(Charophyceae)I Received for Publication November 20, 1987 and in Revised Form January 15, 1988

Plant Physiol. (1988) 87, 78-82 0032-0889/88/87/0078/05/$01 .00/0

Two Class I Aldolases in the Green Alga Chara foetida (Charophyceae)I Received for publication November 20, 1987 and in revised form January 15, 1988

SIGRID JACOBSHAGEN AND CLAUS SCHNARRENBERGER* Institut far Pflanzenphysiologie, Zellbiologie und Mikrobiologie, Freie Universitat Berlin, Konigin-Luise- Str. 12-16a, D-1000 Berlin 33 (West)

ABSTRACT E. gracilis; however, experimental support for a cytosolic class I aldolase, which is different in its properties from the chloroplast Aldolase activity of Chara foetida (Braun) could be separated into a class I aldolase, was not achieved (see also "Discussion"). Very minor (peak I) and a major peak (peak I) by ion-exchange chromatog- early reports claim both class I and class II aldolases being cy- raphy on DEAE-cellulose. Affinity chromatography on P-cellulose re- tosolic (5) or the class II aldolase being plastidic (23). sulted in highly purified aldolase preparations with specific activities of During the course of our studies on aldolases of Chara foetida 3.2 and 4.8 units per milligram protein and molecular subunit masses of we discovered for the first time and describe here two distinct 37 and 35 kilodalton, as shown by SDS-PAGE, for the aldolase of peak class I aldolases in a green alga. This situation is similar to the I and peak II, respectively. Both aldolases belong to class I aldolase since cytosol and chloroplast specific class I aldolases of higher plants. the activity is not inhibited by 1 millimolar EDTA. The Km (fructose-1,6- Since increasing evidence derived from fine structure (16, 20) bisphosphate) values were 0.64 and 13.4 micromolar, respectively. The and sequence homology of 5S rRNA (7) implies a very close aldolase of peak I showed a 6.7 times stronger crossreaction with a specific evolutionary relationship of Charophyceae with higher plants our antiserum against the cytosol aldolase of spinach than with an antiserum finding of two class I aldolases in C. foetida may give a valuable against the chloroplast aldolase of spinach. On the other hand the aldolase clue to the evolutionary origin of higher plant aldolases from the of peak II showed a 5.1 times stronger cross-reaction with the a-plastid- tribe of Charophyceae. aldolase antiserum than with the ar-cytosol-aldolase antiserum. For algae this is the first separation of two class I aldolases. They are similar to the MATERIALS AND METHODS cytosol and chloroplast aldolases in higher plants, but different from a reported class I (Me2+ independent) and class II (Me2+ dependent) al- Plant Material. The green alga Chara foetida (Braun) was dolase in other algae. taken from an open-air culture in the Botanical Garden of Berlin and cleaned from macroscopic debris. Microscopic control showed only a minor contamination with other microorganisms. Separation of Isoenzymes by Chromatography on DEAE-Cel- lulose. Between 10 and 37 g of wet algal material was homoge- nized in three volumes of buffer A (20 mM Tris-HCl [pH 8.6], 10 mM j-mercaptoethanol) for 2 min in an icebath using a Virtis Aldolases from different biological sources are either depend- "45" homogenizer. The homogenate was centrifuged for 15 min ent on divalent ions for activity (class II aldolases) or not (class at 43,000g at 4°C. The supernatant was readjusted to pH 8.6 and I aldolases) (19, 23). Class I aldolases are present in animals and immediately applied to a column (3 x 11 cm) of DE 32-cellulose in higher plants including ferns and mosses (2, 15, 23). Higher (Whatman, Springfield Mill, Maidstone, Kent/England) equili- plants contain even two class I aldolases, one being compart- brated with buffer A. The column was washed with 50 ml of mented in the cytosol and the other in chloroplasts (1, 12, 14). buffer A. The proteins were eluted by a linear gradient of 0 to Class II aldolases occur in fungi and procaryotes (6, 23). In some 0.4 M KCl in buffer A of twice the column volume and subse- procaryotes, such as Escherichia coli, an additional class I type quently by 80 ml of 1.0 M KCl in buffer A. Fractions of 2 ml aldolase can be developed upon growth on pyruvate or lactate were collected, assayed for aldolase activity, and measured for (26). On the other hand, algae appear to have both a class I and KCl concentration by conductimetry. For further a class II aldolase (2, 5, 8, 15, 18, 21-23). In the green algae Affinity Chromatography on Phosphocellulose. pu- Chlamydomonas reinhardii and C. mundana and the eugleno- rification the two aldolases separated by chromatography on phyta Euglena gracilis the class I aldolase appears to be predom- DEAE-cellulose were pooled separately and dialyzed against imidazole 10 mM inant in photoautotrophically grown cells and the class II aldolase buffer B (20 mM [pH 6.5], 8-mercaptoethanol onto a in mixo- or heterotrophically grown cells (2, 5, 18, 21, 22). Only or dithiothreitol). Each aldolase was loaded P-cellulose buffer B. The column in a very early investigation (23) the occurrence of class I and P 11 (Whatman) column equilibrated with x class II aldolase activity was found in the opposite way. The sizes were 2 x 10 cm and 3 12 cm for the aldolase eluting evaluation of the intracellular compartmentation of class I and first and second from DEAE-cellulose, respectively. The col- class II aldolase in algae appears contradictory because of dif- umns were washed each with two column volumes of buffer B ficulties in cell fractionation. The most convincing data suggest and eluted with 0.1 mM fructose-1,6-bisphosphate in buffer B. were is a enzyme (10, 18) and Fractions containing aldolase activity pooled, dialyzed against that the class I aldolase chloroplastic 10 and the class II aldolase a cytosolic enzyme (18). In another paper buffer C (20 mm imidazole [pH 7.0], mM dithiothreitol), (2), some preliminary evidence was achieved for an additional used for enzyme characterization. class I aldolase activity in the cytosol of autotrophically grown Protein was determined with Coomassie brilliant blue G-250 as described by Sedmak and Grossberg (25). I Supported by a grant of the Deutsche Forschungsgemeinschaft. Enzyme Assay. Fructose-bisphosphate aldolase (EC 4.1.2.13) 78 TWO CLASS I ALDOLASES IN CHARA FOETIDA 79 was measured spectrophotometrically at 22°C according to Wu If this method is applied to proteins of C. foetida (Fig. 1) a and Racker (28). One ml of reaction mixture contained 50 mM chromatographic aldolase pattern similar to that of higher plants Tris-HCl (pH 7.5), 4.5 mM MgCl2, 1 mm EDTA, 1 unit each of is obtained, i.e. a small aldolase peak (peak I) preceding a larger triosephosphate isomerase and glycerol-3-P dehydrogenase, 210 aldolase peak (peak II). The activity in peak I represented about /LM NADH, and 2 mm fructose-1,6-bisphosphate trisodium salt 10% and the activity in peak II about 90% of the total activity (Boehringer, Mannheim, FRG). In order to test for class II (average of two experiments). activity the aldolases were incubated in either 1 mm EDTA or The two aldolases were further purified by affinity chroma- 0.1 mm zinc sulfate plus buffer C for 10 min at 37°C and the tography on phosphocellulose up to a specific activity of 3.2 units activity determined subsequently in the above reaction mixture (mg-1 protein) for the peak I aldolase and of 4.8 units (mg-' without both MgCl2 and EDTA. The enzyme activity of 1 unit protein) for the peak II aldolase (Table I). SDS-PAGE showed corresponds to the cleavage of 1 ,umol substrate per min. single protein bands for the two aldolases (Fig. 2). If more protein Immunotitration of Aldolase. The method of immunotitration was applied to the gel, additional minor bands became visible was carried out according to Kessler (9) and as described pre- (not shown). We consider the purity of peak I aldolase to be at viously (12). A constant amount of aldolase activity and 20,ul least 90% and of peak II aldolase to be at least 50%. The mo- of antiserum or dilutions of it were incubated for 5 min. Then, lecular subunit masses were estimated to be 37 and 35 kD for 5 Al of a 12% suspension of protein A-coated Staphylococcus the aldolase of peak I and II, respectively. These molecular aureus Cowan I cells were added. After incubation for 2 min the masses are similar to those of the cytosol and chloroplast aldolase suspension was centrifuged at 14,000g for 1 min in a Biofuge A from higher plants (12, 14). (Heraeus Christ) and the activity in the supernatant was deter- The activity of both aldolases from C. foetida was resistant to mined. For the aldolase of peak I 80 ,l of enzyme were used incubation in 1 mM EDTA. Also, the activity was not increased for incubation with the antisera and 90 ,tl of the supernatant by incubation with 0.1 mm zinc sulfate (Table II). Similarly, an were used for activity measurements. Accordingly, 30 ,ul of en- even higher concentration of 10 mm EDTA had no inhibitory zyme from peak II were used for incubation and 43 Al of the effect on either aldolase. Finally, after incubation with 1 mM supernatant used for activity measurements. The antisera con- EDTA, the activity in the presence of less than 0.1 mm EDTA tained a small amount of aldolase activity that was corrected for was not increased by an excess of 0.5 mM zinc sulfate. These in a blank. It was ascertained previously with aldolase from higher results prove that both aldolases are of the class I type. plants that there is a strong linearity between the amount of Both aldolases exhibit typical Michaelis-Menten kinetics for antiserum and the precipitation of aldolase protein (data not fructose-1,6-bisphosphate. The Km values for this substrate, as shown). obtained from an Eadie-Hofstee plot, were 0.64 and 13.4 AM for PAGE. The method was carried out in 12.5% SDS-polyacry- the aldolase of peak I and II, respectively (data not shown). lamide gels according to Laemmli (13). Subsequent silver staining These values are in the same order as for the cytosol and chlo- was performed according to Merril et al. (17). For determining roplast aldolase in higher plants (24). the molecular subunit masses the following markers were used: In order to evaluate whether or not the two aldolases of C. phosphorylase A (95,000), bovine serum albumin (66,000), oval- foetida may correspond to the cytosol and chloroplast aldolase bumin (45,000), rabbit muscle aldolase (39,500), carbonic an- of higher plants specific antisera against the respective aldolases hydrase (29,000), chymotrypsinogen (25,000), Cyt c (12,500). of spinach leaves were used for immunotitration of the two Chara enzymes (Fig. 3). The data show that 6.7 times more a-chloroplast-aldolase antiserum was needed than of a-cytosol-aldolase RESULTS antiserum to precipitate half of the peak I Chara aldolase. Like- In higher plants the cytosol and chloroplast aldolase can be wise, 9.2 times more a-chloroplast-aldolase antiserum was needed separated by ion-exchange chromatography on DEAE-cellulose. than a-cytosol-aldolase antiserum to precipitate half of the cy-

#01% VI I 0.8e 60 ) E .2 0.6 4- ..54 CL.) FIG.1. Elution profile of the aldolases 4.' 0.4 : from a crude extract of C. foetida upon 0 chromatography on DEAE-cellulose. o 0 ~0 020 0.2 Y x

xI ! °tLI lO 0 20 40 60 80 Fraction number 80 JACOBSHAGEN AND SCHNARRENBERGER Plant Physiol. Vol. 87, 1988 Table I. Purification Scheme of the Two Aldolases from C. foetida Volume Activity Protein Specific Activity PrfctoPurification ml m units ,Ag units mg -fold Crude extract 111 6,470 340 0.17 1.0 DEAE-cellulose (peak I) 18 300 330 0.09 0.5 P-cellulose (peak I) 10 80 2.5 3.20 18.8 DEAE-cellulose (peak II) 26 1,250 350 0.14 0.8 P-cellulose (peak II) 10 310 6.8 4.80 28.2

Table II. Dependence of the Two Aldolases from C. foetida on 1 2 3 Divalent Cations for Activity Incubation Aldolase (Peak I) Aldolase (Peak II) munits Buffer C 0.36 0.78 Buffer C + 1 mM EDTA 0.35 0.77 Buffer C + 0.1 mM Zn2+ 0.36 0.78

_ DISCUSSION The analysis of aldolases in C. foetida has revealed two aldolases which resemble in their properties much the higher plant 66 _ cytosol and chloroplast aldolases. In particular the molecular mass and the affinity to fructose-1,6-bisphosphate in C. foetida are very similar with those reported for the two aldolases in higher plants (12, 14, 24). The immunochemical data imply that the two aldolases of C. foetida and of spinach leaves are ho- mologous enzymes. Since the two higher plant aldolases have 145 been shown previously to be compartmented in the cytosol and in the chloroplasts, respectively (1, 12, 14), it may be concluded that the two aldolases of C. foetida are likewise also compartmented in the cytosol and in the chloroplasts. Admittedly, this is an indirect conclusion. However, considering the constancy of intracellular enzyme and isoenzyme compartmentation it may be legal to extrapolate such a view to the two aldolases of C. foetida as well. The proof of two class I aldolases in C. foetida is the first which is unambiguous. Previously, Bukowiecki and Anderson (2) have claimed evidence for class I aldolase activity in the cytosol and in the plastids of autotrophic Euglena gracilis cells after cell fractionation. We acknowledge the way that these authors de- signed the experimental reasoning for such a statement. How- ever, we want to point out that these authors did not demonstrate any enzyme separation of two class I aldolases from E. gracilis nor could they find any significant difference in the isoelectric ~ ~ ~ ~ ~ | point of the class I aldolase activities in the cytosolic and plastidic cell fraction. We also do not consider the experimental evidence for the compartmentation of class I aldolase activity in the cytosol very conclusive since Chl is no appropriate marker to distinguish between stromal enzymes retained in intact chloroplasts, stromal FIG. 2. SDS-PAGE of the purified aldolases of C. foetida. Proteins enzymes released from chloroplasts during cell fractionation, and were visualized by silver staining. Lane 1, molecular mass markers; lane cytosolic enzymes. 2, aldolase of peak I (62.5 ng); lane 3, aldolase of peak II (170 ng). Peak The aldolases in C. foetida differ from aldolases of other green I corresponds to the aldolase eluting first upon chromatography on DEAE- algae, investigated so far, in their Km (fructose-1,6-bisphosphate) cellulose and peak II to the aldolase eluting second (see Fig. 1.) values. For example, the cytosol aldolase of Chlamydomonas reinhardii and/or C. mundana has a Km (fructose-1,6-bisphosphate) value of 300, M and the chloroplast aldolase of 35 to 40 tosol aldolase of spinach leaves (data not shown). In contrast, ,M. The respective values of the two aldolases in C. foetida are 5.1 times less a-chloroplast-aldolase antiserum was needed than 0.6 and 13 ,Mm. In addition, the cytosolic aldolase in C. reinhardii ca-cytosol-aldolase antiserum to precipitate half of the peak II and C. mundana is reported to be of class II type (5, 22). Such Chara aldolase and 7.6 times less in a control experiment with class II type aldolases have, in general, very high Km values. the chloroplast aldolase of spinach leaves (data not shown). We Likewise, the Km values for the cytosol and chloroplast aldolase therefore conclude that the two aldolases from C. foetida cor- in higher plants are 2 and 10 ,uM, respectively (24). respond to the cytosol and chloroplast aldolase of higher plants. Based on fine structural analyses on the type of spindle for- TWO CLASS I ALDOLASES IN CHARA FOETIDA 81

E 0.4 E 0.8

L 0) 0. 0. L. (A U) L 0.2 04 ._ c 0.L. 9)

U) 0 ~0

0 0.2 1 2 10 20 0 0.02 0.1 0.2 1 2 Amount of antiserum Oul) Amount of antiserum (yuI)

FIG. 3. Immunotitration of the purified two aldolases of C. foetida with specific antisera raised against the cytosol (d) and the chloroplast ( x ) aldolase from spinach leaves. The amount of antiserum yielding 50% precipitation of an aldolase (see arrows) was used as a measure for the reaction of an antiserum with the respective aldolase. The bars represent the standard deviation of three measurements. Left, aldolase of peak I; right, aldolase of peak II (see Fig. 2). mation during mitosis or the type of microtubule association with LITERATURE CITED the basal bodies of flagella (16, 20) green algae can be divided 1. ANDERSON LE, VR ADVANI 1970 Chloroplast and cytoplasmic enzymes. Three into five classes, among them the Chlorophyceae with Chla- distinct isoenzymes associated with the reductive pentose phosphate cycle. mydomonas and the Charophyceae with Chara as representa- Plant Physiol 45: 583-585 2. BUKOWIECKI LE ANDERSON 1974 forms of aldolase and triose tives. In contrast to the the Charophyceae ap- AC, Multiple Chlorophyceae, phosphate isomerase in diverse plant species. Plant Sci Lett 3: 381-386 pear to be very closely related to higher plants. Also, sequence 3. BULLOCK KW, TR DEASON, JC O'KELLEY 1979 Occurrence of glycolate de- analyses of 5S rRNA imply a sequential emergence in evolution hydrogenase and glycolate oxidase in some coccoid, zoospore-producing of Chlamydomonas, other green algae and, finally, Charophy- green algae. J Phycol 15: 142-146 ceae (7). The two aldolases of C. foetida, therefore, fit well into 4. FREDERICK SE, PJ GRUBER, NE TOLBERT 1973 The occurrence of glycolate dehydrogenase and glycolate oxidase in green plants. An evolutionary sur- the view of such an evolutionary relationship among algal classes vey. Plant Physiol 52: 318-323 and higher plants. 5. GUERRINI AM, T CREMONA, EC PREDDIE 1971 The aldolases of Chlamy- Up to now, only few biochemical properties have been found domonas reinhardii. Arch Biochem Biophys 146: 249-255 6. HORECKER 0 CY LAI 1972 Aldolases. In PD The to show variations which correlate with the present view on sys- BL, TsOLAS, Boyer, ed, Enzymes, Ed 3, Vol 7. Academic Press, New York, pp 213-258 tematics and evolution of green algae. In respect to enzymes it 7. HORi H, B LIM, S OSAWA 1985 Evolution of green plants as deduced from was recognized first that glycolate oxidase, a well known enzyme 5S rRNA sequences. Proc Natl Acad Sci USA 82: 820-823 of leaf peroxisomes, is present only in the group of Charophyceae 8. IKAWA T, S ASAMI, K NISIZAWA 1972 Comparative studies on diphosphate while other green algae contain a glycolate dehydrogenase (3, aldolases mainly in marine algae. Proc Int Seaweed Symp 7: 526-531 9. KESSLER SW 1975 Rapid isolation of antigens from cells with a staphylococcal 4). Also, the degradation of urea was found to be accomplished protein A-antibody adsorbent: parameters of the interaction of antibody- by the enzyme urease in Charophyceae and Ulvophyceae, how- antigen complexes with protein A. J Immunol 115: 1617-1624 ever, by the enzymes urea carboxylase and allophanate hydrolase 10. KLEIN U 1986 Compartmentation of glycolysis and of oxidative pentose- in other algae (27). Finally, only and phosphate pathway in Chlamydomonas reinhardii. Planta 167: 81-86 Charophyceae Ulvophyceae 11. KREMER BP, GO KIRST 1982 Biosynthesis of photosynthates and taxonomy form sucrose as sole low mol wt product of photosynthesis while of algae. Z Naturforsch 37c: 761-771 other green algae form glycerol as well (11). Thus, differences 12. KRUGER I, C SCHNARRENBERGER 1983 Purification, subunit structure and im- in the aldolases of C. reinhardii and C. foetida correlate closely munological comparison of fructose-bisphosphate aldolases from spinach and with the variation of some other biochemical properties. corn leaves. Eur J Biochem 136: 101-106 13. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227: 680-685 14. LEBHERZ HG, MM LEADBETTER, RA BRADSHAW 1984 Isolation and char- Acknowledgments-The authors thank the Deutsche Forschungsgemeinschaft acterization of the cytosolic and chloroplast form of spinach leaf fructose for financial support. diphosphate aldolase. J Biol Chem 259: 1011-1017 82 JACOBSHAGEN AND SCHNARRENBERGER Plant Physiol. Vol. 87, 1988

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