Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis

Plant Physiol. (1994) 106: 1137-1 144

Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis

Purification and Characterization

Birgit Pelzer-Reith, Sabine Wiegand, and Claw Schnarrenberger*

lnstitut für Pflanzenphysiologie und Mikrobiologie, Freie Universitat Berlin, Konigin-Luise-Strasse 12-1 6 a, D-14195 Berlin, Germany

l h e plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. l h e class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. l h e native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class I1 aldolase was purified to a specific activity of 21 units/mg by (NH,),S04 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. l h e native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The K,,, (frudose-1,6-bisphosphate) values were 12 PM

for the class I enzyme and 175 p~ for the class I I enzyme. l h e class I I aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Znz+, or 0.5 mM Cuz+. Na+, K+, Rb+, and NH4+ (but not li+ or Cs') enhanced the activity up to 7- fold. After inactivation by EDTA, the activity could be partially restored by MnZ+, Cuz+, or Coz+. A subclassification of class I1 aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.

FBP aldolase is one of the essential enzymes in glycolysis, gluconeogenesis, and the Calvin cycle. Two distinct classes of aldolases have been detected in biological systems (Rich- ards and Rutter, 1961a; Rutter, 1964). Class I aldolases are tetrameric proteins with a molecular mass of about 160 kD. They form a Schiff's base with the substrate and are inactivated by borohydride (NaBH4) but not by metal-chelating agents (Rutter, 1964). Class I1 aldolases are dimers with a molecular mass of about 80 kD and form an endiole as reaction intermediate. The catalytic activity of the latter enzyme depends on divalent metal ions and is, therefore, inhibited by EDTA (Richards and Rutter, 1961b; Rutter, 1964). Class I aldolases are present in animals, higher plants, fems, mosses, and some eukaryotic algae. Class I1 aldolases are confined to more primitive organisms such as bacteria, cy- anobacteria, and fungi. Only a few bacteria possess, in addition or exclusively, a class I aldolase. Algae and protists show a diverse distribution of class I and class I1 aldolases (Rutter, 1964; Antia, 1967; Lebherz and Rutter, 1969; Buko-

* Correspondinz author; fax 49-30-838-4313.

wiecki and Anderson, 1974; Dhar and Altekar, 1986, 1989; for reviews see March and Lebherz, 1992; Schnarrenberger et al., 1992).

Class I aldolases have been sequenced from several animals, protists, plants, and algae. The homology among the eukaryotic aldolases is at least 50% (Schnarrenberger et al., 1994). Recently, the sequence of the class I aldolase of the prokaryote Staphylococcus carnosus was also determined. The latter enzyme differs from the eukaryotic aldolases because it is a monomer of somewhat smaller size (33 kD) than the eukaryotic class I aldolases. Its homology with the eukaryotic aldolases is only 30% (Witke and Gotz, 1993). Therefore, the S. carnosus aldolase has to be considered as ancestral in respect to both oligomeric state and sequence conservation.

Class I1 aldolase sequences exist for the enzymes of yeast, Escherichia coli, and Co ynebacterium glutamicum (Alefounder et al., 1989; Schwellberger et al., 1989; von der Osten et al., 1989). They do not show any homology with class I aldolases. It was concluded that class I1 aldolases developed independ- ently of class I aldolases in evolution (March and Lebherz, 1992). Further, the sequence homology among the class I1 aldolases is only on the order of 37 to 45%. This may indicate a greater variability among class I1 aldolases than among class I aldolases.

Euglena gracilis is one of the few eukaryotic organisms that contains both a class I and a class I1 aldolase (Cremona, 1968; Lebherz and Rutter, 1969; Mo et al., 1973; Karlan and Russell, 1976). As in higher plants, the chloroplast enzyme is of class I type; however, the cytosol enzyme is a class I1 enzyme (Mo et al., 1973). In E . gracilis the class I aldolase is predominately expressed in autotrophically grown cells, whereas the class I1 aldolase is the major enzyme in heterotrophically grown cells (Cremona, 1968; Willard and Gibbs, 1968b; Mo et al., 1973).

In the present paper we describe the purification and characterization of both the class I and class I1 aldolases of E . gracilis. To our knowledge this is the first such documen- tation for a eukaryotic organism. Particularly, an analysis of the class I1 aldolase of E . gracilis should contribute informa- tion inasmuch as this enzyme differs from the enzyme of other eukaryotic taxa (e.g. fungi) or from prokaryotes. These studies are also a necessary prerequisite for cloning and

Abbreviation: FBP, Fru-1,6-bisphosphate. - - - 1137

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1138 Pelzer-Reith et al. Plant Physiol. Vol. 106, 1994

expression studies of the two enzymes. First analyses showed diumal variations in the two enzymes that are regulated at a posttranscriptional level (Pelzer-Reith et al., 1993). Therefore, the class I and class I1 aldolases are interesting not only from an evolutionary point of view, but also as markers for a different metabolic regulation of photosynthesis in the chloroplast and of heterotrophic metabolism in the cytosol.

MATERIALS A N D METHODS

Organism and Growth Conditions

Euglena gracilis strain Z (No 1224-5/25) was obtained from the Sammlung von Algenkulturen of the University of Got- tingen (Germany). Autotrophic cells were grown in 15 L of a medium described by Boger and San Pietro (1967) in trans- parent polycarbonate containers at 27OC and were supplied with 5% Con in air. The cultures were illuminated with 6000 lux of white fluorescent light in 16:8 h 1ight:dark cycles. Heterotrophic cells were grown in complete darkness in a Suc-containing medium described by Hutner et al. (1956).

Enzyme Purification Procedures

Enzyme purification was performed at O to 4OC. Phosphate buffer (10 m K2HP04/KH2P04, pH 7.5, and 10 m /I-mercaptoethanol) was used throughout unless stated otherwise.

E. gracilis cells were harvested at the end of the log phase by centrifugation at 5,OOOg for 15 min. About 45 g of algae (fresh weight) were resuspended in 170 mL of phosphate buffer. Cells were disrupted on ice in 30-mL portions by sonicating 15 to 20 times for 10 s with a B-12 sonifier (Branson Sonic Power Co., Danbury, CT). Sonication resulted in at least 95% ruptured cells. The suspension was centrifuged for 20 min at 12,OOOg.

For purification of the class I aldolase, a crude extract of autotrophic cells was applied to a DE52-cellulose (Whatman Ltd., Maidstone, Kent, UK) column (3 X 14.5 cm). The column was washed with 200 mL of phosphate buffer and proteins were eluted by a linear gradient of 500 mL of O to 250 m KCI. Peak fractions containing aldolase activity were pooled and dialyzed. The enzyme was concentrated by dialysis against PEG 40,000 (Serva, Heidelberg, Germany) and di- luted with 10 m P-mercaptoethanol, pH 7.5, to a conductiv- ity of less than 2.5 millisiemens cm-'. This solution was applied to a phosphocellulose (P11, Whatman Ltd.) column (3 x 14 cm). After washing the column, proteins were eluted with 50 mL of 0.1 m FBP in phosphate buffer. Peak fractions were pooled, dialyzed, and concentrated against PEG 40,000. Three milliliters were applied to a Sephadex G-200 column (2 X 89 cm). The column was run at a flow rate of 11 mL/h and fractions with aldolase activity were pooled. Part of this preparation was applied to a Mono-Q fast protein liquid chromatography (Pharmacia, Freiburg, Germany) column (5 X 50 mm). The column was washed until the was less than 0.001. Proteins were eluted at a flow rate of 2 mL/min by a gradient of 20 mL of O to 250 m KCI. Fractions of 500 pL were collected.

For the purification of the class I1 aldolase, proteins of a crude extract of heterotrophically grown cells were precipitated by adding solid ammonium sulfate to 45% saturation while keeping the pH at 7.5 with 1 M KOH. After !stirring the solution for 1 h on ice, the mixture was centrifuged at 12,OOOg for :30 min and the supematant solution was dialyzed. Pro- teins of this preparation were separated on DE52-cellulose as described above. After dialysis, the aldolase preparation was loaded onto a hydroxylapatite (Bio-Rad) column (3 X 17.5 cm). The colurnn was washed with two column volumes and aldolase was eluted with a linear gradient of 50 to 225 m potassium phosphate, pH 7.5. The peak fraclions were pooled, concentrated by dialysis against PEG 40,000, and in a final step, chromatographed on a Sephadex G-2.00 column as described above.

Enzyme Assays

FBP aldolase was assayed at 2OoC according io Wu and Racker (1959) by measuring the decrease in NADH As4,, in a recording spectrophotometer (Uvicon 810, Kontron Instru- ments, Zürich, Switzerland). The standard reaction medium for total aldolase activity consisted of 50 m Tris/HCI, pH 7.5,4.5 m MgC12, 1 unit each of triosephosphatt! isomerase and glycerol-3-phosphate dehydrogenase, 2 10 phi NADH, 2 m FBP, and 10 to 50 pL of the enzyme solution in a total volume of 1 mL. For determining class I aldolase activity, the enzyme was incubated additionally in 1 m EDTA for 5 or 10 min at 2OoC prior to assaying the activity in the above assay system without MgCl,. The omission of MgC1, had no effect on aldolase activity. The difference between total and class I aldolase activity was defined as class I1 aldolase activity. One unit of aldolase activity was defined as the cleavage of 1 pmol of FBP or oxidation of 2 pmol of NADH per min. All fine chemicals were obtained from Boehringer (Mannheim, Germany).

The pH activity profile was determined in the following buffer systems: Mes/NaOH from pH 5.0 to 8.0, Hepes/NaOH from pH 7.0 to 9.0, and Tris/HCl from pH 7.0 1.0 9.0. The effect of different monovalent and divalent ions ,md of Cys on the class I1 aldolase was determined in the standard assay system without MgC12. The enzyme was incubated for 5 or 10 min in the assay mixture in the presence of the, respective ions before the reaction was started by the addition of NADH (30 pL) and FBP (100 pL). The resulting activities were compared with the activity measured in the standard reaction medium without MgCI2.

Protein was determined with Coomassie brilliant blue G- 250 according to Bradford (1976) and with BSA as standard. KC1 concentrations were determined by conductirnetry.

Electrophoresis, Molecular Mass Determinations, and Antiserum

SDS-PAGE was performed as described by Laenimli (1970) in 12.5% gels with 5% stacking gel. The proteins were visualized by staining with Coomassie brilliant blue 17-250. The calibration proteins for molecular masses were phosphorylase



Class I and Class II Aldolases of Euglena gradlis 1139

Table 1. Purification scheme for the class 1 aldolase of E. gracilis

Step

Crude extractDE52-celluloseP-celluloseSephadex C-200

TotalVolume

mL

28097179

TotalActivity

units5433

4.500.84

Protein

mg

127130.50.042

SpecificActivity

units mg"1

0.432.648.30

20.00

Purification

fold16

1946

Recovery

%1006081.5

(106 kD), BSA (80 kD), ovalbumin (49.5 kD), carbonic an-hydrase (32.5 kD), soybean trypsin inhibitor (27.5 kD), andlysozyme (18.5 kD) (Bio-Rad). Gel electrophoresis under non-denaturing conditions was performed in 4 to 15% gradientgels in IX THE, pH 8.2 (90 rmn Tris, 80 mw H3BO4, 2.5 ITIMEDTA) and 0.6X TBE as running buffer as described byHames (1990). Thyroglobin (669 kD), ferritin (440 kD), cat-alase (232 kD), lactate dehydrogenase (140 kD), and albumin(67 kD) were used as calibration proteins of high molecularmass and were obtained from Pharmacia.

To determine the molecular mass of the native enzymesthe class I aldolase was analyzed by gel filtration on a Bio-Sil HPLC column with thyroglobulin (660 kD), ferritin (500kD), rabbit aldolase (160 kD), a-globulin (158 kD), lactatedehydrogenase (140 kD), ovalbumin (45 kD), and myoglobin(17 kD); the class II aldolase was analyzed on a Sephacryl S-300 column with 0-amylase (200 kD), alcohol dehydrogenase(150 kD), albumin (66 kD), and carbonic anhydrase (29 kD)as standard markers.

Antiserum against the purified native class II aldolase wasraised in a rabbit. Approximately 100 ^g of purified proteinin Freund's complete adjuvant was injected into the legmuscle of an animal. After 3 weeks boosts with 100 /igprotein in Freund's incomplete adjuvant were given threetimes at weekly intervals. Three days after the last boostblood was collected, and after 4 h at room temperature,serum was obtained through centrifugation. The antiserumprecipitated specifically class II but not class I aldolase inan immunopretipitation assay according to Kriiger andSchnarrenberger (1983). In western blots (Towbin et al., 1979)with crude extracts of £. gracilis, only one protein was visu-

18.

alized with the antiserum during SDS-PAGE, but some ad-ditional minor bands were observed during PAGE undernondenaturing conditions.

RESULTS

Purification and Properties of the Class I Aldolase

By measuring total and class I aldolase activity separately,it was found that the chloroplastic class I aldolase activityrepresented 81% of the total aldolase activity in autotrophi-cally grown E. gracilis cells. Heterotrophically grown cellscontained only 7% class I aldolase activity. For this reasonextracts of autotrophically grown cells were used for thepurification of the class I aldolase and extracts of heterotroph-ically grown cells were used for the purification of the classII aldolase. The class I aldolase of E. gracilis (Table I) waspurified about 46-fold by chromatography on DE52-celluloseand phosphocellulose and by gel filtration on Sephadex G-200. The recovery was 1.5% and the specific activity in thefinal preparation was 20 units/mg protein. An analysis bySDS-PAGE (Fig. 1) revealed one peptide with a molecularmass of 40 kD. This value is in good agreement with that forother eukaryotic class I aldolases. A molecular mass of 160kD was determined for the native enzyme by gel filtrationon a Bio-Sil HPLC column (data not shown). The enzymehad a broad pH optimum with a maximum from pH 6.5 topH 9 (Fig. 2). A Km(FBP) value of 12 HM was calculated froma Lineweaver-Burk plot. No effect of mono- or divalent ionsor of Cys was observed.

Figure 1. SDS-PAGE of the purified class I and class II aldolases of£. gracilis. M, Marker proteins; 1, class I aldolase; 2, class II aldolase. Figure 2. pH dependence of the class I aldolase of E. gracilis. www.plantphysiol.orgon April 4, 2019 - Published by Downloaded from

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~_______ ~~

Table II. Purification scheme for the class I / aldolase of E. gracilis

Step Total Volume

Total Activity

Protein Specific Activity

Purification Recovery

mL units mg units ing-' fold %

Crude extract 210 63 56 1.12 1 1 O 0 45% (NH,),SO, supernatant 260 47 39 1.21 1 . 1 75 DE52-cellulose 94 19 1.22 15 13 29 Hydroxylapatite 133 21 1.21 17 15 33 Sephadex G-200 5 7 0.31 22 19 1 1

Purification and Properties of the Class II Aldolase

For the purification of the class I1 aldolase, proteins of a crude extract of heterotrophically grown E. gracilis cells were precipitated by 45% ammonium sulfate. The supernatant solution, which contained class I1 aldolase activity, was used for further purification by chromatography on DE52-cellulose, hydroxylapatite, and Sephadex G-200 (Table 11). The class I1 aldolase was enriched about 19-fold, the recovery was 11%, and the specific activity was 22 units/mg protein. The procedure yielded a single peptide with a subunit size of 38.5 kD as determined by SDS-PAGE (Fig. 1). For the native enzyme a molecular mass of about 80 kD was obtained by gel filtration, indicating a dimeric structure. A distinct pH optimum at pH 7.5 (Fig. 3) was determined and a Km(FBP) value of 175 NM was determined.

Effect of Ions and Cys on the Class II Aldolase

The activity of the class I1 aldolase was strongly influenced by monovalent cations. K', Na+, and Rb' enhanced the activity about 3- to 7-fold; Li+ and Cs' were ineffective. Maximal activity with potassium acetate and potassium chloride was at 0.1 M with only slightly reduced activity at higher concentrations (Fig. 4). If the sulfate salts were used, maximal activity was at 25 to 30 m ~ , the stimulation was not as high as for acetate and chloride salts, and the decline toward

higher concentrations was more pronounced than for the other salts (Fig. 5). In addition, we observed that freshly dialyzed class I1 aldolase kept at O°C for several days grad- ually decreased in activity to less than 30% of the initial activity. Subsequent addition of 50 m~ potassium acetate restored almost fully the initial activity within 5 min. From these data we conclude that alkali ions of mediuin diameter are involved in the regulation of activity by maintaining the enzyme in an active state.

Another inhibitor for the enzyme of E. gracilis is Cys at concentrations higher than 0.8 mM (Fig. 6). The inhibitory effect of Cys cannot be overcome by the addition of 50 m~ potassium acetate. Therefore, the target site for the two molecules on the enzyme must be different.

The class I1 aldolase of E. gracilis is fully active in the absence of divalent metal ions. One millimolar ED TA inhibits the class I1 aldolase of E. gracilis up to 90% during a 5-min preincubation and to more than 95% during a 10-min preincubation, and 5 mM EDTA produced complete inhibition within 2 to 5 min (data not shown). Therefore, the inhibition by EDTA is progressive with time and concentration of EDTA. To test whether or not the inhibition of ):he class I1 aldolase caused changes in the oligomeric state of the protein, we performed western blots with a partially purified enzyme treated with 5 m~ EDTA or 10 m~ Cys. At least in the case of EDTA treatment, the loss of a divalent ion should have

150 - - E . I .- - 5 E - 100 h

> " n

.- .- .- e

P o 50

d

- U -

n i.0 6.0 7.0 R.0 9.0

PH

Figure 3. pH dependence of t h e class I I aldolase of E. gracilis. +, Mes/KOH; El, Mes/NaOH; o, Hepes/KOH; O, Hepes/NaOH.

Potassium ~ C C U I C

-a 'O0 600 i 1 Polnuium chloride

I A

Y .- > 5'. - .- 1 : : -

Sodium c iloride 5 400 - O 3 * 300

O0

Lithium chloride

2

Cesium chliiride

0.0 0.1 O.? 0.3 Salt concentration [ R I ]

Figure 4. Dependence of the class II aldolase activity o f E. gracilis on monovalent ions presented as chloride and acetate jalts.



Class I and Class II Aldolases of Euglena gracilis 1141

okD U

;*> QG to

0.1 0.2Salt concentration [M]

Figure 5. Effect of sulfate ions on the activation of the class IIaldolase of E. gracilis by monovalent ions.

been irreversible for the enzyme. Separation of proteins byPAGE under nondenaturing conditions and immunoblotting(Fig. 7) showed a major band of dimeric aldolase around 80kD and some minor contaminants. The different treatmentswith EDTA and Cys did not change the oligomeric state ofthe enzyme compared with the untreated control enzyme.Thus, it is unlikely that removal of divalent ions by EDTA orinhibition by Cys interferes with the dimeric state of theenzyme.

In the absence of Mg2+ in the standard assay, the class IIactivity was virtually unchanged. No significant activation ofthe class II aldolase activity was observed with Mg2+, Mn2+,Ni2+, or Co2"1" at concentrations of either 0.1 or 1 mM. FeCU,in the range from 100 ^M to 10 HIM, used in some earlierinvestigations on class II aldolase activity, produced a non-specific reaction with NADH in the assay system and wasunsuitable for such studies. At concentrations less than 100MM, FeCl2 did not affect the activity. Cu2+ (data not shown)

669

440

232

140

67

Figure 7. Western blot analysis of class II aldolase without (control)or after treatment with 10 mM Cys or 5 mM EDTA for 20 min.Proteins were separated on a 4 to 15% native gradient gel. Afterblotting and probing with an antiserum against purified native classII aldolase of B. gracilis, proteins were visualized with an anti-rabbitperoxidase-conjugated antibody.

and Zn2+ (Fig. 6) caused almost complete inhibition of theenzyme at concentrations higher than 0.5 mM and no changeat lower concentrations.

In another attempt to analyze the dependence of the classII aldolase on divalent ions, we inactivated the enzyme bydialysis against 1 mM EDTA and 20 mM potassium acetate inenzyme buffer overnight. After 3 to 4 h of dialysis againstthe same solution without EDTA, aliquots of the enzymewere incubated 15 min with divalent ions and tested forenzyme activity. Under these conditions, Mn2+ and Co2+

restored the enzyme activity up to 70% and Cu2+ restored upto 35% of the original activity. Other metal ions (Mg2"1", Fe2+,Zn2+, Ni2+) had no effect (Table III). Therefore, the enzyme

.01 .1 1 [mM] 10Cysteine/zinc concentration

Table III. Reactivation of the aldolase of E. gracilis after inactivationwith 1 mM EDTA in 20 mM potassium acetate and enzyme buffer

Figure 6. The influence of Cys (with and without 50 mM potassiumacetate) and of zinc sulfate on the class II aldolase of E. gracilis.

Chemical

ControlEDTAMn2+

Co2+

Cu2+

Fe2+, Mg2+

Ni2+, Zn2+

Concentration

1 mM1 MM

10 MM100 MM

1 mM1 MM

10 MM

100 MM1 mM1 MM

10 MM

100 MM

10 MM

1 mM

Percent Activityof Control

1005

1313576713113837121148~5f^C




of E. gracilis has a specific binding affinity for Mn2+, Co2+, and Cu2+.

DISCUSSION

The purihed plastidic class I and the cytosolic class I1 aldolase of E. gracilis show the typical structural and catalytic properties of most other eukaryotic class I and class I1 aldolases (for review, see March and Lebherz, 1992). This means that the class I enzyme with a molecular mass of about 160 kD has a tetrameric subunit structure, a broad pH dependence, and a high affinity for the substrate FBP, and that the class I1 aldolase with a molecular mass of 80 kD shows a dimeric subunit structure, a narrow pH dependence, and 10- fold lower affinity for FBP.

The specific activities of the class I and class I1 aldolases are on the order of 20 units/mg protein. The specific activity of the class I aldolase of E. gracilis is therefore about 3-fold higher than that of the chloroplast and cytosol aldolase of higher plants and most green algae (Schnarrenberger et al., 1992). A Km(FBP) value of 2 and 5 ~ L M FBP for the E. gracilis class I aldolase, as reported by Mo et al. (1973) and Karlan and Russell (1976), is lower than our value of 12 ~ L M . The &(FBI') value of 175 ~ L M for the class I1 enzyme is in good agreement with 190 p~ as reported by Mo et al. (1973).

A constitutive property of all class I1 aldolases is the involvement of divalent ions in the class I1 aldolase activity. After inactivation of the E. gracilis enzyme by EDTA, Mn",

Co2+, and Cu2+ restored the activity at least partially, but Mg2+, Fez+, Zn2+, and Ni2+ did not. This implies that Mn2+, Co2+, or Cu2+ is a natural constituent of the enzyme. So far, Fez+ or Zn2+ (or both) were always found to 'De the main divalent ions in addition to some others, such as M e , Mn2+, Co2+, or Cu2+, in the class I1 aldolases of bacteria, fungi, or some algae (see Table IV).

Particularly class I1 aldolases from eukaryotes and prokaryotes show some specific regulatory differences in (a) activation or not by monovalent ions, (b) activation or not by divalent ions, and (c) activation or inhibition by Cys. These criteria will be used to distinguish aldolases front major taxa. First, the class I1 aldolase of E. gracilis showed a requirement for monovalent ions, in this case for K+, Na+, Rb-, and NH,+, but not for Li+ or Cs+, as is also the case with the class I1 aldolases of Saccharomyces cerevisiae, Candida utilis, Clostri- dium perfringens, and of some algae, although with a different specificity for monovalent ions (Bard and Gunsalus, 1950; Richards and Rutter, 1961b; Kowal et al., 1966; Ikawa et al., 1972; Gross et al., 1994). The class I1 aldolasN+containing algae include also the cytosol and plastid enzyme of Cyano- phora paradoxa (Glaucocystophyta). In sum, nci correlation can be found for the activating effect of monovalent ions with major taxa. Because the effects of monovalent ions appear reversible, we think that monovalent ions may function in maintaining the enzyme in a fully active state.

Second, the class I1 enzyme of E. gracilis coukl in no case be further increased in activity by various divalent ions of low ionic strength. Such activation by Fez+, Zn2+, and/or Co2+

Table IV. influence of Cys and of mono- and divalent ions on various class I / aldolasesa n.m., Not measured.

Activation Inhibition Activation Reactivation by K+ by by bY

Effect of Cys

Clostridium perfrin- Stimulation 5 x Fe3+, Cu2+, Niz+ FeZt, Co2+ n.m.

Halobacterium medi- Stimulation n.m. n.m. Fez+ Fe2+, Co2+

Halobacterium vol- Stimulation n.m. n.m. Fe2+ Fe2+, Co2+, Mn2+

Saprospira thermalis' Stimulation' 2x1 n.m. Zn2+, Fez+ n.m. Anacystis nidulans' Stimulation' 1 x' n.m. Fez+, Mn2+ n.m. Prochlorothrix hollan Stimulation I X n.m. n.m. n.m.

Hizikia fusiformish None 3-4x Fe2+, Co2+ n.m. Zn2+, Co2+, Fe2+ Porphyra suborbicu- n.m. n.m. Fe2+ n.m. Zn2+, Co2+, Fez+

Cyanophora paradoxa8

gensb

terraneic,d

caniiCvd

dica8

latah

Cytosol enzyme inhibition 4 x n.m. n.m. Co2+, Mn2+, Zn2+, Mgz+ Plastid enzyme Inhibition 1x n.m. n.m. Co2+, Mn2+, Cu2+, Mg2+

Saccharomyces cerevi- Inhibition 6-35X Znz+, Co2+, Fe2+ n.m. Zn2+, Co2+, Fez+, Cu2--, Ni2+ siae'

Aspergillus niger' Inhibition n.m. n.m. n.m. Zn2+, Co2+, Fe2+, MnZt Candida uti/isk n.m. 3 x n.m. n.m. Zn2+, Mn2+, MgZ+ Fuglena gracilis' Inhibition 5-7x Zn2+, Cuz+ n.m. Mn2+, Co2+, Cuz+

" A class I 1 aldolase of Chlamydomonas mundana (Russell and Gibbs, 1966, 1967) was not included because it might be an artifact (unpublished data by Dr. U. Klein, University of Bonn, and S. Jacobshagen and C. Schnarrenberger, Free University of Berlin). Bard and Cunsalus (1950). Dhar and Altekar (1986). Dhar and Altekar (1989). 'Willard and Gibbs (1968a). ' Other cpnobacteria (Anabaena variabilis, Plectonema sp. Anabaenopsis sp., and Nostoc muscorum) also showed a Cys and Fe2+-dependent activity (Willard and Gibbs, 1968a). 8Gross et al. (1994). ikawa et al. (1972). ' Kobes et al. (1969). Jagannathan et al. (1956). Kowal et al. (1966). ' This paper.



Class I and Class II Aldolases of Euglena gracilis 1143

appears to be a regulatory property and has been observed for bacterial enzymes, but never for eukaryotic enzymes (see Table IV). At high concentrations, e.g. 100 FM, Zn2+ and Cu2+ were inhibitory for the E. gracilis enzyme, probably because of a n unspecific binding. This phenomenon was also reported for the enzymes of S. cerevisiae, C. perfringens, Porphyra suborbiculata, and Hizikia fusiformis.

Third, the inhibition of a class I1 enzyme by Cys was not known before for the enzyme of E. gracilis. None of the eukaryotic class I1 aldolases are activated by Cys, including the enzymes of red and brown algae and the cytosol and plastid enzyme of C. paradoxa (Ikawa et al., 1972; Gross et al., 1994), whereas the enzymes of fungi (Richards and Rutter, 1961b; Kowal e t al., 1966) are inhibited. In contrast, Cys is reported as a necessary factor for the activation of the bacterial and cyanobacterial enzymes (Willard and Gibbs, 1968b; Ikawa et al., 1972; Dhar and Altekar, 1986, 1989).

From the available biochemical data on class I1 aldolases, we propose to distinguish two groups of enzymes, one group covering bacterial and cyanobacterial enzymes that are all activated by Cys and that can be activated considerably by divalent ions of low ionic strength, and another group covering eukaryotic (fungal and algal) enzymes that are inhibited/not activated by Cys or divalent ions (see Table IV). These groupings correlate with systematic taxa and imply conserved features in enzyme structure. Enzymes of both groups show various degrees of activation by monovalent ions and a varying specificity for divalent ions. These latter criteria seem to depend on less-conserved domains in the protein structure.

Received June 20, 1994; accepted July 29,1994. Copyright Clearance Center: 0032-0889/94/106/1137/08.

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Antia NJ (1967) Comparative studies on aldolase activity in marine planktonic algae and their evolutionary significance. J Phycol 3

Bard RC, Gunsalus IC (1950) Glucose metabolism in Clostridium perfringens: existence of a metallo-aldolase. J Bacteriol59 387-400

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Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis

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