- Email: [email protected]
Biosynthesis of 2-O-D-glycerol-α-D-galactopyranoside (Floridoside) in marine Rhodophyceae
Plant Science Letters, 23 (1981)349--357 Elsevier/North-Holland Scientific Publishers Ltd.
349
BIOSYNTHESIS OF 2~)-D-GLYCEROL~-D-GALACTOPYRANOSIDE (FLORIDOSIDE) IN MARINE RHODOPHYCEAE
BRUNO P. KREMER* and GUNTER O. KIRST Universitiit zu K~ln, Seminar fiir Biologie und ihre Didaktik, D-5000 KSln 41 and Technische Hochschule Darmstadt, Institut fiir Botanik, D-6100 Darmstadt (F.R.G.)
(Received March 17th, 1981) (Revision received July 8th, 1981) (Accepted July 15th, 1981)
SUMMARY
Floridoside (2-O-D-glycerol~-D-galactopyranoside) is the main photosynthetic product of many Rhodophyceae. In 14C-fixation pattern glycerol 3-phosphate as well as floridoside phosphate are rapidly labelled. It is assumed, based on their incorporation kinetics, that these compounds are precursors of floridoside. Two enzymes involved in the biosynthesis of floridoside were found to be active in homogenates of Rhodophyceae. Glycerol 3-phosphate dehydrogenase probably catalyzes the formation of glycerol 3-phosphate derived from dihydroxy acetone phosphate. Glycerol 3-phosphate and UDP-galactose are condensed to floridoside phosphate by floridoside phosphate synthetase (UDP-galactose: sn-glycerol-3-pho~ phate-2-D-galactosyl transferase).
INTRODUCTION
Marine and freshwater species of red algae (Rhodophyceae) differ from most other photosynthetic plants by the occurrence of particular heterosides. These are usually accumulated as the main soluble products of photosynthetic carbon reduction. Among them, floridoside has long been known as a typical low-molecular-weight red algal carbohydrate [1,2]. It was identified as a 2~)-D-glycerol-a-D-galactoside [3] and has been/raced in all species investigated in detail except those belonging to the red algal order Ceramic.lee: species of this taxonomic effinity accumulate digeneaside *To whom correspondence should be sent at: ~ 51, D-5300 Bonn 2, F.R.G. Abtzrevistions: BSA, bovine smmm albumin; DHAP, dihydroxy acetone phosphate;
PGA, S - p h m p h c ~ . 0304--4211/81/0000-..0000/$02.75 © 1981 Ek~r~-r/North-Hoiland Scientific P u b i M ~ Ltd.
350 (mannosidoglycerate) [4,5]. When exposed to H14CO~, floridoside receives strong 14C-labelling after relatively short, term photosynthesis [4,6,7]. Floridoside content in marine algae is usually in the range 1.5--8% on a dry-weight basis [8]. It has become evident from kinetic tracer studies using radiocarbon that both constituents of floridoslde, galactose as well as glycerol, are 14C-labeiled during photosynthesis and are thus obviously derived from intermediates of the reductive pentose phosphate cycle. However, no further experimental data have hitherto been presented to give a detailed account to the biosynthetic pathway of floridoside. The present study is mainly concerned with the further characterization of some intermediates and enzymes presumably involved in the biosynthesis of glycerolgalactoside in red algae. MATERIAL AND METHODS Plant material Dumontia incrassata (O.F. Mi~ller) Lamour., Chondrus crispus Stackh., Cystoclonium purpureum (Lightf.) Batt., Corallina officinaUs L., and Porphyra umbilicalis (L.) J. Ag. were collected during low tide from their intertidal habitats on the rocky shores of Helgoland (North Sea, Germany). All specimens were carefully checked and maintained in seawater flow tanks at environmental temperatures (5--10°C) under continuous light irradiance. All plants were used for the experiments within 12 h of collection. Lomentaria umbeUata, (H.u.H.) Yendo, Corallina officinalis L., and Catenella nipea Zan. were collected from Port Jackson (Sydney). Cleaning and maintenance of the algae, H14CO3-fixation experiments and identification of labelled compounds were done as described by K i n t and Bisson [7]. Enzyme extraction Selected thallus samples of 1--2 g fresh weight were rapidly deep-frozen in liquid N2 and homogenized by grinding in a mortar with qtmrtz sand. To the still frozen mixture 1 g Polyclar AT and 4 nfl Tris--C1 buffer (0.1 M, pH 7.2) as well as detergent (Triton X-100) were added to give a 0.5--1% solution (v/v). The extraction buffer contained ~ A (10 ~mol), D-araboasco~bate (10 #tool), ~ (10 ~mot), dithioth~itol (10 t~mol). The thoroughly homogenized mixture was then c e n t t ~ at 2--4°C and 40 000 × g for 15 rain. The r e c i t i n g m p e m e t m t was ~ as a paeticle-free crude enzyme extract end umd for enzyme t e ~ without further purification. The enzyme extracts were stored at O°C for not longer than I h. Enzyme mmys Glycerol-8-pholphate dahydrogmm~ (EC 1.1.1.8). T h e preswi~ o f this
351 enzyme was investigated by two different procedures. In a first set of experiments the test system contained 1.2 ml Tris--C1 buffer (0.3 M, pH 7.5), 0.1 ml MgCI2 (10 #tool), 0.2 ml NADH (1 #tool) and 0.1--0.5 ml enzyme extract. The reaction was started by adding 0.2 ml dihydroxyacetone phosphate (= 2/~mol; prepared by hydrolysis and ion-exchange chromatography from dihydroxyacetone phosphate dimethylketal as cyclohexylammonium salt according to manufacturer's advice (Boehfinger)). Enzyme reaction was followed by recording decrease in absorbance at 366 nm in a spectrophotometer. In a further series 0.5 ml Tris--Cl buffer (0.3 M, pH 7.5) were incubated at 20°C with 0.1 ml NADH (5 #tool), 0.1 ml NADPH (5 #mol), 0.1 ml ATP (2 #tool), 0.1 ml MgC12 (10 #mol) and 14C-labelled 3-phosphoglycemte (approx. 2.5 × l 0 s cpm; prepared from H14C~ incubated photosynthesizing algae (Kremer 1978c)) as well as phosphoglycerate kinase, glyceraldehyde phosphate dehydrogenase, and triose phosphate isomerase (Boehringer; approx. 200 units/experiment) for 3 h. To 100 #1 of this mixture were added 10/~1 of algal enzyme extract and the reaction continued for 10--60 min. The incubation was stopped by 20 #1 acetic acid (6 N). The reaction products were further analyzed by thin-layer chromatography (see below). Floridoside phosphate synthase (EC number not yet assigned) was measured by the following procedure. The test system contained 50/~1 Tris-C1 buffer (same concentration and cofactors as extraction buffer, but additionally 1% bovine serum albumin (BSA) and 10 mM NaF), 10 #1 uridine~tiphospho-galactose (2 #tool) (Sigma) and 5--10 ~1 enzyme extract. The reaction was started with 10/~1 [i4C] glycerol 3-phosphate (-~ 0.5 #Ci ~ 18.5 kBq; Amersham Buchler CFB 171) and stopped by addition of 10/~1 acetic acid.
Analysis of reaction products To confirm the identity of particular reaction products, the complete test solutions were analysed by thin-layer chromatography on cellulose (MN 300; layer thickness 350--500 #m) using solvent systems as detailed earlier [7,10].
RESULTS AND DISCUSSION
During short-term photosynthesis in H14CO~ glycerol as well as galactose, the precursors of galactosylglyceride, become labelled at appreciable rates. About 3--7% of the 14C incorporated into EtOH-soluble compounds after 30 s were recovered from glycerol (Fig. 1). The percentage of glycerol declined within 2 min to 0.5--2~ (Fig. I and Table I), indicating a rapid turnover of the glycerol pool. This was also confirmed by puise~hase experiments using Lomentaria umbeUata (Table II). It is obvious that, following a labelling pulse, the amounts of 14C confined to glycerol are rather low, whereas floridoside receives strong 14C-labelling. Glycerol is therefore not
352
A
30
%
t
20" oo- ~
10
~
~
~
~
FIo
..n ....
FiO-®
i.................... o Glycerol 30-
o
°/o
I t o~
B tt s"r'l~
20 o ~" ", t~
tO
/ I Fio
"',~
\o~ °°'o---. o_
i
"'-- ................... ~ FIo-(~ -,
Glycerol 1'0 •
5
t
(mini
Fig. 1. Time course of H~4CO;-fixation. Labelling of floridoside (Flo), floridoside phosphate (Flo-P), and glycerol in % of total ~4C in the EtOH-soluble fraetion. Fixation rates into EtOH-soluble compounds were: 3.3 * 0.3 ~mol CO 2 g-1 fresh wt. I 0 min -I (Corollino) and 3.0 ± 0.4 , m o l CO t g-1 fresh wt. 10 rain -~ (Lomentorio).
an accumulated photosynthate. It has to be mentioned, however, that the actual content of free glycerol and galactose might be masked by inevitable hydrolysis offloridos/de during the separation procedure by ion-exchange resins (see below). More attention was therefore directed towards the characterization of an enzyme providing the biosynthesis of the C3-unit of floridoside. TABLE I 14C-INCORPORATION INTO PRECURSORS OF FLORIDOSIDE AND FLORIDOSIDE A F T E R 5 MIN P H O T O S Y N T H E J ~ (MEANS OF 3 TO 5 REPLICATES ± S.D.) FIX), FLORIDOffIDE; F L O P , F L O R I ~ ] I PHOaPHATE. Species
Catene[~ repen# Lomentar~ umbellafa CoraUina o l ~ ' i ~ s l k
% of EtOH4oluble compounds Hexose-P
Fio-P
Flo
Galsetose
Glycerol
18.8 ± 2.4 5.7 ± 0.6
15.3 ± 1.8 18.7 ± 8.7
8.4 ± 1.6 15.7 ± 4.4
5.1 ± 0.5 2.1 ± 0.6
0.9 ± 0.I 2.3 ± 0.8
11.5 ± 0.7
17.4 ± 0.4
25.0 ± 4.8
1.9 ± 0.I
2.8 ± 1.0
353 TABLE II L O M E N T A R I A UMBELLATA. 1'C-LABELLING O F SOLUBLE PHOTOSYNTHATES FOLLOWING A 14C-FEEDING PULSE (5 MIN) IN % OF TOTAL EtOH-SOLUBLE 14C.ACTIVITY F o r abbrevations see Table I. Means of three replicates. Chase period (min)
Compound
Hexose-P FIo-P Floridoside Glycerol
5
I0
11
7
9 24 1
7.5 34 0.8
15
8
10 38 0.6
Appropriate results are exemplified in Fig. 2a for Dumontia incrassata. When the triose phosphate dihydroxy acetone phosphate (DI4AP) is present as a substrate, it is immediately reduced by an algal enzyme (extract) to a glycerol phosphate at the expense of NADH. Linevxity of this reaction is observed at least over 15 min. However, the turnover rate of DHAP under the given experimental conditions is relatively low. This might possibly be due to instability of the substrate preparations. In addition the actual substrate concentration might be considerably less than that calculated from the previous preparation steps. Very similar findings have been obtained with all other red algal species investigated in this connection. In order to confirm the identity of the reaction product 14C-labeUed ft. I Or) I
A 40
o
-r 30 t~
300
B
200
@
o 20
,,.;.
Z (.)
"5 10 E
100
o"
c
rn
1
5
10
15 t (rain)
io
30
60
Fig. 2. Activity of glycerol-3-phosphate dehydrogenase in marine red algae. (a) Enzymatic conversion of the triose phosphate DHAP to glycerol 3-phosphate by an enzyme extract from Dumontia inerassata e x p r e ~ e d as N A I T formed. (b) Incorporation of z4C from triose phosphate (originally supplied as [z4C]PGA) into glycerol 3-phosphate by an enzyme extract from Cystoclonium purpureum.
354
3-phosphoglycerate (PGA) was applied and its subsequent enzymatic conversion was followed. Analyses of the reaction mixture by means of thinlayer chromatography revealed that appreciable amounts of the 14C originally applied are located in glycerol 3-phosphate (-- L-~-glycerophosphate = sn-glycero-3-phosphate). This c o m p o u n d is distinguishable chromatographically from glycerol 2-phosphate (= L~-glycerophosphate = sn-glycero2-phosphate), which was n o t 14C-labelled in this treatment. About 12% of the 14C-activity determined for 3-PGA on the thin-layer chromatograms is confined to glycerol 3-phosphate after 60 min incubation. The time-course of 14C-labelling of this c o m p o u n d by an enzyme extract from Cystoclonium purpureum is shown in Fig. 2b. ~4C-labelling of free glycerol was n o t observed in this assay. Taking into account these findings it might be suggested that in red algae the enzyme providing the conversion of triose phosphate (immediately derived from the RPP cycle) to glycerol phosphate is a L-glycerol-3-pho~ phate dehydrogenase. This enzyme has long been known as Baranowskienzyme from heterotrophic organisms and has more recently been found to be involved in glycerol biosynthesis in the green alga Dunaliella tertiolecta [11]. Its basic function in representatives of the Rhodophyceae is to provide sufficient amounts of glycerol phosphate for further use in the heteroside formation. Though Bean and Hassid [9] report on appreciable amounts of [14C]glycerol phosphate exceeding those of [ 14C]floridoside after 2 rain photosynthesis, this intermediate is mostly n o t pooled. Labelled glycerol p h o ~ phate could n o t be identified unequivocally in the experiments described here. Precursors of floridoside like glycerol (mentioned already above), galactose, floridoside phosphate and floridoside itself are labelled very early and hence the relative a m o u n t exceeds by far the percentage of the few other compounds (i.e. alanine, glycine), which were labelled also after very short times (Fig. 1, Table I). However, the absolute content of free glycerol and glycerol phosphate estimated quantitatively together with floridoside [8] is low and was always less than 0.05% on a dry wt. basis. UDP-galactose has been traced among short-term labelled 14C-photosynthates of the red algae Iridophycus flaccidum [9]. Floridoside phosphate is consistently found among the photosynthetically ~4C-labelled phosphate esters in several species of marine Rhodophyceae. Data for Catenell¢, Lomentaria and Corallina are given in Table I and Fig. 3. After 45 min of H14CO3fixation Grateloupia filicina incorporated 22% of the 14C (EtOH-soluble fraction) into floridoside phosphate, 29% into floridoside and 1% into glycerol, the values for Hypnea valentiae were 3%, 38% and 1% respectively [ 7]. Therefore it seems reasonable to assume an enzyme performing a transfer of the nucleoside on glycerol 3-phosphate to yield floridoside phosphate. The results of appropriate experiments are shown in Fig. 4. When 14Clabelled glycerol 3-phosphate and UDP-galactose were present in the test
355
~=- Glycerol Glycerol
FIo
Fru 1""'4 GA I--'4 Glu
FIo-P
Hexose-P G-6-P H
start
start Ct
L
Co
Fig. 3. Autoradiographs of TLC-separated EtOH-extracts of CateneUa (Ct), Lomentaria (L), and CoralUna (Co) after 5 rain photosynthesis of H14CO;. Compounds identified: Flo, floridoside, polysaccharides (start) on the right; positions of marker substances (on the left): G-6-P, glucose-6-phosphate; Glu, glucose; GA, glyceric acid; Fru, fructose. o" CO 0 ,
200
• 20 pl Enzyme extract o 10p[
~
0
"o 1 5 0 o
e
~
LL ,._
100.
0
•- ~
Q
50
m _J |
o -
1
5
10 t (mini
15
F~. 4. Oystoelonium purpureum~ ~ t y
20 and proportionality of an ensyms trims-
feting UDP..Smlaeko~ on [s*O]glyee¢ol 8 - ] p h o ~ l m t e t h u yid,41,~_ YlceidosJde phoephs~.
356
_,j~je G-3- P ~ ~ U
RPP-cycle l,&
~SUDP-Gal.
CH2OH HO,~-----O CH2OH : 2-glycerol-3-(~----~ ~ O--CH I O (Floridosidephosphate) ~ ~ I Pi OH CH20H ~-Gatactosyl-
Floridoside Fig. 5. Scheme of biosynthesis of floridoside. Abbreviations: RPP-cycle = reductive pentose phosphate cycle; G-3-P = glycerol-3-phosphate. Enzymes tested: ( ~ Glycerol phosphate dehydrogenase (EC 1.1.1.3); 2 ~ floridoside phosphate synthase.
system and were incubated with an enzyme extract, biosynthesis of floridoside phosphate in vitro was readily achieved: time-dependent amounts of radiocarbon were recovered from floridoside phosphate when the reaction mixture was subjected to thin-layer chromatographic separation. Traces of 14C-activity were also found in the region of free floridoside. Linearity and proportionality of this reaction was obtained with enzyme preparations from all red algae so far investigated in detail. The enzyme system (Fig. 4) obviously catalyzes the linkage of the Cl-atom of galactose with the C2atom of glycerol by means of a transferase reaction (see Fig. 5). This action seems to be very similar to that previously described for the biosynthesis of iso-floridoside in Po teriochromonas [ 12]. In close analogy to findings from this unicellular chrysomonad we propose that the enzyme should be termed floridoside phosphate synthase, which is a UDP-galactose: sn-glycerol-3-phosphate 2-a-D-galactosyl transfemse.
POR~ a.
[] DUMONTIA
OHONDI~S
b.
CORAkClNA
CVSlrO~_____ONIJI~
C.
[]
10 t 10 2 10 3 k Bq t4C in Floridoside phosphate Fig. 6. Activity in vitro of floridoside phosphate synthase obtained from differesst marine red slpe. ~ ~ used re,e: (a) f : fresh wt. h " ; (b) rag': ehl. a h " ; (c) 100 ~ :
protein h-:.
357
Rates of floridoside phosphate synthase in vitro that have been obtained with extracts from a variety of marine red algae (Fig. 6) are lower than those reported from chrysomonads [12]. On the whole, they are n o t sufficient to account for the rates of floridoside biosynthesis that were observed in vivo during photosynthesis [4,5,8]. This may be due to the assay procedure which might be insufficient and hence requires further improvement. A major difficulty is the high instability of this enzyme system, which is also known from iso-floridoside phosphate synthase in Poteriochromonas [12]. However, qualitatively the enzyme data that are hitherto available fit into the framework derived from analysis of 14C-assimilate patterns very well. They thus substantiate a proposal for the individual steps of floridoside biosynthesis discussed by Bean and Hassid [9]. AKCNOWLEDGEMENT
This work was supported by the Deutsche Forschungsgemeinschaft and an Australian Research Grant. Technical assistance by Christina Zander and Michelle Stokes is gratefully acknowledged. We thank J.A.C. Smith for reading of the manuscript. REFERENCES 1 H. Colin and J. Augier, C.R. Acad. Sci., 208 (1939) 1450. 2 J.S. Craigie, Storage products, in: W.D.P. Stewart (Ed.), Algal physiology and Biochemistry, BlackweU Scientific Publication, Oxford, 1974, p. 206. 3 E.W. Putman and W.Z. Hassid, J. Am. Chem. Soc., 76 (1954) 2221. 4 B.P. Kremer, Can. J. Bot., 56 (1978) 1655. 5 B.P. Kremer, Mar. Biol., 48 (1978) 47. 6 J.S. Craigie, J. McLachlan, R.D. Tocher, Can. J. Bot., 46 (1968) 605. 7 G.O. Kirst and M.A. Bisson, Aust. J. Plant Physiol., 6 (1979) 539. 8 G.O. Kirst, Phytochemistry, 19 (1980) 1107. 9 R.C. Bean and W.Z. Hassid, J. Biol. Chem., 212 (1955) 411. 10 B.P. Kremer, in J.A. Hellebust and J.S. Craigie (Eds.), Handbook of Phycological Methods II, Cambridge University Press, 1978, p. 270. 11 K. Wegmann, Ber. Deutsch. Bot. Ges., 92 (1979) 43. 12 H. Kauss and B. Schobert, FEBS Lett., 19 (1971) 131.
349
BIOSYNTHESIS OF 2~)-D-GLYCEROL~-D-GALACTOPYRANOSIDE (FLORIDOSIDE) IN MARINE RHODOPHYCEAE
BRUNO P. KREMER* and GUNTER O. KIRST Universitiit zu K~ln, Seminar fiir Biologie und ihre Didaktik, D-5000 KSln 41 and Technische Hochschule Darmstadt, Institut fiir Botanik, D-6100 Darmstadt (F.R.G.)
(Received March 17th, 1981) (Revision received July 8th, 1981) (Accepted July 15th, 1981)
SUMMARY
Floridoside (2-O-D-glycerol~-D-galactopyranoside) is the main photosynthetic product of many Rhodophyceae. In 14C-fixation pattern glycerol 3-phosphate as well as floridoside phosphate are rapidly labelled. It is assumed, based on their incorporation kinetics, that these compounds are precursors of floridoside. Two enzymes involved in the biosynthesis of floridoside were found to be active in homogenates of Rhodophyceae. Glycerol 3-phosphate dehydrogenase probably catalyzes the formation of glycerol 3-phosphate derived from dihydroxy acetone phosphate. Glycerol 3-phosphate and UDP-galactose are condensed to floridoside phosphate by floridoside phosphate synthetase (UDP-galactose: sn-glycerol-3-pho~ phate-2-D-galactosyl transferase).
INTRODUCTION
Marine and freshwater species of red algae (Rhodophyceae) differ from most other photosynthetic plants by the occurrence of particular heterosides. These are usually accumulated as the main soluble products of photosynthetic carbon reduction. Among them, floridoside has long been known as a typical low-molecular-weight red algal carbohydrate [1,2]. It was identified as a 2~)-D-glycerol-a-D-galactoside [3] and has been/raced in all species investigated in detail except those belonging to the red algal order Ceramic.lee: species of this taxonomic effinity accumulate digeneaside *To whom correspondence should be sent at: ~ 51, D-5300 Bonn 2, F.R.G. Abtzrevistions: BSA, bovine smmm albumin; DHAP, dihydroxy acetone phosphate;
PGA, S - p h m p h c ~ . 0304--4211/81/0000-..0000/$02.75 © 1981 Ek~r~-r/North-Hoiland Scientific P u b i M ~ Ltd.
350 (mannosidoglycerate) [4,5]. When exposed to H14CO~, floridoside receives strong 14C-labelling after relatively short, term photosynthesis [4,6,7]. Floridoside content in marine algae is usually in the range 1.5--8% on a dry-weight basis [8]. It has become evident from kinetic tracer studies using radiocarbon that both constituents of floridoslde, galactose as well as glycerol, are 14C-labeiled during photosynthesis and are thus obviously derived from intermediates of the reductive pentose phosphate cycle. However, no further experimental data have hitherto been presented to give a detailed account to the biosynthetic pathway of floridoside. The present study is mainly concerned with the further characterization of some intermediates and enzymes presumably involved in the biosynthesis of glycerolgalactoside in red algae. MATERIAL AND METHODS Plant material Dumontia incrassata (O.F. Mi~ller) Lamour., Chondrus crispus Stackh., Cystoclonium purpureum (Lightf.) Batt., Corallina officinaUs L., and Porphyra umbilicalis (L.) J. Ag. were collected during low tide from their intertidal habitats on the rocky shores of Helgoland (North Sea, Germany). All specimens were carefully checked and maintained in seawater flow tanks at environmental temperatures (5--10°C) under continuous light irradiance. All plants were used for the experiments within 12 h of collection. Lomentaria umbeUata, (H.u.H.) Yendo, Corallina officinalis L., and Catenella nipea Zan. were collected from Port Jackson (Sydney). Cleaning and maintenance of the algae, H14CO3-fixation experiments and identification of labelled compounds were done as described by K i n t and Bisson [7]. Enzyme extraction Selected thallus samples of 1--2 g fresh weight were rapidly deep-frozen in liquid N2 and homogenized by grinding in a mortar with qtmrtz sand. To the still frozen mixture 1 g Polyclar AT and 4 nfl Tris--C1 buffer (0.1 M, pH 7.2) as well as detergent (Triton X-100) were added to give a 0.5--1% solution (v/v). The extraction buffer contained ~ A (10 ~mol), D-araboasco~bate (10 #tool), ~ (10 ~mot), dithioth~itol (10 t~mol). The thoroughly homogenized mixture was then c e n t t ~ at 2--4°C and 40 000 × g for 15 rain. The r e c i t i n g m p e m e t m t was ~ as a paeticle-free crude enzyme extract end umd for enzyme t e ~ without further purification. The enzyme extracts were stored at O°C for not longer than I h. Enzyme mmys Glycerol-8-pholphate dahydrogmm~ (EC 1.1.1.8). T h e preswi~ o f this
351 enzyme was investigated by two different procedures. In a first set of experiments the test system contained 1.2 ml Tris--C1 buffer (0.3 M, pH 7.5), 0.1 ml MgCI2 (10 #tool), 0.2 ml NADH (1 #tool) and 0.1--0.5 ml enzyme extract. The reaction was started by adding 0.2 ml dihydroxyacetone phosphate (= 2/~mol; prepared by hydrolysis and ion-exchange chromatography from dihydroxyacetone phosphate dimethylketal as cyclohexylammonium salt according to manufacturer's advice (Boehfinger)). Enzyme reaction was followed by recording decrease in absorbance at 366 nm in a spectrophotometer. In a further series 0.5 ml Tris--Cl buffer (0.3 M, pH 7.5) were incubated at 20°C with 0.1 ml NADH (5 #tool), 0.1 ml NADPH (5 #mol), 0.1 ml ATP (2 #tool), 0.1 ml MgC12 (10 #mol) and 14C-labelled 3-phosphoglycemte (approx. 2.5 × l 0 s cpm; prepared from H14C~ incubated photosynthesizing algae (Kremer 1978c)) as well as phosphoglycerate kinase, glyceraldehyde phosphate dehydrogenase, and triose phosphate isomerase (Boehringer; approx. 200 units/experiment) for 3 h. To 100 #1 of this mixture were added 10/~1 of algal enzyme extract and the reaction continued for 10--60 min. The incubation was stopped by 20 #1 acetic acid (6 N). The reaction products were further analyzed by thin-layer chromatography (see below). Floridoside phosphate synthase (EC number not yet assigned) was measured by the following procedure. The test system contained 50/~1 Tris-C1 buffer (same concentration and cofactors as extraction buffer, but additionally 1% bovine serum albumin (BSA) and 10 mM NaF), 10 #1 uridine~tiphospho-galactose (2 #tool) (Sigma) and 5--10 ~1 enzyme extract. The reaction was started with 10/~1 [i4C] glycerol 3-phosphate (-~ 0.5 #Ci ~ 18.5 kBq; Amersham Buchler CFB 171) and stopped by addition of 10/~1 acetic acid.
Analysis of reaction products To confirm the identity of particular reaction products, the complete test solutions were analysed by thin-layer chromatography on cellulose (MN 300; layer thickness 350--500 #m) using solvent systems as detailed earlier [7,10].
RESULTS AND DISCUSSION
During short-term photosynthesis in H14CO~ glycerol as well as galactose, the precursors of galactosylglyceride, become labelled at appreciable rates. About 3--7% of the 14C incorporated into EtOH-soluble compounds after 30 s were recovered from glycerol (Fig. 1). The percentage of glycerol declined within 2 min to 0.5--2~ (Fig. I and Table I), indicating a rapid turnover of the glycerol pool. This was also confirmed by puise~hase experiments using Lomentaria umbeUata (Table II). It is obvious that, following a labelling pulse, the amounts of 14C confined to glycerol are rather low, whereas floridoside receives strong 14C-labelling. Glycerol is therefore not
352
A
30
%
t
20" oo- ~
10
~
~
~
~
FIo
..n ....
FiO-®
i.................... o Glycerol 30-
o
°/o
I t o~
B tt s"r'l~
20 o ~" ", t~
tO
/ I Fio
"',~
\o~ °°'o---. o_
i
"'-- ................... ~ FIo-(~ -,
Glycerol 1'0 •
5
t
(mini
Fig. 1. Time course of H~4CO;-fixation. Labelling of floridoside (Flo), floridoside phosphate (Flo-P), and glycerol in % of total ~4C in the EtOH-soluble fraetion. Fixation rates into EtOH-soluble compounds were: 3.3 * 0.3 ~mol CO 2 g-1 fresh wt. I 0 min -I (Corollino) and 3.0 ± 0.4 , m o l CO t g-1 fresh wt. 10 rain -~ (Lomentorio).
an accumulated photosynthate. It has to be mentioned, however, that the actual content of free glycerol and galactose might be masked by inevitable hydrolysis offloridos/de during the separation procedure by ion-exchange resins (see below). More attention was therefore directed towards the characterization of an enzyme providing the biosynthesis of the C3-unit of floridoside. TABLE I 14C-INCORPORATION INTO PRECURSORS OF FLORIDOSIDE AND FLORIDOSIDE A F T E R 5 MIN P H O T O S Y N T H E J ~ (MEANS OF 3 TO 5 REPLICATES ± S.D.) FIX), FLORIDOffIDE; F L O P , F L O R I ~ ] I PHOaPHATE. Species
Catene[~ repen# Lomentar~ umbellafa CoraUina o l ~ ' i ~ s l k
% of EtOH4oluble compounds Hexose-P
Fio-P
Flo
Galsetose
Glycerol
18.8 ± 2.4 5.7 ± 0.6
15.3 ± 1.8 18.7 ± 8.7
8.4 ± 1.6 15.7 ± 4.4
5.1 ± 0.5 2.1 ± 0.6
0.9 ± 0.I 2.3 ± 0.8
11.5 ± 0.7
17.4 ± 0.4
25.0 ± 4.8
1.9 ± 0.I
2.8 ± 1.0
353 TABLE II L O M E N T A R I A UMBELLATA. 1'C-LABELLING O F SOLUBLE PHOTOSYNTHATES FOLLOWING A 14C-FEEDING PULSE (5 MIN) IN % OF TOTAL EtOH-SOLUBLE 14C.ACTIVITY F o r abbrevations see Table I. Means of three replicates. Chase period (min)
Compound
Hexose-P FIo-P Floridoside Glycerol
5
I0
11
7
9 24 1
7.5 34 0.8
15
8
10 38 0.6
Appropriate results are exemplified in Fig. 2a for Dumontia incrassata. When the triose phosphate dihydroxy acetone phosphate (DI4AP) is present as a substrate, it is immediately reduced by an algal enzyme (extract) to a glycerol phosphate at the expense of NADH. Linevxity of this reaction is observed at least over 15 min. However, the turnover rate of DHAP under the given experimental conditions is relatively low. This might possibly be due to instability of the substrate preparations. In addition the actual substrate concentration might be considerably less than that calculated from the previous preparation steps. Very similar findings have been obtained with all other red algal species investigated in this connection. In order to confirm the identity of the reaction product 14C-labeUed ft. I Or) I
A 40
o
-r 30 t~
300
B
200
@
o 20
,,.;.
Z (.)
"5 10 E
100
o"
c
rn
1
5
10
15 t (rain)
io
30
60
Fig. 2. Activity of glycerol-3-phosphate dehydrogenase in marine red algae. (a) Enzymatic conversion of the triose phosphate DHAP to glycerol 3-phosphate by an enzyme extract from Dumontia inerassata e x p r e ~ e d as N A I T formed. (b) Incorporation of z4C from triose phosphate (originally supplied as [z4C]PGA) into glycerol 3-phosphate by an enzyme extract from Cystoclonium purpureum.
354
3-phosphoglycerate (PGA) was applied and its subsequent enzymatic conversion was followed. Analyses of the reaction mixture by means of thinlayer chromatography revealed that appreciable amounts of the 14C originally applied are located in glycerol 3-phosphate (-- L-~-glycerophosphate = sn-glycero-3-phosphate). This c o m p o u n d is distinguishable chromatographically from glycerol 2-phosphate (= L~-glycerophosphate = sn-glycero2-phosphate), which was n o t 14C-labelled in this treatment. About 12% of the 14C-activity determined for 3-PGA on the thin-layer chromatograms is confined to glycerol 3-phosphate after 60 min incubation. The time-course of 14C-labelling of this c o m p o u n d by an enzyme extract from Cystoclonium purpureum is shown in Fig. 2b. ~4C-labelling of free glycerol was n o t observed in this assay. Taking into account these findings it might be suggested that in red algae the enzyme providing the conversion of triose phosphate (immediately derived from the RPP cycle) to glycerol phosphate is a L-glycerol-3-pho~ phate dehydrogenase. This enzyme has long been known as Baranowskienzyme from heterotrophic organisms and has more recently been found to be involved in glycerol biosynthesis in the green alga Dunaliella tertiolecta [11]. Its basic function in representatives of the Rhodophyceae is to provide sufficient amounts of glycerol phosphate for further use in the heteroside formation. Though Bean and Hassid [9] report on appreciable amounts of [14C]glycerol phosphate exceeding those of [ 14C]floridoside after 2 rain photosynthesis, this intermediate is mostly n o t pooled. Labelled glycerol p h o ~ phate could n o t be identified unequivocally in the experiments described here. Precursors of floridoside like glycerol (mentioned already above), galactose, floridoside phosphate and floridoside itself are labelled very early and hence the relative a m o u n t exceeds by far the percentage of the few other compounds (i.e. alanine, glycine), which were labelled also after very short times (Fig. 1, Table I). However, the absolute content of free glycerol and glycerol phosphate estimated quantitatively together with floridoside [8] is low and was always less than 0.05% on a dry wt. basis. UDP-galactose has been traced among short-term labelled 14C-photosynthates of the red algae Iridophycus flaccidum [9]. Floridoside phosphate is consistently found among the photosynthetically ~4C-labelled phosphate esters in several species of marine Rhodophyceae. Data for Catenell¢, Lomentaria and Corallina are given in Table I and Fig. 3. After 45 min of H14CO3fixation Grateloupia filicina incorporated 22% of the 14C (EtOH-soluble fraction) into floridoside phosphate, 29% into floridoside and 1% into glycerol, the values for Hypnea valentiae were 3%, 38% and 1% respectively [ 7]. Therefore it seems reasonable to assume an enzyme performing a transfer of the nucleoside on glycerol 3-phosphate to yield floridoside phosphate. The results of appropriate experiments are shown in Fig. 4. When 14Clabelled glycerol 3-phosphate and UDP-galactose were present in the test
355
~=- Glycerol Glycerol
FIo
Fru 1""'4 GA I--'4 Glu
FIo-P
Hexose-P G-6-P H
start
start Ct
L
Co
Fig. 3. Autoradiographs of TLC-separated EtOH-extracts of CateneUa (Ct), Lomentaria (L), and CoralUna (Co) after 5 rain photosynthesis of H14CO;. Compounds identified: Flo, floridoside, polysaccharides (start) on the right; positions of marker substances (on the left): G-6-P, glucose-6-phosphate; Glu, glucose; GA, glyceric acid; Fru, fructose. o" CO 0 ,
200
• 20 pl Enzyme extract o 10p[
~
0
"o 1 5 0 o
e
~
LL ,._
100.
0
•- ~
Q
50
m _J |
o -
1
5
10 t (mini
15
F~. 4. Oystoelonium purpureum~ ~ t y
20 and proportionality of an ensyms trims-
feting UDP..Smlaeko~ on [s*O]glyee¢ol 8 - ] p h o ~ l m t e t h u yid,41,~_ YlceidosJde phoephs~.
356
_,j~je G-3- P ~ ~ U
RPP-cycle l,&
~SUDP-Gal.
CH2OH HO,~-----O CH2OH : 2-glycerol-3-(~----~ ~ O--CH I O (Floridosidephosphate) ~ ~ I Pi OH CH20H ~-Gatactosyl-
Floridoside Fig. 5. Scheme of biosynthesis of floridoside. Abbreviations: RPP-cycle = reductive pentose phosphate cycle; G-3-P = glycerol-3-phosphate. Enzymes tested: ( ~ Glycerol phosphate dehydrogenase (EC 1.1.1.3); 2 ~ floridoside phosphate synthase.
system and were incubated with an enzyme extract, biosynthesis of floridoside phosphate in vitro was readily achieved: time-dependent amounts of radiocarbon were recovered from floridoside phosphate when the reaction mixture was subjected to thin-layer chromatographic separation. Traces of 14C-activity were also found in the region of free floridoside. Linearity and proportionality of this reaction was obtained with enzyme preparations from all red algae so far investigated in detail. The enzyme system (Fig. 4) obviously catalyzes the linkage of the Cl-atom of galactose with the C2atom of glycerol by means of a transferase reaction (see Fig. 5). This action seems to be very similar to that previously described for the biosynthesis of iso-floridoside in Po teriochromonas [ 12]. In close analogy to findings from this unicellular chrysomonad we propose that the enzyme should be termed floridoside phosphate synthase, which is a UDP-galactose: sn-glycerol-3-phosphate 2-a-D-galactosyl transfemse.
POR~ a.
[] DUMONTIA
OHONDI~S
b.
CORAkClNA
CVSlrO~_____ONIJI~
C.
[]
10 t 10 2 10 3 k Bq t4C in Floridoside phosphate Fig. 6. Activity in vitro of floridoside phosphate synthase obtained from differesst marine red slpe. ~ ~ used re,e: (a) f : fresh wt. h " ; (b) rag': ehl. a h " ; (c) 100 ~ :
protein h-:.
357
Rates of floridoside phosphate synthase in vitro that have been obtained with extracts from a variety of marine red algae (Fig. 6) are lower than those reported from chrysomonads [12]. On the whole, they are n o t sufficient to account for the rates of floridoside biosynthesis that were observed in vivo during photosynthesis [4,5,8]. This may be due to the assay procedure which might be insufficient and hence requires further improvement. A major difficulty is the high instability of this enzyme system, which is also known from iso-floridoside phosphate synthase in Poteriochromonas [12]. However, qualitatively the enzyme data that are hitherto available fit into the framework derived from analysis of 14C-assimilate patterns very well. They thus substantiate a proposal for the individual steps of floridoside biosynthesis discussed by Bean and Hassid [9]. AKCNOWLEDGEMENT
This work was supported by the Deutsche Forschungsgemeinschaft and an Australian Research Grant. Technical assistance by Christina Zander and Michelle Stokes is gratefully acknowledged. We thank J.A.C. Smith for reading of the manuscript. REFERENCES 1 H. Colin and J. Augier, C.R. Acad. Sci., 208 (1939) 1450. 2 J.S. Craigie, Storage products, in: W.D.P. Stewart (Ed.), Algal physiology and Biochemistry, BlackweU Scientific Publication, Oxford, 1974, p. 206. 3 E.W. Putman and W.Z. Hassid, J. Am. Chem. Soc., 76 (1954) 2221. 4 B.P. Kremer, Can. J. Bot., 56 (1978) 1655. 5 B.P. Kremer, Mar. Biol., 48 (1978) 47. 6 J.S. Craigie, J. McLachlan, R.D. Tocher, Can. J. Bot., 46 (1968) 605. 7 G.O. Kirst and M.A. Bisson, Aust. J. Plant Physiol., 6 (1979) 539. 8 G.O. Kirst, Phytochemistry, 19 (1980) 1107. 9 R.C. Bean and W.Z. Hassid, J. Biol. Chem., 212 (1955) 411. 10 B.P. Kremer, in J.A. Hellebust and J.S. Craigie (Eds.), Handbook of Phycological Methods II, Cambridge University Press, 1978, p. 270. 11 K. Wegmann, Ber. Deutsch. Bot. Ges., 92 (1979) 43. 12 H. Kauss and B. Schobert, FEBS Lett., 19 (1971) 131.