OA) is given. B. Conditions as for A, but phospholi(Apase C was omitted during the pre-incubation. Synthesis of monogalactosyldiacylglycerol (wn). C. Conditions as for A, but phosphatidylcholine and pre-incubation with phospholipase C were omitted. Monogalactosyldiacylglycerol synthesis (O.).

195 down,

rived from standard 15 min incubations are therefore somewhat lower than maximal intitial rates. Fig. 2 also gives the time courses from two control experiments. Omission of the pre-incubation with phospholipase C resulted in a markedly reduced monogalactolipid production (Fig. 2, curve B), probably as a consequence of dilution of endogenous diacylglycerol substrate with the added phosphatidylcholine. Omitting phosphatidylcholine and phospholipase (Fig. 2, curve C) resulted initially in a quite similar rate of monogalactosyldiacylglycerol synthesis. Apparently UDPgalactose: 1,2-diacylglycerol galactosyltransferase was already saturated with substrate lipid without the addition of external diacylglycerol, in accordance with the relatively high diacylglycerol concentration of the envelopes used in this experiment (in contrast to the membranes used in the experiment of Table I: see legends to Table I and Fig. 2). After about 40 min of incubation synthesis slowed

because

of depletion

of the endogenous

diacylglycerol. Under assay conditions, UDPgalactose: 1,2-diacylglycerol galactosyltransferase activity was linear up to 70 pg envelope protein (Fig. 3A). With higher amounts of protein it deviated from linearity, and the contribution of contaminating galactolipid: galactolipid galactosyltransferase activity increased. Increasing the amount of soybean phosphatidylcholine resulted in a slight decrease of UDPgalactose: 1,2-diacylglycerol galactosyltransferase activity (Fig. 3B). The contribution of galactolipid: galactolipid galactosyltransferase was suppressed from 15 l.tg phosphatidylcholine and more. It was necessary to use well-purified phosphatidylcholine, otherwise UDPgalactose: 1,2-diacylglycerol galactosyltransferase activity strongly decreased with higher phosphatidylcholine concentrations. In short-term incubations the maximal rate was found at about 50°C (Fig. 3C), but

150

i0

i

nmol slo$xan

PC +JO”

&4-A----A--. I

20

80

10 temp. (“Cl

01

!

03

I

0.5 rd4 UOPGal

Fig. 3. UDPgalactose: 1,2-diacylglycerol galactosyltransferase assay characteristics. A. Variation in the amount of envelope membranes. B. Variation in the amount of soybean phosphatidylcholine. C. Variation of incubation temperature. D. Variation in UDPgalactose concentration. Standard conditions were: envelope membranes (20 pg protein) mixed with liposomes of soybean phosphatidylcholine (70 pg) were preincubated with phospholipase C (0.2 units), and incubated with 0.12 mM UDP[‘4C[galactose for 15 min at 30 o C (pH 7.2). Enzymatic activities are as defined in Table I. UDPgalactose: 1,2-diacylglycerol galactosyltransferase 0) and galactolipid: galactolipid galactosyltransferase (A -A) activities are given. (O-

196

the maximum shifted to 30’ C in longer-term incubations (not shown). The ratio of UDPgalactose: 1,Zdiacylglycerol galactosyltransferase and galactolipid: galactolipid galactosyltransferase activities was not affected by temperature, hence, a lower temperature of 30°C was chosen for standard condition. Dependence on UDPgalactose concentration is shown in Fig. 3D. The apparent K, value was quite constant in three experiments, 40 k 5 PM (S.D., n = 3) whereas the V,,, value was more variable, 135 f 27 nmol/mg per h. Enzymatic activities varied with storage time of the membranes [lo], and purity of the phosphatidylcholine used (see above). The pH optimum for UDPgalactose: 1,2-diacylglycerol galactosyltransferase was around pH 7. The pH curve was rather asymmetrical, with sub-optimal activities of up to at least pH 9 (Fig. 4). Activity of galactolipid: galactolipid acyltransferase was not detected. This acyltransferase, which is a well-documented enzyme of the chloroplast envelope [23], produces 6-O-acylmonogalactosyldiacylglycerol from monogalactosyldiacylglycerol [24]. High rates of this enzyme are usually measured during incubation of envelope membranes with UDP[‘4C]galactose at acid pH [22,23].

nmol / mg protein I h

200

1W

0

5

6

1

6

9 PH

Fig. 4. UDPgalactose: 1Jdiacylglycerol galactosyltransferase assay, effect of pH. Envelope membranes (21 pg protein) sonicated with liposomes of soybean phosphatidylcholine (75 pg), treated with phospholipase C (0.2 units) and incubated with UDP[t4C]galactose in various buffers. Buffer systems used were: 35 mM Mes/NaOH (pH 5.1-7.0), and 35 mM Tricine/NaOH (pH 7.0-8.9) (final conditions). Symbols are as in Fig. 3 (SD., n = 3).

Since fairly specific assays are available now for the measurement of UDPgalactose: 1,2-diacylglycerol galactosyltransferase (this paper) and galactolipid: galactolipid galactosyltransferase [14,15] activities, it is possible for the first time to ascribe the influence of effecters unequivocally to either of the galactosyltransferases. Effects of metal ions and inhibitors on UDPgalactose: I,2-diacylglycerol galactosyltransferase and galactolipid: galactolipid galactosyltransferase activity The influence of various metal ions on UDPgalactose: 1,2-diacylglycerol galactosyltransferase and galactolipid: galactolipid galactosyltransferase activity was measured using the respective assays (Table II). Most tested ions were ineffective on UDPgalactose: 1,2-diacylglycerol galactosyltransferase, only Mg*+ and Mn2’ were stimulatory. These moderate stimulations are to be expected for an enzyme which uses a uridine diphosphate as a substrate. UDPgalactose: 1,2-diacylglycerol galactosyltransferase activity proceeded at a fairly high rate in the absence of of EDTA had only a Mg*+, and the addition slight effect on the enzyme (Table III)..Whereas 1 mM Zn” was somewhat stimulatory (Table III), 10 mM concentrations of Zn*’ were inhibitory to UDPgalactose: 1,2-diacylglycerol galactosyltransferase, as were Fe2+ and Cd*+ (Table II). Stimulation of galactolipid: galactolipid galactosyltransferase by Mg 2+ has been noted before [14]. Table II shows that Mn2+, Ba*+ and Ca*+ are also very potent stimulators of galactolipid: galactolipid galactosyltransferase. Maximal stimulation of Mg2+ and Ca*+ was at 10 mM concentrations, and the simultaneous presence of both cations did not result in a further increase of the activity (not shown). Fe*+ and Co*+ were weakly active, and Zn2’ and Cd*+ were completely inactive. However, as is shown in Table IV, Zn*+ and Cd*+ were inhibitory to galactolipid: galactolipid galactosyltransferase in the presence of Mg*+. The influence of several potential inhibitors of galactolipid synthesis is presented in Tables III and IV. Various o-galactose derivatives had no clear effect (Table III). Low concentrations of UDPGlc, UMP and UDP were inhibitory to UDPgalactose: 1,2-diacylglycerol galactosyltrans-

197

TABLE

TABLE

II

INFLUENCE OF CATIONS FERASE ACTIVITIES

ON

GALACTOSYLTRANS-

UDPgalactose: 1,2-diacylglycerol galactosyltransferase (UDGT) activity was measured with 20 ng envelope protein, 76 pg dioleoylphosphatidylcholine, and UDP[ 3H]galactose. Indicated cations were present during the incubation as their chloride salts (final concentrations). Galactolipid: galactolipid galactosyltransferase (GGGT) was assayed with [i4C]monogalactosyldiacylglycerol as substrate. Incubations contained 20 pg envelope protein. The total amount of monogalactolipid (externallly added plus endogenous) during the incubations was about 450 nmol/mg protein. MgCl, was replaced by the chloride salts of the indicated cations (final concentrations). Values are means * S.D. of three experiments. UDPgalactose: 1,2-diacylglycerol galactosyltransferase activity (120 + 12 nmol/mg protein per h) and galactolipid: galactolipid galactosyltransferase activity (1050 * 40 nmol/mg protein per h) under standard conditions were arbitrarily set at 100 for each experiment. Cation

added

UDGT

GGGT

100 180*10 213_+13 91* 4 125& 9 87i 8 38* 6 1+ 0.5 35* 4 98+10 98i 8 104* 4

5*3 100 11619 115i_4 114k6 15&2 32*4 6i2 5&l 2513 23+4 25+5

(mM) None Mg’+ (10) Mn*+ (10) Baa’ (10) Ca” (10) coz+ (10) Fe’+ (10) Zn2+ (10) Cd’+ (10) NH: (50) K+ (50) Na+ (50)

ferase, but not to galactolipid: galactolipid galactosyltransferase. UDP especially is a quite effective inhibitor of UDPgalactose: l,Zdiacylglycerol galactosyltransferase (Table IV). UDPgalactose had no effect on galactolipid: galactolipid galactosyltransferase activity, i.e., on the synthesis of di- and higher galactolipids (Table III). Concerning the influence of sulfhydryl reagents, both galactosyltransferases were inhibited by low concentrations of 4-(hydroxymercuri)benzoic acid, but were insensitive towards 2-iodoacetamide. In contrast to the indirect measurements of Mudd et al. [25], we found UDPgalactose: 1,2-diacylglycerol galactosyltransferase more sensitive to N-ethylmaleirnide than was galactolipid: galactolipid galactosyltransferase. Of the in-

III

POTENTIAL INHIBITORS FERASE ACTIVITIES

OF

GALACTOSYLTRANS-

UDPgalactose: 1,2-diacylglycerol galactosyltransferase (UDGT) activity was measured with 18 pg envelope protein, 50 pg dioleoylphosphatidylcholine and UDP[ 3H]galactose. Final concentrations of agents are given in mM (in parenthesis). Galactolipid: galactolipid galactosyltransferase was assayed with [‘4C]monogalactosyldiacylglycerol as substrate, 18 /.rg envelope protein and 10 mM MgCl,. The total amount of monogalactolipid present was 640 nmol/mg protein. Values are means & S.D. of three experiments. UDPgalactose: 1,2-diacylglycerol galactosyltransferase activity (152 f 13 nmol/mg protein per h) and galactolipid: galactolipid galactosyltransferase activity (1220*35 nmol/mg protein per h) under standard conditions were set arbitrarily on 100 for each experiment. Addition

(mM)

None D-Galactose (5) 1-Thio-/?-D-galactopyranose p-Nitrophenyl-P-D-galactopyranoside (5) a o-Nitrophenyl-P-D-galactopyranoside (5) UDPgalactose (0.5) UDPGlc (0.5) UMP (0.5) UDP (0.15) 5-Fluoro-uracil(5) 2-lodoacetamide (5) N-Ethylmaleimide (0.5) 4-(HydroxymercurQbenzoic EDTA (1) Oleic acid (0.3) Cholesterol (5) Ethanol (5%, v/v) Zn2+ (1)

(5)

acid (0.01)

UDGT

GGGT

100 106k 8 114+10

100 lOOk 93*

2 4

103k

7

lOO+

6

115* _

9

94*10 101* 1071 11Ok 113* IlO? 96* 78+ 5* lOlk3 103+ 92k 25+

63k 2 83k 5 21* 5 104* 7 106k 6 43+ 4 35* 6 85+3 42k5 84k 2 54i 5 126i 10

7 5 5 7 5 3 2 1

3 3 2

a p-Nitrophenyl-a-D-galactopyranoside and o-galactose diethyl dithioacetal showed quite similar activities of UDPgalactose: 1,2-diacylglycerol galactosyltransferase and of galactolipid: galactolipid galactosyltransferase.

hibitory effects of Cd2+ and Zn2’ (Table IV) Zn2+ especially is interesting, since, within a certain concentration range, it acts as a specific inhibitor for galactolipid: galactolipid galactosyltransferase, e.g., at 1 mM Zn2+ this enzyme was inhibited 75%, whereas UDPgalactose: 1,2-diacylglycerol galactosyltransferase was stimulated 25%. Inhibition of galactolipid synthesis by oleic acid has been reported in spinach envelope membranes [7]. In our separate assays, UDPgalactose:

198 TABLE

IV

50% INHIBITION CONCENTRATIONS FOR GALACTOSYLTRANSFERASES

OF INHIBITORS

UDPgalactose: 1,2-diacylglycerol galactosyltransferase (UDGT) and galactolipid: galactolipid galactosyltransferase (GGGT) were assayed as described in Table III with inhibitors added in various concentrations. Concentrations resulting in 50% inhibition of enzymatic activity are presented. Inhibitor

UDGT

GGGT

UDP UMP UDPGlc

30 PM 2.5 mM 1.5 mM

8 10 -

4-(Hydroxymercuri)benzoic N-Ethylmaleimide ZnCl z CdCl z Ethanol (v/v)

acid

7 0.3 6 8 7%

PM mM mM mM

5 5 0.6 2

=

mM mM

PM mM mM mM

25%

a Galactolipid: galactolipid galactosyltransferase was tested in the presence of 10 mM MgCl,, as in Table III.

1,2-diacylglycerol galactosyltransferase activity was inhibited 57% by 0.3 mM oleic acid, and galactolipid: galactolipid galactosyltransferase was not affected at all (Table III). Since 0.3 mM oleic acid was equivalent to 0.5 mg oleic acid/mg envelope protein, its influence on UDPgalactose: 1,2-diacylglycerol galactosyltransferase probably is irrelevant. A membrane stabilizer like cholesterol had little effect on the activity of either of the galactosyltransferases, even at a concentration of 5 mM (i.e., 10.7 mg/mg envelope protein). Likewise, the solubilizer ethanol had remarkably little influence, especially on galactolipid: galactolipid galactosyltransferase activity (Table Iv). Effects of molecular species of lipid substrates on UDPgalactose: 1,2-diacylglycerol galactosyltransferase and galactolipid: galactolipid galactosyltransferase activity The possibility to discriminate between the galactosyltransferase activities opened the way to examine the influence of particular lipid substrates. Specificity of the enzymes was studied with various molecular species of diacylglycerol (for UDPgalactose: 1,2-diacylglycerol galactosyltransferase) and of monogalactosyldiacylglycerol

(for galactolipid: galactolipid galactosyltransferase). UDPgalactose: 1,2-diacylglycerol galactosyltransferase used all tested molecular species of diacylglycerol, which were made in situ by phospholipase C treatment of the mixture of envelope

TABLE

V

UDP-GALACTOSE: 1,2-DIACYLGLYCEROL GALACTOSYLTRANSFERASE (UDGT) AND GALACTOLIPID: GALACTOLIPID GALACTOSYLTRANSFERASE (GGGT) ACTIVITY WITH VARIOUS MOLECULAR SPECIES OF LIPIDIC SUBSTRATE Incubations for UDPgalactose: 1,2-diacylglycerol galactosyltransferase contained 19 pg envelope protein and various molecular species of phosphatidylcholine as indicated, each 125 pg. Pre-incubation with 0.5 units of phospholipase C was during 20 min. UDPgalactose: 1,2-diacylglycerol galactosyltransferase values are means of monogalactosyldiacylglycerol (MGDG) synthesis *S.D. measured from three experiments. Synthesis with 18 : O/18 : O-PC (118 * 12 nmol/mg protein per h) was arbitrarily set on 100 for each experiment. Lipid mixtures for the measurement of galactolipid: galactolipid galactosyltransferase were prepared by firstly mixing hexaene [ I4C]monogalactosyldiacylglycerol or hydrogenated [ I4C]monogalactosyldiacylglycerol with 2 eq of unlabeled hexaene monogalactosyldiacylglycerol, and next solubilizing these with 6 eq of desoxycholate. Galactolipid: galactolipid galactosyltransferase incubations with the solubilized mixtures (total monogalactosyldiacylglycerol content was 1140 nmol/mg envelope protein) were for 10 min at 30 o C (see Experimental procedures). The conversion of labeled hexaene [‘4C]monogalactosyldiacylglycerol (1640 + 40 nmol/mg protein per h, + S.D., n = 3) was arbitrarily set on 100 for each experiment. Abbreviations as in Table I. Enzyme

Species of lipid substrate

Synthetic product(s) and rate of formation

UDGT

Diacylglycerol from:

MGDG

18:0/18:0-PC 16 : O/16 : O-PC 10 : o/10 : O-PC 16:0/18:1-PC 18 : l/16 : O-PC 18:1/18:1-PC 18 : 2/18 : 2-PC egg-PC a soybean-PC a

100 79&15 55k12 63k 4 75*11 93*17 71* I 74+14 59* 7

MGDG hexaene monoene/saturated

DGDG + T(e)GDG 100 23*10

GGGT

a Mean number of double bonds in one molecule of egg-PC was 1.2 and in soybean-PC 2.7.

199

membranes and liposomes of various molecular species of phosphatidylcholine (Table V). The molecular species of the produced monogalactosyldiacylglycerol corresponded quite well with the species of phosphatidylcholine, as is shown in Fig. 5 (lanes c-h). For instance, the same species of monogalactosyldiacylglycerol was found when UDPgalactose: 1,2-diacylglycerol galactosyltransferase was assayed with dioleoylphosphatidylcholine and UDP[ l4 Clgalactose, as when di[ I4 Cloleoylphosphatidylcholine was used with unlabeled UDPgalactose (Fig. 5, lanes g-h). The highest 1,2-diacylglycerol galactoU DPgalactose: syltransferase activities were measured with disstearoyl- and dioleoylphosphatidylcholine (Table V). Increase of the desaturation degree of the phospholipid, and shortening of the chain length of the acyl residues resulted in a somewhat lower activity. So, under the experimental conditions used, UDPgalactose: 1,2-diacylglycerol galactosyltransferase reacts with a whole range of species

of diacylglycerol, but has a slight preference for more saturated substrates containing long-chain fatty acids. Galactolipid: galactolipid galactosyltransferase activity was tested with galactose-labeled hexaene monogalactosylglycerol and galactose-labeled hydrogenated monogalactosyldiacylglycerol as substrates. The latter consisted of saturated and monoene species (Fig. 5, lanes a-b). Since solubilization of the hydrogenated monogalactolipid was difficult, it was mixed with two equivalents of hexaene monogalactolipid before incorporation into the membranes (see legend of Table V). Conversion by galactolipid: galactolipid galactosyltransferase of the hydrogenated (partially saturated) monogalactosyldiacylglycerol was considerably lower than that of hexaene monogalactosyldiacylglycerol (Table V). This indicates that this enzyme has a preference for hexaene species as galactosyl acceptor for the conversion into di- , tri- and tetragalactosyldiacylglycerol.

18:0/18:0

18:2/18:2

18:2/18:2

18:3/16:3 18:3/18:3

a

b

C

d

e

f

9

h

Fig. 5. UDPgalactose: 1,2-diacylglycerol galactosyltransferase and galactolipid: galactolipid galactosyltransferase: molecular species of monogalactosyldiacylglycerol. Radioactive labeled monogalactosyldiacylglycerol from different origins was purified by TLC and then subjected to argentation TLC (0.73 kBq) (see Experimental procedures). Autoradiograms are shown of the separated molecular species. Identification was according to literature [12,20]. Expt. 1. Species of monogalactosyldiacylglycerol used as substrate for the galactolipid: galactolipid galactosyltransferase assay. (a) monogalactosyldiacylglycerol obtained from incubation of envelope membranes with UDP[14C]galactose and 10 mM MgCI, for 3 h. This type of monogalactosyldiacylglycerol was normally used for the galactolipid: galactolipid galactosyltransferase assay. (b) As (a), but after hydrogenation over Pd-charcoal. Expt. 2. Species of monogalactosyldiacylglycerol obtained from the UDPgalactose: 1.2.diacylglycerol galactosyltransferase assay. (c) Assay with UDP[“‘C]galactose in combination with 16 :0/18 : l-PC; (d) m combination with 18 :0/18 :O-PC; (e) with soybean-PC. Expt. 3. Species of monogalactosyldiacylglycerol obtained from the UDPgalactose: 1,2-diacylglycerol galactosyltransferase assay. (f) Assay with UDP[i4C]galactose plus 18 : 2/18 : 2-PC; (g) assay with UDP[i4C]galactose plus 18 : l/18 : l-PC: (h) with UDPgalactose plus di[ i4C]oleoylphosphatidylcholine.

200

Such a preference was confirmed by analysis of the (small amounts of) digalactosyldiacylglycerol which were synthetized in the UDPgalactose: 1,2diacylglycerol galactosyltransferase assay with dipalmitoylglycerol and distearoylglycerol. Whereas the produced monogalactolipid in these cases was greatly enriched in the 16 : O/16 : 0 or 18 : O/18 : 0 combination as expected (e.g., Fig. 5, lane d), the simultaneously produced digalactolipid consisted mainly of unsatured, hexaene molecular species (not shown).

The assay for UDPgalactose: 1,2-diacylglycerol galactosyltransferase described in this paper basically consists of two steps. Firstly, diacylglycerol is generated from phosphatidylcholine which was incorporated into the envelope membranes, and secondly, it is used for monogalactosyldiacylglycerol synthesis by the galactosyltransferase. Dome et al. [27] have already demonstrated that similar treatment of envelope membranes with phospolipase C results in a fast degradation of phosphatidylcholine endogenously present in the membranes, and also in a slower degradation of the endogenous phosphatidylglycerol. Indeed, under our assay conditions, externally added and endogenous phosphatidylcholine are both hydrolyzed by phospholipase C, as could be shown by HPLC analysis (which did not detect phosphatidylglycerol) [19]. Therefore, several sources of diacylglycerol are present in the assay mixture: diacylglycerol generated from endogenous phosphatidylcholine and (probably) phosphatidylglycerol, endogenous diacylglycerol and finally diacylglycerol derived from the added (soybean) phosphatidylcholine. In our experiments, the endogenous diacylglycerol sources were typically 330, 170 and 100 nmol/mg envelope protein, respectively. In comparison to these sources, the amount of added phosphatidylcholine (4450 nmol/mg protein) constituted a 13 to 44-fold excess, and permitted the production by UDPgalactose: 1,2diacylglycerol galactosyltransferase activity of molecular species of monogalactosyldiacylglycer01, which are quite similar to the species of the added phosphatidylcholine (Fig. 5). The specificity of the assay for monogalactolipid synthesis

under different conditions was about 92% (Table I), indicating that the activity of both galactosyltransferases was largely decoupled under assay conditions. However, small amounts of di- , triand tetragalactolipid were still produced. Complete inactivation of contaminating galactolipid: galactolipid galactosyltransferase activity could only be obtained after destruction of the enzyme by treating the chloroplast membranes with the proteinase thermolysin [15,16]. Since our goal was to measure UDPgalactose: 1,2-diacylglycerol galactosyltransferase and galactolipid: galactolipid galactosyltransferase activites in the same batch of envelope membranes, such a treatment was undesirable. The present results indicate that the former can now be assayed with sufficient, though incomplete, specificity without the use of proteinase. In unmodified envelope membranes, the presence of magnesium permits considerably higher rates of UDPgalactose-derived UDPgalactose: L2diacylglycerol galactosyltransferase and galactolipid: galactolipid galactosyltransferase activities (Table I, Exp. B). In the assay of the latter, much higher rates of activity were measured (e.g., see legends of Tables II and III and Ref. 15), which represent the conversion of total monogalactosyldiacylglycerol. However, the galactolipid: galactolipid galactosyltransferase-catlyzed formation of di- , tri- and tetragalactosyldiacylglycerol as given in Table I (Expt. B) represents only the conversion of newly labeled monogalactosyldiacylglycerol, and does not include unlabeled monogalactolipid conversion. Application of the assays for the two galactosyltransferases revealed a number of different properties. However, the results obtained must be evaluated carefully, because of the unnatural composition and physical state of the membranes under assay conditions. For instance, distorted membrane structures can be expected as a consequence of the high amounts of diacylglycerol generated in the UDPgalactose: 1,2-diacylglycerol galactosyltransferase assay; and the activity of galactolipid: galactolipid galactosyltransferase is affected by the presence of detergent in the assay [14]. Nevertheless, the results obtained can be compared easily with the scarcely available data about the enzymes in their natural membranes (see below), and thus both assays promise to be useful

201

tools in future purification studies of the enzymes. In most studies of galactolipid synthesis with unmodified chloroplast membranes, production of mono- , di- , tri- and tetragalactolipid were measured together. Under such conditions, a possible specificity of effecters for mono- or oligogalactolipid synthesis could be derived mainly from differences in the distribution of the synthetized galactolipids. Conclusions which have been drawn in the literature about monogalactolipid synthesis are generally in agreement with the present results. For example, Mudd et al. [4] found an optimal pH of 7.2 in acetone powders from spinach chloroplasts, and an optimal temperature of 50 o C. Likewise, pH optima of around 8 have been reported for monogalactolipid synthesis in spinach [16,28] and pea [ll] chloroplast envelope membranes (compare Fig. 4). Various authors have published apparent K, values for UDPgalactose comparable to the present ones: for spinach K, values of about 38 PM [7,13] or 90 PM [28] are given, and for pea membranes the K, was approx. 36 PM [ll]. Moreover, apparent inhibitors for monogalactolipid synthesis in envelope membranes such as UDP, UMP, UDPGlc and sulfhydryl reagents [7,25,29] were similarly effective in the UDPgalactose: 1,2-diacylglycerol galactosyltransferase assay. As regards the assay for galactolipid: galactolipid galactosyltransferase, the pH optimum of 6-7 [14] is in good agreement with optima estimated elsewhere with unmodified envelope membranes [13,28]. Furthermore, the effects of cations found in the galactolipid: galactolipid galactosyltransferase assay (Table II) are very similar to their effects on the activity the enzyme as measured in intact spinach chloroplasts [30]. The inhibition experiments (Tables III and IV) may give some insight into the molecular mechanism of the galactosyltransferases. Neither of the enzymes seems to have a high affinity for the galactosyl part of their substrates, although UDPgalactose: 1,2-diacylglycerol galactosyltransferase can distinguish between UDPgalactose and UDPGlc (Table III); it seems to recognize especially the uridine-phosphate moiety of its substrate. UDP is a very effective inhibitor of UDPgalactose: 1,Zdiacylglycerol galactosyltransferase. The lack of effect of uridine-phosphate derivatives on galactolipid: galactolipid galacto-

syltransferase activity is not unexpected. The inhibition at very high concentrations of these derivatives (Table IV) may be due to chelation or precipitation of magnesium. Further, the lack of any effect of UDPgalactose in the galactolipid: galactolipid galactosyltransferase assay demonstrates clearly that a direct galactosylation of monogalactosyldiacylglycerol by UDPgalactose can not be measured in our experiments (Table III). The presence of sulfhydryl groups seems essential for both galactosyltransferases, although the accessibility for some sulfhydryl reagentia must be different for the each enzyme. The assays for UDPgalactose: 1,2-diacylglycer01 galactosyltransferase and galactolipid: galactolipid galactosyltransferase indicate very different kinetic properties of the galactosyltransferases. It has been shown before that the activity of the latter in envelope membranes is strongly dependent on the concentration of monogalactolipid. Saturation of the enzyme was observed above 3300 nmol/mg protein, and maximal velocity was 2700 nmol/mg protein per h [15]. In contrast, the I$,, of UDPgalactose: 1,2-diacylglycerol galactosyltransferase appears to be much lower, about 135 nmol/mg per h (Fig. 3). This enzyme seems saturated at low concentration of diacylglycerol. Since it was difficult to obtain envelope membranes limited in diacylglycerol, velocities of UDPgalactose: 1,2-diacylglycerol galactosyltransferase under substrate-limiting conditions could only be estimated. For instance, from Fig. 2 (curve C) the amount of diacylglycerol in the membranes at half-maximal velocity is calculated at about 40 nmol/mg protein. Similar values can be calculated from data in the literature [6,31]. Such a low value may indicate a relatively high affinity of UDPgalactose: 1,2-diacylglycerol galactosyltransferase for its lipidic substrate, as compared to galactolipid: galactolipid galactosyltransferase. High initial rates of UDPgalactose-derived galactolipid synthesis of about 3000 nmol/mg protein per h have been measured in unmodified spinach envelope membranes, where both galactosyltransferases were active [6,7,13]. Considering the values of the separately determined velocities of both, such high rates suggest that galactolipid: galactolipid galactosyltransferase is the rate controlling enzyme in the cooperative system. It should be

202

pointed out that the high velocities with unmodified envelope preparations were obtained from short-term incubations (1 min) with UDP[ l4 Clgalactose, where only monogalactosyldiacylglycerol was labeled. However, such observation does not necessarily mean that only UDPgalactose: 1,2-diacylglycerol galactosyltransferase is active and exclude galactolipid: galactolipid galctosyltransferase activity, but simply means that the concentration of labeled, newly produced monogalactolipid is negligible in comparison to the endogenous monogalactolipid concentration. Indeed, we have shown previously that galactolipid: galactolipid galactosyltransferase is strongly dependent on the monogalactolipid concentration

[W Although our results indicate that all of the digalactosyldiacylglycerol arises by action of galactolipid: galactolipid galactosyltransferase, the cooperativity of UDPgalactose: 1,2-diacylglycerol galactosyltransferase and galactolipid: galactolipid galactosyltransferase may be questioned in view of the localization of the latter in the outer envelope membrane of spinach chloroplasts [15,16], and of the former in the inner spinach envelope membrane [9,10]. In our opinion, however, the cooperativity observed both in envelope membrane preparations [13,14] and in intact spinach chloroplasts [30] can be understood if one assumes that galactolipid: galactolipid galactosyltransferase is an integral membrane protein of the outer envelope, which has an active site on the ES, facing the chloroplast inside, and a proteinase sensitive site on the PS, facing the cytosol, as was demonstrated by Dorne et al. [16]. In our experiments we found that a UDPga1,Zdiacylglycerol galactosyltransferase lactose: had a certain preference for more saturated lipid substrates, and galactolipid: galactolipid galactosyltranserase a preference for unsaturated, hexaene species of monogalactolipid as galactosyl acceptor. Such specificities can be derived also from data in the literature with unmodified membranes. When spinach envelope membranes were labeled with UPD[i4C]galactose, Siebertz et al. [20] found labeled oligoene and hexaene species of monogalactosyldiacylglycerol, but most of the labeled digalactosyldiacylglycerol and nearly all of the triand tetragalactosyldiacylglycerol species were

hexaene. Further, in in vivo experiments where envelopes were isolated from spinach leaves after labeling with 14COZ, oligoene species of both monoand digalactosyldiacylglycerol were recovered, which were nearly uniformly labeled. For the (oligoene) digalactolipid this included labeling of the inner and the outer galactosyl group. Label of the simultaneously found hexaene digalactosyldiacylglycerol, in contrast, was nearly exclusively in the outer galactosyl moiety. Thus, both types of experiments indicate that oligoene diacylglycerol species are good substrates for the first galactosylation enzyme, and that hexaene species of monogalactosyldiacylglycerol are preferential galactosyl acceptors for digalactolipid synthesis, in agreement with our data. Our results point to an aspecific activation of galactolipid: galactolipid galactosyltransferase by a number of metal ions, in contrast to UDPgalactose: 1,Zdiacylglycerol galactosyltransferase (Table II). Such activation may be due to an indirect effect of the cations on the membrane lipids, rather than to a direct effect on the enzyme. Effects of cations on galactolipids are known from the literature. For instance, studies with membraneous lipids from chloroplasts and from Cyanobacteria demonstrated phase separation between the non-bilayer forming monogalactosyldiacylglycerol and the remaining lipids after the addition of MgCl, or NaCl [32,33]. Likewise, it has been suggested that cations induce changes in the lateral surface pressure of the lipids in the chloroplast membranes [34]. It is possible that similar cation effects in our experiments make monogalactosyldiacylglycerol better available as a substrate for galactolipid: galactolipid galactosyltransferase. So far detailed biochemical studies on monoand digalactolipid synthesis have been performed mainly with the 16 : 3-plant [35] spinach. Now it is necessary to extend those studies to other plant species, notably 18 : 3-plants. As yet, the metabolic way and the regulation of galactolipid synthesis in other plants than spinach are only poorly understood [3]. Acknowledgements

We wish Laeyendecker

to

thank G. Bijgemann for experimental and

and Th. technical

203

assistance, and J.H. Veerkamp (Dept. of Biochemistry, Nijmegen) for reading the manuscript. References 1 Douce, R., Block, M.A., Dome, A.-J. and Joyard, J. (1984) in Subcellular Biochemistry, Vol. 10 (Roodyn, D.B., ed.), on. l-84. Plenum Press, New York 2 Gounaris, K. and Barber J. (1983) Trends Biochem. Sci. 8, 378-381 J.F.G.M. and Heemskerk, J.W.M. (1987) in 3 Wintermans, Plant Lipids: Biochemistry, Structure and Function (Stumpf, P.K., Mudd, J.B. and Nes, W.D., eds.), Plenum Press, New York, in the press 4 Mudd, J.B., Van Vliet, H.H.D.M. and Van Deenen, L.L.M. (1969) J. Lipid. Res. 10, 623-630 5 Deuce, R. and Guillot-Salomon, T. (1970) FEBS Lett. 11, 121-124 6 Joyard, J. and Deuce, R. (1976) Biochim. Biophys. Acta 424. 125-131 J.F.G.M. (1979) FEBS 7 Van Besouw, A. and Wintermans, Lett. 102, 33-37 M., Heinz, E., McKeon, T. and Stumpf, P.K. 8 Frentzen, (1983) Eur. J. Biochem. 129, 629-636 9 Block, M.A., Dome, A.-J., Joyard, J. and Deuce, R. (1983) J. Biol. Chem. 258, 13281-13286 J.W.M., Bogemann, G. and Wintermans 10 Heemskerk, J.F.G.M. (1985) B&him. Biophys. Acta 835, 212-220 11 Cline, K. and Keegstra, K. (1983) Plant Physiol. 71,366-372 Z. Phys12 Siebertz, M. and Heinz, E. (1977) Hoppe-Seyler’s iol. Chem. 358, 27-34 J.F.G.M. (1978) Bio13 Van Besouw, A. and Wintermans, chim. Biophys. Acta 529, 4453 J.W.M., Bogemann, G. and Wintermans, 14 Heemskerk, J.F.G.M. (1983) Biochim. Biophys. Acta 754, 181-189 J.W.M., Wintermans, J.F.G.M., Joyard, J., 15 Heemskerk, Block, M.A., Dome, A.-J. and Deuce, R. (1986) Biochim. Biophys. Acta 877, 281-289 16 Dome, A.-J., Block, M.A., Joyard, J. and Deuce, R. (1982) FEBS Lett. 145, 30-34

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