Glyoxylate cycle enzymes of the glyoxysomal membrane from cucumber cotyledons

ARCHIVES

OF

BIOCHEMISTRY

Glyoxylate

AND

BIOPHYSICS

236-248

181,

(1977)

Cycle Enzymes of the Glyoxysomal Cucumber Cotyledonsly WOLFRAM

Biochemie

(Fachbereich

KGLLER Chemie), Received

AND

HELMUT

Philipps-Universitit, November

Membrane

from

KINDL Marburg,

Germany

12, 1976

Glyoxysomes were isolated from etiolated cotyledons of cucumber seedlings. After separation of matrix proteins from the glyoxysomal membranes, enzymes were solubilized from the membranes by 100 mM MgCl, and purified by sedimentation velocity centrifugation, ion exchange chromatography, and separation on hydroxylapatite. Malate synthase, citrate synthase, and malatc dehydrogenasethe three enzymes of the glyoxylate cycle which were primarily membrane bound in this type of microbody-were thus obtained in a homogeneous form, as judged by sodium dodecyl sulfate-gel electrophoresis. Enzymatically active malate synthase, as obtained by solubilization of membrane proteins, behaved on Sepharose 6B columns as a protein with a molecular weight of about 70,000 and is characterized by an acidic isoelectric point. Malate synthase aggregates in the presence of Mg2+ and glyoxylate, yielding an active octamer with an alkaline isoelectric point and a molecular weight of about 540,000. Upon sodium dodecyl sulfate-gel electrophoresis, a subunit molecular weight of 63,000 was estimated. Citrate synthase exists as a dimer (molecular weight of 100,000) and tetramer (molecular weight of 200,000) and exhibits the same subunit molecular weight as the liver enzyme (46,000). Malate dehydrogenase was found to have a molecular weight similar to the microbody catalase (about 225,000), while for the single peptide chain a value of approximately 34,000 was determined.

Glyoxysomes house enzymes which are responsible for fatty acid degradation, the glyoxylate cycle, and purine degradation (2-4). Until now, biochemical studies have primarily concentrated on the physiological role of the glyoxylate cycle enzymes. To our knowledge, these enzymes have so far not been purified to homogeneity using isolated glyoxysomes or microbody subfractions as starting material. To provide a better understanding of the functioning of these enzymes and with the aim to use the proteins as probes for biosynthetic studies, one would like to have the glyoxylate cycle enzymes available in a purified form. It was our intention to characterize the part of the enzymes which

is associated with the membranes of the glyoxysomes (5, 6). Since citrate synthase and malate dehydrogenase are found in several compartments of the cells, an isolation of glyoxysomes appeared, therefore, to be a prerequisite for further purification. In the case of malate synthase, the glyoxysomal membrane guarantees a homogeneous starting material for the preparation of a single form of the enzyme, although one has to expect low yields under these conditions. In the case of malate dehydrogenase, another way has already been taken to the same goal: separation of multiple forms and subsequent attribution to certain organelles (7, 8). The present paper deals with the characteristics of malate synthase, citrate synthase, and malate dehydrogenase, which comprise a high percentage of the glyoxysomal protein. It was the purpose, thus, to accumulate sufficient information for all of

*This is paper No. 4 in a series on plant microbody proteins. Number 3 of this series is Ludwig, B., and Kindl, H. (1976) Hoppe Seyler’s 2. Physiol. Chem. 357, 393-399. *This investigation was supported by the Deutsche Forschungsgemeinschaft. 236 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0003-9861

ENZYMES

OF

GLYOXYSOMAL

the further studies aimed at the elucidation of biogenesis and assembling of glyoxysomes . MATERIALS

AND

METHODS

(1) Isolation ofglyoxysomes and other organelles. Seeds of Cucumis satiuus (Chinesische Schlangengurken) were surface sterilized and germinated at 25°C. Crude homogenates were prepared from cotyledons (600 g) of &day-old seedlings by blending (3 x , 10 s) in a homogenizer. The grinding medium contained 150 mM Tri&HCl (pH 7.5), 10 mM KCl, 1 mM EDTA, 1 mM MgCl,, and 25% (w/w) sucrose. Portions (100 g) of cotyledons were homogenized in 100 ml of medium. The homogenates were squeezed through two layers of Miracloth, the residue was extracted again with grinding medium, and the combined extracts were centrifuged at 10006 for 10 min. The supernatant thus obtained was then centrifuged at 13,OOOg for 30 min. The pellet, representing a mixture of mitochondria, proplastids, and glyoxysomes, was suspended in grinding medium (about 100 ml) and the organelles were separated by centrifugation in a zonal rotor (Ti-15 or Ti-14, Beckman) as described elsewhere (91. Small gradients in the SW 27 rotor were run for 5 h and were then fractionated by collecting successive 1.2-ml samples from an ISCO Model 680 density gradient fractionator connected to a fraction collector. All large scale preparations in the zonal rotors were fractionated as previously described (9). Fractions were assayed for protein concentration, sucrose concentration, and marker enzyme activity. (2) Glyoxysom,al membranes and their enzymes. Glyoxysomal membranes were obtained from the isolated glyoxysomes by osmotic shock: For every 10 ml of glyoxysomal suspension, 5 ml of buffer (50 rnru Tris-HCl, pH 8.5) was added and left standing for 30 min and then centrifuged at 42,000 rpm (Beckman Ti-60 rotor) for 60 min. The supernatant was used as a source of matrix enzymes, while the pellet was suspended in buffer and again sedimented at 42,000 rpm. This pellet is referred to as “glyoxysomal membranes.” They were then suspended in 5 ml of TrisHCl buffer using a Potter homogenizer and mixed with 1 ml of a mixture containing 600 rnxr MgC12, 12 mM sodium glyoxylate, and 6 mM sodium oxalacetate for 30 min at 4°C. Subsequently, the suspension was centrifuged at 42,000 rpm for 30 min and the supernatant, representing a high percentage of the membrane-bound malate synthase, citrate synthase, and malate dehydrogenase, was further puri3 Abbreviations used: Tris, methyl)-1,3-propanedioh SDS, fate; DEAE, diethylaminoethyl; yethylj-1-piperazineethanesulfonic boxymethyl.

2-amino-2-(hydroxysodium dodecyl sulHepes, 4-(2-hydroxacid; CM, car-

MEMBRANES

237

fied as follows. The remaining pellet was again treated with MgCl, in the same way and finally assayed for tightly bound membrane proteins. All steps described here were followed by SDS-gel electrophoresis as described below. (3) Separation of proteins by sedimentation uelocity centrifugation. Samples (1.5 ml each) of the enzyme mixture obtained by salt treatment were layered onto sucrose gradients consisting of 1.0 ml of 50% sucrose, 1.0 ml of 40% sucrose, 1.5 ml of 37% sucrose, 2.5 ml of 35% sucrose, 1.0 ml of 30% sucrose, 1.0 ml of 25% sucrose, and 2.5 ml of 20% sucrose; the zonal centrifugation was performed in a SW 40 rotor (Beckman) at 40,000 rpm for 45 h at 4°C. A density gradient fractionator and a fraction collector were utilized to fractionate and collect 0.6-ml samples. The samples were assayed for malate synthase, citrate synthase, catalase, and malate dehydrogenase. Samples of enzyme mixtures together with various internal molecular weight standards were also run. Absorbance at 280 nm was monitored by an ISCO UA-4 absorbance monitor and sucrose density was determined refractometrically. f4,l Separation on Sepharose 6B. As an alternative to zonal centrifugation, the proteins were separated by molecular sieving on a Sepharose 6B column (50 x 2.5 cm). Five and one-half milliliters of the solubilized mixture of associated enzymes was placed on the column, previously equilibrated with elution buffer, and eluted with a 20 mM Tris-HCl buffer, pH 8.0, containing 10 mM MgCl,, 200 mM KCl, 1 mM oxaloacetate, and 2 mM glyoxylate. (5) Chromatography on DEAE-Sephadex. Prior to the anion exchange chromatography, the enzyme preparation was freed from salts by pouring the preparation over a small column (1.5 x 20 cm) with Sephadex G-50. The protein-rich fractions were placed onto a column filled with DEAE-Sephadex A25 previously equilibrated with 20 mM Tris-HCl (pH 8.0), 50 mM KCl, and 1 mM oxaloacetate. After washing with 20 ml of the same buffer, 80 ml of a gradient of 50-300 mM KC1 was used to elute the proteins. Prior to fractionation, each test tube in the fraction collector was supplied with 50 ~1 of 6 M KC1 containing 40 mM oxaloacetate. This leads to a final concentration of 200 mM KC1 and 1 mM oxaloacetate in each fraction immediately after fractionation. (6) Chromatography on CM-Sephadex. The salt concentration of protein mixtures was reduced by passing the mixtures through Sephadex G-50. To elute the proteins from the CM-Sephadex column (1.0 x 18 cm), first, a 10 mM Hepes buffer, pH 8.0, containing 50 mM NaCl and 1 mM oxalacetate was used and, second, a salt gradient of 50-400 mM NaCl was applied. (7) Chromatography on hydroxylapatite. A column (1.0 x 22 cm) was filled with hydroxylapatite (Biogel HTP, Bio-Rad Lab.) suspended in 10 mM potassium phosphate, pH 7.8, and 200 mM KCl. Cit-

238

KGLLER

AND

modifications. The reaction mixture contained, in a total volume of 1.0 ml, 100 rnzi Tris-HCl, pH 8.0, 0.05% Triton X-100 (in the case of membrane suspensions only), 0.10 rnM Ellman reagent, 5.0 rniu MgCl,, 4 mM glyoxylate, 60 ELM acetyl-CoA, and a suitable amount of enzyme. For the assay ofcitrate synthase (EC 4.1.3.7) (17), the following chemicals were included in the incubation mixture (1.0 ml): 0.1 mM 5,5’-dithiobis-2-nitrobenzoate, 1.3 mM oxalacetate, 60 FM acetyl-CoA, 0.1 M Tris-HCl, pH 8.0, and 0.05% Triton X-100 (with membranes only). NADH-cytochrome c reductase (EC 1.6.2.4) was tested according to Lord et al. (18) following the increase of oxidized cytochrome c at 550 nm. Protein was precipitated with trichloroacetic acid and then estimated by the method of Lowry et al. (19) with corrections described by Gerhardt and Beevers (20) if necessary.

rate synthase-containing fractions were put on this column and washed with 15 ml of equilibration buffer. Elution was then performed with a gradient of lo-400 mru phosphate containing 200 mr.r KCl. Fractions were collected in test tubes already containing 50 ~1 of 30 mM oxalacetate. (8) Zsoelectric focusing. Isoelectric focusing was performed in sucrose gradients at 2°C by using a minor modification of the method of Vesterberg (10). Current was supplied at 3 mA until the voltage had risen to 400 V, the voltage was then maintained at the level of 1000 V for 45 h. Gradients were fractionated and assayed for enzymes. (9) SDS-gel electrophoresis. Enzymes or membrane fractions were dissolved in a SDS-urea mixture according to Weber et al. (111 and then adjusted to 0.1% SDS by dialysis. Electrophoresis was performed in a slab gel apparatus (12) using a continuous SDS system described by Ludwig and Kind1 (9). The resolving gel (0.3 x 20 cm) was 5% acrylamide with net cross-linking of 0.5%. The samples were allowed to enter this gel at 5 mA and were then subjected to 20 mA until the bromophenol blue marker had moved to within 1 cm of the bottom of the resolving gel. Calibration curves were obtained as described earlier (9). Protein was stained for 10 h with 0.02% Coomassie blue in a mixture of 60 g of trichloroacetic acid, 70 ml of acetic acid, 200 ml of methanol, and 800 ml of H,O. The gels were destained in methanol/acetic acid overnight. (10) Enzyme assays. All enzymes were assayed spectrophotometrically at 25°C. The incubation mixture for the test of fumarase (EC 4.2.1.2) contained 1.5 ml of 100 mM Tris-HCl, pH 7.5, 0.2 ml of 1% Triton X-100, and 0.2 ml of enzyme solution. After preincubation for 15 min, the reaction was started with 0.1 ml of a 1 M solution of r,-malate and the formation of fumarate was followed at 240 nm. Malate dehydrogenase (EC 1.1.1.37) was tested according to Ochoa (13). Cytochrome c oxidase (EC 1.9.3.1) (14) and catalase (EC 1.11.1.6) (15) were assayed as previously described. For the estimation of malate synthase (EC 4.1.3.21, the procedure described by Hock and Beevers (16) was employed with minor

RESULTS

The purification of the three membrane enzymes shared similar initial steps: preparation of glyoxysomal membranes, solub&&ion, and fractionation of associated proteins. Subsequently, each protein was purified to homogeneity separately and characterized further. Preparation of Glyoxysomes ma1 Subfractions

OF ENZYMES

(SPECIFIC

FRACTIONS

Fractions Gradient superna~.;t~.O9-

Catalase (U/mg) Fumarase (mU/mg) Citrate synthase (mU/mg) Malate synthase (mU/mg) Malate dehydrogenase (U/mgl

I

ACTIVITIES) BETWEEN CENTRIFUGATION

Enzymes

1222 26 160 225 32

and Glyoxyso-

An essential prerequisite for an unequivocal assignment to a certain intracellular compartment of the enzymes to be isolated was a purified organelle fraction. Accordingly, glyoxysomes were isolated by isopycnic density gradient centrifugation and characterized by marker enzymes. It was important to use a certain shape of the sucrose gradient to achieve an optimal separation between mitochondria and glyoxysomes. A quantitative survey of the

TABLE DISTRIBUTION

KINDL

Mitochondria (1.17-1.19) 175 399 532 164 60

OBTAINED

(densities,

BY ISOPYCNIC

GRADIENT

g/cm”)

Intermediary region (1.22-1.23) 1170 41 308 762 38

Glyoxysomes (1.23-1.26) 2920 <5 626 1630 48

ENZYMES

OF

GLYOXYSOMAL

ing for (a) a soluble fraction representing the matrix enzymes and (b) a membrane pellet. Then, peripheral proteins were solubilized from the membrane by increasing the ionic strength; we used 100 mM MgCl,. Treatment with various detergents, e.g., Triton X-100 or cholate, was not effective in solubilizing the membrane enzymes. This agrees with findings about the properties of certain glyoxysomal enzymes from castor bean endosperm (21). The electrophoretic analysis of the subfractionation of glyoxysomes into matrix enzymes, peripheral (i.e., salt solubilized) enzymes, and integral proteins of the membrane is summarized in Fig. 1. The balance sheet of the operations, as far as the interesting enzymes are concerned, is shown in Table II.

distribution of the marker enzymes (Table I> makes it evident that the glyoxysomal fraction was free of contaminating mitochondria. Glyoxysomes were subsequently broken by osmotic shock and centrifuged provid-

malate

synthase

catalase citrate

synthase

malate dehydrogenase

Separation cording

TABLE (I)

Fraction Supernatant after osmotic shock Supernatant after membrane washing Supernatant after solubilization with MgClz Pellet after solubilization Total

recovery

OF ENZYMES

AND

PROTEIN

Designation Matrix Peripheral ciated) Membranes

(assoenzymes

of the Solubilized Enzymes to Their Molecular Weight

Ac-

A reasonably good separation of the major proteins obtained by Mg2+ treatment of the membrane could be achieved by zonal centrifugation. This procedure seemed to be complicated, as malate synthase was found to exist as two species differing in molecular weight. Systematic investigations indicated that, for a useful procedure, it is necessary to maintain malate synthase as a high molecular weight form, This was accomplished by adding Mg2+ and glyoxylate to gradients or elution buffers. A representative example of a prepara-

FIG. 1. SDS-electrophoretic analysis of membrane pellets and supernatants after subfractionation of glyoxysomes. M, marker proteins on the gel, were as follows: phosphorylase (muscle, 95,000); serum albumin (bovine, 68,000); catalase (liver, 58,000); ovalbumin (43,000); alcohol dehydrogenase (yeast, 37,000); carbonate dehydratase (erythrocytes, 29,000); cytochrome c (muscle, 12,400). 1, glyoxysomes; 2, matrix; 3, untreated membrane (prior to MgCl, treatment); 4, supernatant after washing the membranes with buffer a second time; 5, MgCl,-solubilized proteins; 6, membrane remaining after MgCl, treatment (showing probable integral proteins).

DISTRIBUTION

239

MEMBRANES

II AFTER

Catalase

SUBFRACTIONATION

Malate ByIlthC3Se

OF GLYOXYSOMEP

Citrate synthase

Malatc dehydrogenase

Protein

73

16

27

25

69

19

1

3

8

16

6

69

63

54

6

2

14

7

13

9

87

95

51

92

120

a Glyoxysomes were disrupted osmotically by dilution with buffer. The membranes were separated from the soluble fraction (matrix proteins) by centrifugation. The pellet was suspended in buffer and centrifuged again (supernatant after membrane washing). Finally, the resulting pellet was resuspended, treated with 100 mM MgCIZ, and sedimented (supernatant and pellet after solubilization).

240

KC)LLER

4

8

AND

12

KINDL

16

20

24 fraction

SDS-gel electrophoresis

-t

no.

468 *58

FIG. 2. Separation of associated enzymes by zonal centrifugation. Using tubes for a SW 40 rotor, 1.5 ml of solubilized protein was layered onto a sucrose gradient. Equivalents (50-100 rg) of the fractions (0.6 ml) were concentrated and analyzed by SDS-gel electrophoresis. Patterns of the proteins in the respective fractions are given in the lower part of the figure. B-m, malate synthase (1 relative unit = 15 pmol x min-1 x fraction-‘); A-A, citrate synthase (1 relative unit = 9 pm01 X min-’ X fraction-‘); *----*, catalase (1 relative unit = 7 mm01 x min-1 x fraction-‘); 0, sucrose concentration (percentage, w/w); O-U, malate dehydrogenase (1 relative unit = 500 pm01 X min-’ X fraction-‘).

tive separation of salt-solubilized proteins were also detected in the fraction of matrix by zonal centrifugation (Fig. 2) and on proteins (Fig. 1). Sepharose 6B (Fig. 3) is shown. The analyPurification and Some Properties of the sis of the fractions by SDS-gel electrophoEnzymes resis is presented in the two Figures. Malate synthase was already highly pure after (a) Malate synthase. Zonal centrifugathis purification step. Both enzymatically tion afforded, in most cases, homogeneous active species of citrate synthase derive malate synthase. This was observed in from the monomer with a molecular most of our experiments (Fig. 2, fraction weight of 46,000. Proteins having subunit 19). But if the enzyme thus obtained was molecular weights of 22,000 and 18,000 not sufficiently pure, as judged by SDS-gel were coinciding with the profile of the high electrophoresis, chromatography on CMmolecular weight form of citrate synthase. Sephadex might be used to separate negliThese proteins were not only found upon gible amounts of malate dehydrogenase, subfractionation of peripheral proteins but catalase, and a protein (see Fig. 2, electro-

ENZYMES

OF

GLYOXYSOMAL

phoresis of fraction 15) with a molecular weight of about 200,000 and a subunit molecular weight of about 20,000. The steps used in the purification of malate synthase from glyoxysomes and the results obtained are summarized in Table III. If associated proteins were solubilized from the glyoxysomal membranes and chromatographed on Sepharose 6B, malate synthase was found to be present as an enzymatically active monomer with a molecular weight of approximately 70,000 (Fig. 4). Systematic studies using sedimentation velocity centrifugation revealed that, in the presence of glyoxylate and Mg2+, the enzyme is converted to an oligo-

MEMBRANES

241

mer. While the zonal centrifugation in the presence of Mg2+ and glyoxylate (Fig. 5) led to malate synthase preparations with specific activities of 20-25 pm01 X min-’ X (mg of protein)-‘, the same procedure in the absence of these compounds yielded the high molecular weight form, besides a low molecular weight form, exhibiting a considerably lower specific activity of about 4 pm01 x min-’ x (mg of protein)-‘. Both species of the enzyme are enzymatitally active. The phenomenon of two forms showing different molecular weights on molecular sieving or zonal centrifugation was paralleled by the behavior of malate synthase

f 0 > .c 0 Yi L

FIG. 3. Separation of solubilized proteins by gel permeation chromatography on Sepharose 6B. Equivalents (50-100 pg) of the fractions (3.0 ml) were concentrated and analyzed by SDSgel electrophoresis. Patterns of the proteins in the respective fractions are given in the lower part of the figure. m---m, malate synthase (1 relative unit = 15 pmol x min-’ x fraction-‘); A-A, citrate synthase (1 relative unit = 9 Fmol x min-’ x fraction-‘); O-0, malate dehydrogenase (1 relative unit = 500 pmol x min-’ x fraction-‘).

242

KGLLER

AND KINDL

TABLE PURIFICATION SkP

S’Y m

Crude extract Crude mitochondrial fraction Glyoxysomes Solubihzation Zonal centrifwation

* c : ._: ‘r 5L

Total activity (U) 2849 1251

1440 110 240 6 4.4

536 174 133

1.0

0.5

65

95

125

III

OF MALATE PI-0th

ml

FIG. 4. Chromatography on Sepharose 6B of malate synthase and citrate synthase in the absence of glyoxylate and MgZ+. VO, void volume; m---H, malate synthase; A-A, citrate synthase.

on CM-Sephadex, on which two species again can be observed. Malate synthase alters its properties drastically depending on both pretreatments and the substances added to the elution buffer. Upon chromatography on CM-Sephadex (Fig. 6), a high molecular weight fraction from the sedimentation velocity centrifugation gave only one form, binding as cation (“alkaline malate synthase”). When Mg2+ and glyoxylate were omitted, however, a crude membrane protein fraction afforded two forms of the enzyme: one not bound to the cation exchange Sephadex at 60 mM Na+ (“acid malate synthase”), and a second species behaving as a cation at pH 7.0 (Fig. 6). High recoveries after ion exchange chromatography were achieved only when glyoxylate and Mg2+ were present in equilibration buffer and eluants. The properties of crude malate synthase during isoelectric focusing (Fig. 7) were in full accordance with these data. An alka-

(mg/ml) 17.0 15.0

-

1.4 3.1 1.2

SYNTHASE

Specific activity W/mg) 0.12 0.76

Yield (%)

1.63 9.34 25.1

19 6 4.6

100 44

p”f’fic$on n 1 6.5 14.1 81 209

line malate synthase, p1 = 9.1, and one or two acid forms (PI= 5.4) were prepared in this way. On the basis of experiments with the homogeneous protein, we attribute the isoelectric point of pH 9.1 to the high molecular form of malate synthase. The subunit molecular weight of the enzyme, as determined by SDS-gel electrophoresis, is 63,000. Molecular weight determinations by the method of Martin and Ames (22) using catalase, fumarase, and pig heart citrate synthase as markers exhibited an oligomer with a molecular weight of about 540,000 (18.6 S) besides a small activity peak of 79,000 (5.2 S). The K, (acetyl-CoA) was determined to be 11 PM. (b) Citrate synthczse. Citrate synthase was obtained from molecular sieving with high specific activity, the 100,000 molecular weight form prevailing over the 200,000 molecular weight form. Chromatography on hydroxylapatite (Fig. 8) enabled us thus to purify the enzyme to near homogeneity. The corresponding data are summarized in Table IV. The K, (acetyl-CoA) was determined to be 60 PM. If citrate synthase preparations were taken after zonal centrifugation, some accompanying malate dehydrogenase was further separated by chromatography on Sepharose 6B. The purified enzyme exhibited a subunit molecular weight of 46,000. The molecular weights of the active dimer and the tetramer were determined to be 100,000 (6.0 S) and 200,000 (9.2 S), respectively. Isoelectric focusing indicates that an “alkaline form” (p1 = 8.8) and “acidic forms” (p1 = 5.4) exist. Applying CM-Sephadex chromatography, we also showed that two species can exist (Fig. 9).

ENZYMES

OF GLYOXYSOMAL

It is noteworthy to emphasize the stabilization of citrate synthase by 200 mM KCl, bovine serum albumin (1 mg/ml), or oxaloacetate (1 mM). When citrate synthase preparations were left standing for 6 days at 4°C in the presence of stabilizing agents, the activity of the 100,000 molecular weight form was reduced by 50%, while the activity of the high molecular weight form was not altered. The binding of citrate synthase to the glyoxysomal membrane could be rather specifically influenced by Mg2+. While KC1 at the same ionic strength solubilized citrate synthase and malate synthase to the same extent, with MgC12, citrate synthase was preferentially washed off the membrane (Table V). (c) Malate dehydrogenase. Upon solubilization from the glyoxysomal membrane, 50% of the malate dehydrogenase activity was lost, although other procedures did not affect the activity of the enzyme to a similar extent. When we used a 1.0

I-

243

MEMBRANES NaCl 1.0

0.5

b

NaCl

20

40 fraction

no.

FIG. 6. CM-Sephadex column chromatography of malate synthase: (a) purified 18.6 S malate synthase (48 pmol x min-I); (b) fraction of solubilized membrane proteins (70 pmol x min-9. Glyoxylate and MgCl* were present in a, but absent in b. Fraction volume, 1.5 ml; protein (O----O), mg x fraction+; 1 relative unit (H-M), 7 pm01 X min-’ X fraction-‘.

0.5

20

30

40 % su
FIG. 5. Analytical separation of solubilized membrane proteins and marker enzymes by zonal centrifugation: (a) A mixture of solubilized proteins was layered onto a sucrose gradient (15-4556, w/w) containing 2 mM glyoxylate and 5 mM MgCl,. (b) Proteins were separated on a sucrose gradient made in a 50 mM Tris-HCl buffer. O----O, fumarase; M-B, malate synthase; *-*, catalase.

zones from the sedimentation velocity centrifugation which were rich in catalase and malate dehydrogenase, the dehydrogenase could be purified by chromatography on CM-Sephadex (Fig. 10). The total purification procedure is outlined in Table VI. Malate dehydrogenase showed virtually the same elution pattern, by zonal centrifugation, as the glyoxysomal catalase. The latter enzyme, in turn, was not distinguishable from the peroxisomal enzyme (23) as far as molecular weight (Fig. 2) and subunit molecular weight (Fig. 1) were concerned. Accordingly, an oligomer molecular weight of 225,000 was estimated from these data. The homogeneous protein exhibits a subunit molecular weight of 34,000 and occurs, hence, in our preparations predominantly as a hexamer. Figure 11 shows the SDS-gel electrophoretie characteristics of the purified en-

244

KGLLER

AND

KINDL

zymes, malate synthase, citrate synthase, and malate dehydrogenase.

brane, were purified to homogeneity. Besides structural proteins, e.g., the protein with a subunit molecular weight of 63,000 DISCUSSION (trace 6 in Fig. 1) described earlier (91, and Three enzymes of the glyoxylate cycle, probably two proteins which exhibited a all attached to the glyoxysomal mem- molecular weight of about 200,000 and subunits of 22,000 and 18,000 (Fig. 11, these l ..; three enzymes represent the quantita. tively dominant protein components of the 1.0. ..j. 4 -aPH membrane. In accordance with findings ~j~\ *.z with glyoxysomes from castor bean endo! ‘6 i \..:“” : sperm (5, 6) or cotyledons (24), the enzymes were preferentially solubilized with : ; .i salt, while nonionic detergents at low concentrations were not effective. With this, -‘!’ .\\ I ‘\5 4 .-.-.-,-.‘-we use the operational distinction between 20 40 60 10 salt-solubilized peripheral enzymes and ‘rOrtionno. integral proteins. The three MgCl,-solubiFIG. 7. Isoelectric focusing of malate synthase. A lized enzymes, the purification of which we portion of solubilized membrane enzymes was apreport here, may, therefore, be considered plied to the column. Fractions, 1.0 ml; pH (0); 1 to be peripheral enzymes organized at the relative unit malate synthase (W-m), 300 nmol x inner surface of glyoxysomes by ionic min-’ X fraction-‘. forces (5, 6). Alternatively, we can interpret the results in the light of findings (25) that malate synthase was electron micro0.0s scopically detected in the granular matrix II ; of glyoxysomes. From this point of view, ?. , one could assume that malate synthase, malate dehydrogenase, citrate synthase, i and lipids constitute the matrix granules. DO1 From the total activities of a membrane fraction, the molecular weights, and the specific activities of homogeneous enzymes, one can calculate (Table VII) that FIG. 8. Purification of citrate synthase on hythe molar ratio of the three enzymes on the droxylapatite. Fraction volume, 1.5 ml; protein (O), mg x fraction-‘; A-A, citrate synthase (1 relamembrane is 1.30 molecules of malate syntive unit = 20 pm01 X min-’ x fraction-‘). thase (octamer), 1.00 molecule of citrate .:

as-

n ..*

.

.

t

-.

\

I

_

TABLE PURIFICATION SkP Crude extract Crude mitochondrial fraction Glyoxysomes Solubilization Zonal centrifugation DEAE-Sephadex umn Hydroxylapatite umn

Y”;; m

Total activity= m

Protein (mg/ml)

1440 110

3269 1545

17.0 15.0

0.13 0.96

Yield

(%)

yFm&on n

100 47

1 7

133

11 6.4 4.2 4.1

8 87 149 478

94

0.13

107.90

2.9

830

col-

11.9

col-

6.7 of acetyl-CoA,

Specific activity (U/mg)

1.04 11.24 19.30 62.10

350 209 138

costs

SYNTHASE

1.4 3.1 1.1 0.18

240 6 6.5

a Due to the high

IV

OF CITRATE

the standard

assay

was run

at half

saturation

(60 PM).

ENZYMES

OF GLYOXYSOMAL

CoA into the glyoxylate cycle and recycling coenzyme A to the fatty acid-activating system which is membrane bound, too. This concept may apply to glyoxysomes in general, since malate synthase and citrate synthase are considered to be membrane bound in glyoxysomes of plants (5, 6, 24). Microbodies from maize scutellum seem to be a notable exception: Their malate synthase is already solubilized in the presence of 10 mM Hepes buffer and is considered to be rather a matrix enzyme (26). The same enzyme was reported to exist only as protein with a molecular weight of 500,000 (27). The enzyme from castor bean endosperm similarly was found to exhibit a high molecular weight on zonal centrifugation (28). On the contrary, the glyoxysoma1 enzyme from cucumber cotyledons is mainly (Fig. 5b) or exclusively (Fig. 4) found as a monomer with a molecular weight of about 70,000. This discrepancy between the value of the minimal molecular weight (63,000) determined by SDS-gel electrophoresis (Fig.

synthase (tetramer), and 0.61 molecule of malate dehydrogenase (hexamer). If one assumes that malate dehydrogenase is a tetramer under in uiuo conditions, the above ratios come close to 1:l:l. On the basis of these data, it is tempting to speculate (cf. 6) that the three enzymes act as a functional unit responsible for coenzyme A-dependent processes: channeling acetyl-

FIG. 9. CM-Sephadex column chromatography of citrate synthase. Fraction volume, 1.5 ml; protein (GO), mg X fraction-‘; (A-A), citrate synthase (1 relative unit = 3 pm01 X min-’ X fraction-‘). TABLE PREFERENTIAL 5mrd

10

IUM

Citrate synthase

1.2

2.6

M&L 17.2

Malate synthase

0.5

0.4

0.6

%lM

V

SYNTHASE BY MeZ+” Percentage aolubilizstion upon treatment with

SOLUBILIZATION

EIIZpe

M&L

245

MEMBRANES

a Catalytic activities of the solubilized untreated glyoxysomal membrane.

OF CITRATE 20 InM

4.7

MdX 39.8

0.7

4.2

3kF,M

5.8

30 InM M&L 71.6

5.3

28.1

%r

15.9

40 nlM M&L 84.8

26.1

38.6

“%lM

‘“%Y 26.5

enzymes are given as percentage of the total activities

NaCI

(M)

0

fraction

no.

FIG. 10. Chromatographic purification of malate dehydrogenase on a CM-Sephadex column. Fraction volume, 1.5 ml; protein (O-O), mg x fraction-‘; O-O, malate dehydrogenase (1 relative unit = 300 wmol X min-’ X fraction-r).

31.6 of the

KGLLER

246

AND KINDL

TABLE PURIFICATION

Step Crude extract Crude mitochondrial fraction Glyoxysomes Solubilization Zonal centrifugation CM-Sephadex column

V~luI~ m 1,440 110 240 6 6.5 3.9

OF

Total~activity

VI

MALATE

DEHYDROGENASE

273,620 68,420

Protein (mg/ml) 17.0 15.0

15,744 3,473 2,766 1,209

1.4 3.1 0.7 0.09

Specific activity (U/mg) 11.2 41.5 47.9 186.7 607.8 3,444

Yield (%o) Fu,rifc&on n 100 25 5.8 1.3 1.0 0.5

1 3.7 4.3 16.7 54.3 307

not correlate these two species with different forms of aggregation. The loss of malate synthase activity upon exposure to various separation procedures may be interpreted as not being entirely due to the inactivation but may reflect an alteration in the stage of aggregation and configuration which entails a decrease in enzymatic activity. The specific activity of 25 pm01 X mine1 X (mg of protein)-’ reported here for the purified enzyme agrees closely with the values of Schmid et al. (30) found with homogeneous preparations of yeast malate synthase characterized by a molecular weight of 170,000. Malate synthases with signiflcantly lower specific activities have already been prepared from plants (28, 31). The control on the interconversion of the FIG. 11. SDS-gel electrophoresis of purified enzymes. 1, malate synthase (100 pg); 2, malate syn- two active forms of citrate synthase, the thase (10 pg); 3, malate dehydrogenase (100 pg); 4, dimer (molecular weight of lOO,OOO), and malate dehydrogenase (mitochondrial, pig heart); 5, the tetramer (molecular weight of citrate synthase (pig heart); 6, citrate synthase (100 200,000), is not easy to explain. It seems ~cg); M, marker proteins (see legend to Fig. 1). that high concentrations of protein cause an alteration in favor of the tetramer, 9) and the value estimated previously by while more diluted citrate synthase prepacomposed of the diBrown et al. (29) may be interpreted as rations are primarily being due to differences in calibrating the mer. With respect to molecular weight and electrophoretic procedure. Supplementation of the medium with subunit molecular weight, the glyoxysoglyoxylate and Mg2+ could shift the equi- mal citrate synthase shows a close resemlibrium to the oligomer form on zonal cen- blance to the enzyme from eucaryotes, e.g., pig heart (321, which seemed to be trifugation (Fig. 2) as well as on molecular distinguishable from the Escherichia coli sieving chromatography. The oligomer enzyme (33). As far as the molecular species seems to be the alkaline malate weight is concerned, the data determined synthase, as the high molecular weight for the cucumber enzymes are difficult to 18.6 S form gives, upon CM-Sephadex chromatography, only one activity peak reconcile with the 65,000 reported for the molecular weight of partially purified attributable to the cationically charged protein. Servettaz et ~2. (27) also observed glyoxysomal citrate synthase from maize two bands on disc electrophoresis, but did scutellum (34). The specific activity of the

ENZYMES

OF

GLYOXYSOMAL TABLE

CORRELATION

OF MOLAR

RATIOS

VII BETWEEN

Calculation (1) Activities in 1 mg of a purified glyoxysomal (from Table I) (2) Specific activity of the homogeneous protein (3) Pure protein in the fraction (calculated from (4) 1 nmol of enzyme (oligomere) (5) Amount of enzyme in 1 mg of the glyoxysomal (calculated from 3 and 4)

247

MEMBRANES

THE THREE

ENZYMES Ma1Et2yn-

Malate dehydrogenase

1.09 u

1.75 u

48U

110 10.0 100 100

25 70.0 540 130

Cit;;siynfraction

1 and 2) fraction

homogeneous glyoxysomal enzyme as reported here [llO pm01 x min-’ x (mg of protein)-7 is higher than that of the hitherto highest purified plant enzyme (351, the mitochondrial citrate synthase from Phaseolus vulgaris 151 pm01 x mine1 x (mg of prot.&P]. Malate dehydrogenase from cucumber glyoxysomes shows properties not directly comparable with other malate dehydrogenases. It behaves as a cation at pH 7.0 and was prepared as a hexamer (molecular weight of 225,000), while the enzyme from spinach leave peroxisomes (7) has a molecular weight of 70,000 and an isoelectric point of pH 5.65. Most likely the dehydrogenase described here corresponds to the one investigated by Walk and Hock (36). All malate dehydrogenases so far known seem to be characterized by a subunit molecular weight of about 35,000. The investigations of the three membrane enzymes enable us to identify some of the prominent peptide bands upon SDSgel electrophoretic analysis of glyoxysomes or glyoxysomal subfractions (cf. Fig. 1). Together with subunit molecular weights of catalase (54,000) (23), isocitrate lyase (SS,OOO>,crotonase (75,000), and thiolase (45,000) (Frevert and Kindl, unpublished work), these results make possible a quick and reliable view of the protein components of a cell compartment. REFERENCES 1. LUDWIG, B., AND KINDL, H. (1976) Hoppe Seyler’s Z. Physiol. Chem. 357, 393-399. 2. FREDERICK, S. E., GRUBER, P. J., AND NEWCOMB, E. H. (1975) Protoplasma 84, l-29. 3. TOLBERT, N. E. (1971)Annu. Rev. Plant Physiol. 22, 45-74. 4. VIGIL, E. L. (1973) Sub-Cell. B&hem. 2, 237285.

Ulmg /.Lg Pg pm01

U/mg pg /-G pm01

3500 13.7 225 61

U/mg pg pg pmol

5. HUANG, A. H. C., AND BEEVERS, H. (1973) J. Cell Biol. 58, 379-389. 6. BIEGLMAYER, C., GRAF, J., AND Rum, H. (1973) Eur. J. B&hem. 37, 553-562. 7. ZSCH~CHE, W. C., AND TING, I. P. (1973) Arch. B&hem. Biophys. 159, 767-776. 8. HOCK, B. (1973) Planta 110, 329-344. 9. LUDWIG, B., AND KINDL, H. (1976) Hoppe Seyler’s Z. Physiol. Chem. 357, 177-186. 10. VESTERBERG, 0. (1971) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 22, pp. 389-412, Academic Press, New York. 11. WEBER, K., F'RINGLE, J. R., AND OSBORN, M. (1973) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 26, pp. 327, Academic Press, New York. 12. STECIEMANN, H. (1972)Z. Anal. Chem. 261,388391. 13. OCHOA, S. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 735-739, Academic Press, New York. 14. SCHNARRENBERGER, C., OESER, A., AND TOLBERT, N. E. (1971) Plant Physiol. 48, 566-674. 15. LOCK, H. (1962) in Methoden der enzymat. Analyse (Bergmeyer, H.-U., ed.), pp. 885-894, Verlag Chemie, WeinheimlBergstr. 16. HOCK, B., AND BEEVERS, H. (1966) Z. PfZunzenphysiol. 55, 405-414. 17. SRERE, P. A., BRAZIL, H., AND GONEN, L. (1963) Actu Chem. Scund. 17, 129-134. 18. LORD, J. M., KAGAWA, T., MOORE, T. S., AND BEEVERS, H. (1973) J. CeZZBioZ. 57,659-667. 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL., R. J. (1951) J. Biol. Chem. 193, 265-275. 20. GERHARDT, B., AND BEEVERS, H. (1968) Anal. B&hem. 24, 337-352. 21. BIEGLMAYER, C., NAHLER, G., AND Rurs, H. (1974) Hoppe Seyler’s Z. Physiol. Chem. 355, 1121-1128. 22. MARTIN, R. G., AND AMES, B. N. (1961) J. Biol. Chem. 236, 1372-1379. 23. SCHIEFER, S., TEIFEL, W., AND KINDL, H. (1976) Hoppe Seyler’s Z. Physiol. Chem. 357,163-175.

248

KOLLER

24. HUANG, A. H. C. (1975) Plant Physiol. 55, 870876. 25. TRELEA~E, R. N., BECKER, W. M., AND BURKE, J. J. (1974) J. Cell Biol. 60, 483-495. 26. LONGO, G. P., BERNASCONI, E., AND LONGO, C. P. (1975) Plant Physiol. 55, 1115-1119. 27. SERVETTAZ, O., FILIPPINI, M., AND LONGO, C. P. (1973) Plant Sci. Z&t. 1, 71-80. 28. BREIDENBACH, R. W. (1969) Ann. N. Y. Acad. Sci. 168, 342-347. 29. BROWN, R. H., LORD, J. M., AND MERRETT, M. J. (1974) Biochem. J. 144, 559-566. 30. SCHMID, G., DURCHSCHLAG, H., BIEDERMANN, G., EGGERER, H., AND JAENICKE, R. (1974)

AND

KINDL

Biochem. Biophys. Res. Commun. 58,419-426. 31. YAMAMOTO, Y., AND BEEVERS, H. (1961) Biochim. Biophys. Acta 48, 20-25. 32. WV, J. Y., ANDYANG, J. T. (1970)5. Biol. Chem. 245, 212-218. 33. TONG, E. K., AND DUCKWORTH, H. W. (1975) Biochemistry 14, 235-241. 34. BARBARESCHI, D., LONGO, G. P., SERVETTAZ, O., ZULIAN, T., AND LONGO, C. P. (1974) Plant Physiol. 53, 802-807. 35. GREENBLATT, G. A., AND SARKISSIAN, I. V. (1973) Phytochemistry 12, 1249-1254. 36. WALK, R.-A., AND HOCK, B. (1976) Eur. J. Biothem. 71, 25-32.