Purification and properties of NADP+-dependent isocitrate dehydrogenase from the corpus luteum

219

Biochimica et Biophysica Acta, 1034 (1990) 219-227 Elsevier BBAGEN 23297

Purification and properties of NADP +-dependent isocitrate dehydrogenase from the corpus luteum Gary Thomas Jennings, John Willoughby Sadleir and Patricia Margaret Stevenson Department of Biochemistry, The University of Western Australia, Nedlands (Australia) (Received 21 November 1989)

Key words: NADP ÷ dependent isocitrate dehydrogenase; Corpus luteum; Protein purification; Protein blotting; Western blotting; (ovary)

Cytoplasmic NADP +-dependent isocitrate dehydrogenase (isocitrate:NADP + oxidoreductase (decarboxylating), EC 1.1.1.42) was purified 290-fold from the 15000 × g supernatant fraction of porcine corpora lute& The major purification step was by anion-exchange chromatography with an FPLC mono P column. Enzyme lability was overcome by including Mg 2+, DL-isocitrate, dithiothreitol and glycerol in the elution buffers. The molecular weight of the denatured enzyme was found to be 48 000 by SDS-polyacrylamide gel electrophoresis. The Stokes' radius was estimated to be 3.7 nm by gel filtration and the isoelectric point was 4.8 as determined by chromatofocusing. The purified enzyme had a specific activity of 57.8 units/mg and a broad optimal pH for activity from 7.5 to 9.0. The K m for the substrates DL-isocitrate and NADP + were 13 and 12 pM, respectively. Polyclonal antibodies were raised against the purified enzyme. Protein (Western) blotting showed an immunological similarity between the cytoplasmic enzyme of the ovary, liver, adrenal gland and heart. A difference was demonstrated between the ovarian enzyme and the heart mitochondrial enzyme. The substrate turnover number and M r of the ovarian enzyme were similar to those found for the enzyme from the liver and adrenal gland.

Introduction

The activity of cytoplasmic NADP+-dependent isocitrate dehydrogenase (isocitrate : NADP ÷ oxidoreductase (decarboxylating), EC 1.1.1.42) increases markedly during the hormone-induced development of the ovary. This increase in activity coincides with the development of the corpus luteum and the associated increase in steroid hormone synthesis [1,2]. The enzyme is a useful marker of ovarian differentiation. Isocitrate dehydrogenase is thought to provide NADPH for cholesterol and fatty acid synthesis and for fatty acid chain elongation and desaturation reactions in the cytoplasm [1,2]. Isocitrate dehydrogenase has been purified from many mammalian tissue sources including the heart [3-5] liver [6,7], adrenal [8] and mammary gland [9]. The enzyme is predominantly mitochondrial in the heart and cytoplasmic in the other tissues including the ovary.

Abbreviations: SDS, sodium dodecyl sulphate; FPLC, fast protein liquid chromatography; Kay, distribution coefficient; Rs, Stokes' radius. Correspondence: P.M. Stevenson, Department of Biochemistry, The University of Western Australia, Nedlands 6009, W.A., Australia.

The mitochondrial and cytoplasmic enzymes appear to be different isoenzymes [10,11]. The ovarian cytoplasmic isocitrate dehydrogenase has only been partially purified to date and its properties investigated with crude enzyme preparations [2]. The aim of this study was to purify the cytoplasmic NADP+-dependent isocitrate dehydrogenase from the porcine corpus luteum, characterise some of its physical, kinetic and immunological properties and compare it to the enzyme from different tissues.

Materials and Methods

Pepstatin A, phenylmethylsulphonyl fluoride, NADP +, DL-isocitrate, nitro blue tetrazolium and phenazine methosulphate were obtained from Sigma (St. Louis, MO, U.S.A.). Polybuffer 74, Sephadex G-25 and CM-Sephadex C-50 were purchased from Pharmacia (Uppsala, Sweden). Bio-Rad (Australia) supplied the electrophoresis reagents, nitrocellulose membranes and molecular weight standards. The horseradish peroxidase-conjugated sheep anti-rabbit antibody was from Silenus Laboratories (Melbourne, Australia). All other reagents were of analytical grade.

0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

220

Enzyme assays During the purification, the activity of isocitrate dehydrogenase was monitored by assay at 25 ° C in a 3 ml reaction mixture composed of 0.1 M Tris-HC1 (pH 8.4), 3 mM MgCI2, 0.5 mM N A D P + and enzyme. The reaction was initiated by adding DL-isocitrate to the test cuvette to give a final concentration of 1.5 mM. The rate of N A D P H production was recorded at 340 nm on a Gilford Response spectrophotometer. Enzyme assays for kinetic and immunological studies were performed as above at 3 0 ° C in 0.1 M Tris-HC1 (pH 8.0) on a Varian DMS 80 split beam recording spectrophotometer. Kinetic studies were performed with varying cofactot, substrate and ion concentrations as indicated in the results. For determination of the pH optimum, buffers were maintained at an ionic strength of 0.1 by adding NaC1 [12]. Desalting samples The purified enzyme used for kinetic studies was desalted to remove small ions by microcentrifuge desalting with a Sephadex G-25 column [13]. Protein estimation Protein concentrations were determined by the Ponceau S method [14]. Protein purification Isocitrate dehydrogenase was purified from porcine corpora lutea as described by Stevenson et al. [2] with the following modifications. Corpora lutea were homogenised in 5 vol. of 0.25 M sucrose, 5-mM EDTA, 1 # M Pepstatin A and 1 mM Hepes-KOH (pH 7.2). After homogenisation, phenylmethylsulphonyl fluoride was added in 200 #1 of acetone to give a final concentration of 1 mM. The homogenate was centrifuged at 15 000 x g for 20 min. Ammonium sulphate precipitation. Isocitrate dehydrogenase was precipitated from the above supernatant using a saturated solution of (NH4)2SO 4 adjusted to p H 7.2 with Na2CO 3. The enzyme activity that precipitated between 45 and 70% saturation was collected by centrifuging at 15 000 x g for 30 min. Heat treatment. The pellet was dissolved in 0.1 M triethanolamine-KOH (pH 7.4), 1 mM DL-isocitrate and 10 mM MgC12 and incubated at 6 0 ° C for 30 min. The solution was cooled on ice and the heat denatured protein was removed by centrifuging at 15 000 x g for 10 min. Batchwise cation exchange. The supernatant was dialysed for 18 h against 10 mM trisodium citrate-HC1 (pH 6.2) and 5% w / v glycerol with two changes of the buffer. The enzyme solution was then treated in a batchwise step with CM-Sephadex C-50 equilibrated with the same buffer. The mixture was stirred on ice for 15 min, the gel removed by filtration and the super-

natant taken to 70% saturation with ( N H 4 ) 2 S O 4. The precipitate was collected by centrifuging at 15000 X g for 30 min. FPLC anion exchange. The precipitate from the above step was dissolved in a minimal volume of buffer A (20 mM triethanolamine-KOH (pH 7.6), 2 mM dithiothreitol, 1 m M MgC12, 1 mM DL-isocitrate, and 5% w / v glycerol) and dialysed for 18 h against the same buffer. FPLC was carried out using equipment purchased from Pharmacia and under the recommended conditions of separation [15]. Between 1.0 and 2.0 mg of protein was applied to a mono P (HR 5/20) anion exchange column pre-equilibrated with buffer A. Protein was eluted with a continuous salt gradient from 0 to 350 mM KC1 in buffer A at a flow rate of 0.5 ml/min. The column eluant was monitored for protein at 280 nm and fractions were assayed for isocitrate dehydrogenase activity. The optimal p H for the separation of components by anion exchange was determined by the electrophoretic titration method of Radola [16].

Gel electrophoresis Discontinuous SDS-polyacrylamide gel electrophoresis was carried out by the procedure of Laemmli and Favre [17]. Slab gels were prepared (16 × 14 × 0.15 cm) with a 3 cm, 4% polyacrylamide stacking gel and a 10% polyacrylamide separating gel. Gels were run at 25 mA. Nondenaturing polyacrylamide gel electrophoresis was performed using a Bio-Rad mini protean slab cell apparatus. A 7.5% polyacrylamide separating gel (pH 8.8) was used with a 4.0% polyacrylamide stacking gel (pH 6.8). The gel was run at 200 V for 45 rain at 4°C. After electrophoresis, isocitrate dehydrogenase activity was detected by a modification of the method of Reeves et al. [18]. The gel was incubated for 1 h at room temperature in 0.1 M Tris-HC1 (pH 8.4), 0.5 mM N A D P ÷, 3 mM MgC12, 1.5 mM DL-isocitrate, 65 # M phenazine methosulphate and 12 /,M nitro blue tetrazolium. Gels were stained for protein with Coomassie brilliant blue R-250. Determination of the native molecular weight The molecular weight of the native enzyme was estimated using a FPLC superose 12 (HR 10/30) gel filtration column pre-equilibrated with 100 mM triethanolamine-KOH (pH 7.4), 2 mM dithiothreitol, 1 mM MgC1 z, 1 m M DL-isocitrate and 5% w / v glycerol. Isocitrate dehydrogenase isolated by anion exchange was dialysed against the same buffer and 25/,g applied to the column. The column eluant was monitored for protein at 280 nm and fractions were assayed for isocitrate dehydrogenase activity. The molecular weight standards thyroglobulin, y-globulin, bovine serum albumin, ovalbumin, myoglobin and vitamin B-12 were used to calibrate the column.

221

Determination of the tsoelectric point Purified isocitrate dehydrogenase was dialysed against 25 mM Bis-Tris-HC1 (pH 6.0), 2 m M dithiothreitol, 1 mM MgC12, 1 mM DL-isocitrate and 5% w / v glycerol and applied to the FPLC mono P ( H R 5/20) column. The column was pre-equilibrated with the above buffer. Approx. 100/,g of isocitrate dehydrogenase was applied to the column and eluted with a p H gradient from 6 to 4, generated by applying 10% v / v polybuffer 74-HC1 (pH 4.0), 2 mM dithiothreitol, 1 mM MgC12, 1 mM DL-isocitrate and 5% w / v glycerol to the column. The pH of the eluant was measured by a flow-through p H electrode, protein was monitored at 280 nm and fractions were assayed for isocitrate dehydrogenase activity.

Comparison of NADP+-dependent isocitrate dehydrogenase in oarious tissues of the superovulated rat Preparation of tissue fractions. Superovulated ovaries, adrenal glands, livers and hearts were obtained from gonadotropin induced female Wistar rats [1]. The tissues were homogenised in 4 vol. of ice-cold 0.25 M sucrose with a loose fitting teflon glass homogeniser. Homogenates were centrifuged at 800 X g for 10 rain and the pellet was discarded. The supernatant was centrifuged at 15 000 x g for 20 rain to give a fraction representative of the cytosol. Mitochondria were obtained from the heart tissue by resuspending the 15 000 x g pellet in 0.25 M sucrose and centrifuging at 8000 x g for 10 rain. This procedure was repeated twice to wash the mitochondria. Mitochondria were sonicated for three periods of 20 s on ice. Aliquots of the cytoplasmic and heart mitochondrial samples obtained above were stored immediately at - 8 0 ° C for protein blot analysis and the remainder was assayed for isocitrate dehydrogenase activity and protein concentration. Preparation of antisera. NADP+-dependent isocitrate dehydrogenase, purified from the porcine corpus luteum as above was used as antigen to produce antisera. Two rabbits were bled to obtain preimmune sera, then injected subcutaneously at four sites on the back of the neck with the antigen (175 #g) mixed 1 : 2 with Freunds

complete adjuvant. Booster injections of antigen (75 #g) mixed 1 : 2 with Freunds incomplete adjuvant were given 3, 8 and 12 weeks after the primary injection. The titre of the antisera 2 weeks after the final injection was found to be 1 in 7.107 by an ELISA assay [19] using purified isocitrate dehydrogenase as the antigen. Protein blotting procedure. Tissue fractions and the purified samples of isocitrate dehydrogenase were subjected to discontinuous SDS-polyacrylamide gel electrophoresis using an 11% separating gel. The separated proteins were transferred to a nitrocellulose membrane using a Bio-Rad Trans Blot semi dry transfer cell, run at 275 mA for 2 h. The transfer buffer consisted of 48 mM Tris, 39 mM glycine and 20% methanol (pH 9.2). The blocking of non-specific binding sites on the nitrocellulose was carried out as described by Tuckey and co-workers [20]. Protein bands were visualised by incubating the membrane with antisera diluted 1 : 500 and horseradish peroxidase-conjugated sheep anti-rabbit antibody [21]. Protein bands were quantitated by densitometric scanning with a Bio-Rad model 620 video densitometer set in the reflectance mode. The concentration of isocitrate dehydrogenase in the various tissues was determined from standard curves constructed from simultaneous blots of purified isocitrate dehydrogenase from the porcine corpus luteum. Results

Purification Isocitrate dehydrogenase was purified 290-fold from the 15 000 x g supernatant fraction of the porcine corpus luteum (Table I). The recovery was 42% and purified enzyme had a specific activity of 57.8 units/mg. A 90% recovery of enzyme activity was obtained from the heat treatment step when performed in the presence of 1 mM ot-isocitrate and 10 mM MgC12 compared to a 30% recovery in their absence. The greatest loss of activity was during the CM-Sephadex batch treatment. We found that a 10 m M citrate buffer preserved more activity than a 1 m M citrate buffer [2]. Electrophoretic titration of the post CM-Sephadex sample showed max-

TABLE I

Summary of the purification of isocitrate dehydrogenase from the porcine corpora lutea Step 15000 × g supernatant Ammonium sulphate fract. (45-70%) Heat treatment CM-Sephadex C-50 batch treatment FPLC mono P anion-exchange step

Total activity (units)

Total protein (mg)

410

2 070

0.20

1

100

350 330

420 260

0.83 1.3

4 6

86 80

250

130

1.9

10

60

290

42

170

3.0

Specific activity (units/mg)

58

Purification (fold)

Yield (%)

222 The purified enzyme was stable for 12 weeks when stored at - 2 0 ° C in 20 m M triethanolamine-KOH (pH 7.6) with the stabilising agents.

Physical properties Molecular weight of denatured enzyme. The molecular

1

2

3

4

5

6

7

8

Fig. 1. SDS-polyacrylamidegel showing the purification and Mr of NADP +-dependent isocitrate dehydrogenase from the porcine corpus luteum. Lane 1: standards, from top to bottom bovine serum albumin (M, 66000), ovalbumin (Mr 45000), soy bean trypsin inhibitor (Mr 24000) and fl-lactalbumin (Mr 18000). Lane 2, 15000×g supernatant; lane 3, post-ammonium sulphate treatment; lane 4, post-heat treatment; lane 5, post-CM-Sephadex C-50 treatment; lane 6, isocitrate dehydrogenase from non-denaturing polyacrylamidegel. Lanes 7 and 8, post-mono P treatment, 10/*g and 18/tg, respectively.

imum separation of protein bands occurred at p H 7.6. This p H was used for the mono P anion-exchange step, which gave a 29-fold purification. The enzyme was found to be very labile to column chromatographic techniques. Reasonable recoveries and maintenance of enzyme activity could only be obtained by adding stabilising agents (2 m M dithiothreitol, 1 m M MgC12, 1 m M DL-isocitrate and 5% w / v glycerol) to the separation buffers. After FPLC mono P anion-exchange step isocitrate dehydrogenase was apparently homogenous as judged by SDS-polyacrylamide gel electrophoresis (Fig. 1) and non-denaturing gel electrophoresis. There was a single heavily stained band on the SDS-polyacrylamide gel (Fig. 1, lanes 7 and 8) which was positively identified as being isocitrate dehydrogenase as foilows. Purified enzyme was electrophoresed on non-denaturing polyacrylamide and the gel cut into two pieces. One was stained for isocitrate dehydrogenase activity and the position of the enzyme was noted. The section of gel containing isocitrate dehydrogenase was excised and homogenised in deionised water. The protein was separated from the gel by centrifugation and applied to a SDS-polyacrylamide gel (Fig. 1, lane 6).

weight of isocitrate dehydrogenase was determined under denaturing conditions by SDS-polyacrylamide gel electrophoresis (Fig. 1). From a calibration plot of log M r against the relative mobility, using three separate purifications, the M r of the enzyme was found to be 48 000 + 1000 (S.D., n = 3). Gel filtration. The molecular weight of the native enzyme was determined by gel filtration using the FPLC superose 12 column. F r o m a calibration curve of log M r plotted against Kav, the M r of isocitrate dehydrogenase in 100 m M triethanolamine-KOH (pH 7.4) and stabilising agents (2 m M dithiothreitol, 1 m M MgC12, 1 m M DL-isocitrate and 5% w / v glycerol) was estimated to be 72 000. The Stokes' radius was estimated by plotting the Rs of the marker proteins against Kav [22]. A value of 3.7 n m was obtained. The recovery of enzyme activity was 98%. Gel filtration was peformed in the absence of the stabilising agents to examine their effects on the molecular weight of isocitrate dehydrogenase. When Mg 2÷ and DL-isocitrate were absent from the buffer, the M r of isocitrate dehydrogenase was not altered, however the activity and the protein peak were both reduced. The recovery and peak size were further reduced when this experiment was performed in the presence of 5 m M EDTA. When all of the stabilising factors were omitted, only 7% of the activity was recovered and the protein peak reduced in size. Each of the stabilising compounds appeared to contribute to the stabilisation of enzyme activity as sequential removal of these agents resulted in a cumulative loss of activity. However, the loss of activity was not accompanied by a change in the M r of isocitrate dehydrogenase. The Kay of the calibration proteins did not change during these experiments. When gel filtration was performed under denaturing conditions, in the presence of 6 M guanidine hydrochloride, isocitrate dehydrogenase eluted as a single peak after the marker protein bovine serum albumin, i.e., the M r was apparently less than the 72 000 found above. Changing the ionic strength of the elution buffer from a 100 m M triethanolamine-KOH (pH 7.4) to a 20 m M triethanolamine buffer in the presence of all of the stabilising agents, also caused a reduction in the apparent M r of isocitrate dehydrogenase. The enzyme was similarly affected when the experiment was performed in the presence of 100 m M diethanolamine-HC1 (pH 9.0) with stabilising agents. There was 98% recovery of activity for both these experiments. Isoelectric point. Fig. 2 shows an elution profile for purified isocitrate dehydrogenase subjected to analytical

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Kinetic properties Determination of pH optimum. Purified isocitrate dehydrogenase was active over a p H range of 6.0-10.0 (Fig. 3). There was a b r o a d p H o p t i m u m for the reaction from 7.5 to 9.0. The ionic strength of the different buffers was maintained at 0.1 over the p H range tested. The p H profile for the enzyme was similar for Tris and glycylglycine buffers in the p H range 7.0-9.0. The p H profile of the enzyme does not appear to be affected b y 70 60 O3

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the charge on the conjugate base of the Tris and glycylglycine, which are - 1 and 0, respectively. Tris buffer at a p H of 8.0 was chosen for further kinetic work. K,, for substrate and cofactor. The K m of isocitrate dehydrogenase for the substrate oL-isocitrate was 13 /~M and for the cofactor N A D P ÷ was 12 /~M. These values were determined f r o m double-reciprocal plots. Isocitrate dehydrogenase isolated from other sources [9,23] is specific for the D-I-isocitrate isomer. If this were true for isocitrate dehydrogenase isolated from the porcine corpus luteum, and assuming the L-enantiomer is not inhibitory, the K m for D + isocitrate would be 6.5 /~M. The effect of divalent cations on enzyme activity. The divalent cations M g 2+ and M n 2÷ were found to activate isocitrate dehydrogenase whereas Ca 2+ inhibited activity (Fig. 4a). High concentrations of Mg 2+ and M n 2÷ also inhibited activity. M n 2+ was able to activate the enzyme at a concentration 10-fold lower than that for Mg 2+. The m a x i m u m enzyme activity in the presence of M n 2+ was 34% greater than that for M g 2+ at the same concentration. The enzyme showed 10% of m a x i m u m activity in the absence of added divalent cations and no activity in the presence of 5 m M E D T A . The low activity observed in the absence of added cations probably reflects trace a m o u n t s of divalent metal ions in the buffer c o m p o n e n t s or b o u n d to the enzyme. Stevenson and co-workers [2], using a crude enzyme preparation, reported an enhancement of isocitrate dehydrogenase activity when Ca 2+ was included in the assay in a ratio 1 • 100 with Mg 2+. This effect of Ca 2+ and M g 2÷, acting in concert, was re-investigated using purified enzyme. W h e n the Ca 2+ concentration was increased in the presence of a constant a m o u n t of Mg 2+ the enzyme activity decreased (Fig. 4b). Lowering the Mg 2+ concentration in the assay also lowered the concentration of Ca 2+ necessary for inhibition. The greatest change in the activity of the enzyme occurred when the Mg2+//Ca2+ ratio changed from 10 : 1 to 1 : 1, i.e., a 10-fold increase in the Ca 2+ concentration resulted in a 50% inhibition of enzyme activity. The enhancement of activity reported previously [2] was not observed for experiments with the purified enzyme. The activation found with the crude enzyme preparation m a y have been caused by a Ca 2+ mediated secondary factor which is absent f r o m the purified enzyme. Lag phase. U n d e r certain conditions the purified enzyme exhibited a lag phase at the beginning of the reaction which lasted up to 3 rain. This lag was apparent when the reaction was started by the addition of DL-isocitrate and the protein concentration in the assay was less than or equal to 0 . 1 / ~ g / m l . The concentration of the enzyme in the stock solution did not affect the lag when the reaction was initiated by the addition of enzyme. The lag phase was also observed at low con-

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Fig. 5. Protein blot showing the distribution of NADP + dependent isocitrate dehydrogenase in various tissues of the superovulated rat. Lane 1, purified isocitrate dehydrogenase from the porcine corpus luteum (0.12 /~g); lane 2, 15000xg supernatant fraction from the superovulated rat ovary (5.4 ~g protein); lane 3, liver 15000×g supernatant fraction (18 #g). Lane 4, adrenal gland 15000x g supernatant fraction (9.7/~g); lane 5, heart 15000 x g supernatant fraction (4.5 ~tg); lane 6, heart mitochondrial fraction (2.7/~g).

Fig. 4. The effect of (a) cation concentration and (b) altering the Ca2+ concentration in the presence of Mg2+ on the activity of isocitrate dehydrogenase. The activity of purified isocitrate dehydrogenase was measured in a 3 ml reaction mixture composed of 0.1 M Tris-HC1 (pH 8.0) and 0.5 mM NADP ÷ with (a) MnC12 (A), MgC12 (O) and CaC12 (D), added in a volume of 20 btl or (b) CaC12 and either 1.0 mM MgC12 (A) or 0.1 mM MgC12 (e). The assay was initiated by adding DL-isocitrateto the test cuvette to give a final concentration of 1.5 mM. The enzyme sample was microcentrifuge desalted against 20 mM triethanolamine-KOH (pH 7.6), 2 mM dithiothreitol and 5% w/v glycerol. Each data point represents the mean of three separate experiments.

Comparison of NADP+-dependent isocitrate dehydrogenase from various tissues by protein blotting Specificity of antisera. The antisera used in the pro-

centrations of the activating ion e.g., when the Mg 2+ c o n c e n t r a t i o n was less t h a n 1.0 m M a n d when M n 2÷ was less t h a n 10 /tM. The lag observed with 0.1 m M Mg 2÷ could be reduced b y p r e i n c u b a t i n g the e n z y m e with Mg 2+ a n d DL-isocitrate a n d starting the reaction with N A D P +. The lag phase was n o t observed at low DL-isocitrate or low N A D P ÷ c o n c e n t r a t i o n s i n the presence of 1 m M Mg 2+. The a d d i t i o n of Ca 2÷ to the assay i n d u c e d a n d increased the lag period. W h e n Ca 2÷ was included in the assay at a c o n c e n t r a t i o n greater t h a n or equal to 0.1 m M i n the presence of 1 m M Mg 2+ a lag was observed. The lag phase, that was n o r m a l l y observed when the Mg 2÷ c o n c e n t r a t i o n was 0.1 mM, was greatly increased when Ca 2+ was included i n the assay at a c o n c e n t r a t i o n of 1 ~tM a n d above.

tein b l o t t i n g procedure was shown to be specific for isocitrate dehydrogenase. The antisera detected a single b a n d in the tissue fractions which corresponded to the b a n d observed with the purified p r o t e i n (Fig. 5). Preimm u n e sera did n o t detect a b a n d with the purified or tissue samples. W h e n the antisera was i n c u b a t e d with purified isocitrate dehydrogenase before a d d i t i o n to the assay, the i n t e n s i t y of the b a n d s detected was markedly reduced (results n o t shown). Tissue distribution. A positive b a n d was detected for isocitrate d e h y d r o g e n a s e in the cytoplasmic fractions of the s u p e r o v u l a t e d rat ovary, liver a n d adrenal gland (Fig. 5). The b a n d s h a d the same M r (48000) as the purified e n z y m e from the porcine corpus luteum. F a i n t b a n d s were also observed with the heart cytoplasmic a n d m i t o c h o n d r i a l fraction. If the m i t o c h o n d r i a were not washed the i n t e n s i t y of the b a n d observed for this fraction increased suggesting the b a n d m a y be due to c o n t a m i n a t i o n by the cytoplasmic enzyme.

225 T A B L E II

Comparison of NADP +-dependent isocitrate dehydrogenase in various tissues of the superovulated rat Results are expressed as the mean + S.D. from three separate experiments. Tissue

Fraction

Specific activity (units/mg)

Enzyme concentration (nmol/mg)

Substrate turnover number

(s -1) Ovary Liver Adrenal Heart Heart

cytosol cytosol cytosol cytosol mitochondria

0.80-t-0.18 0.19 + 0.00 0.20 + 0.02 0.50 + 0.16 1.0 +0.1

1.1 +0.3 0.32 + 0.03 0.25 + 0.03 0.12 + 0.03 <0.01

12+1 10 + 1 13 + 3 71 + 9 >1700

Enzyme concentration and specific activity. The standard curves used to estimate the concentration of isocitrate dehydrogenase by protein blotting were linear up to 5 pmol of purified protein with a correlation coefficient of 0.99. The concentration of isocitrate dehydrogenase was highest in the cytoplasmic fraction of the superovulated rat ovary, while specific activity was greatest for the heart mitochondrial fraction (Table II). Substrate turnover number. Substrate turnover numbers were calculated using the M r value of 48 000 for the enzyme. The assay conditions used for measuring the activity of the enzyme in the different tissues, were close to optimum e.g. p H (see Discussion). The substrate turnover numbers for the enzyme from the ovary, liver and adrenal gland were similar. The high turnover number observed for the heart cytoplasmic fraction may be due to mitochondrial breakage resulting from the vigorous homogenisation required to disrupt this tissue. Since our antisera reacts poorly with the heart mitochondrial enzyme this would result in enzyme activity which is not detected by the blotting procedure, hence giving a high turnover number. Discussion

The modified method of purification of N A D P + dependent isocitrate dehydrogenase from the porcine corpus luteum gave homogeneous enzyme with a level of purification four times that reported previously [2]. The major improvements on the old method, which gave only partial purification, were the inclusion of the FPLC mono P anion-exchange step and the stabilisation of enzyme activity. The lability of the ovarian enzyme to column chromatographic techniques reported previously was minimised by including the stabilizing agents Mg 2+, DL-iSOcitrate, glycerol and dithiothreitol. This was a necessary development for the purification of the enzyme. The lability of isocitrate dehydrogenase to CM-Sephadex C-50 treatment is not unique to the ovarian enzyme;

loss of activity during a similar treatment at low temperatures was reported by McFadane and co-workers [5] for the ox heart mitochondrial enzyme. Instability at low ionic strength and high losses of activity during chromatographic separation procedures have also been observed during the purification of the enzyme from heart, liver and mammary gland [5,6,9,11]. However, the lability of the enzyme to purification procedures has not been reported by all workers [4,7,8]. The M r of 48 000 determined for the denatured protein by SDS-polyacrylamide gel electrophoresis is within the range of values obtained for all the NADP+-depen dent isocitrate dehydrogenases isolated to date. This method of size estimation has given values ranging from 46 000 to 58 000 [5,6,8,9]. The M r of 72000 for the native enzyme from the corpus luteum is similar to the M r values of 75 000 and 76 000 obtained, by gel filtration, for the enzymes from the porcine and bovine liver, respectively [6,7]. This value seems anomalous when the M r determined by SDS-polyacrylamide gel electrophoresis is considered, i.e., it is not indicative of a monomer or a dimer structure. However, gel filtration separates proteins by size and shape and these properties are not necessarily related to the M r. The determination of M r from the Kav can lead to an error of 80% [24]. The elution behaviour of a protein during gel filtration is better related to the Stokes' radius than the M r. The Rs of 3.7 nm for ovarian isocitrate dehydrogenase is close to the Rs values of 4.1, 3.7 and 3.9 obtained for the enzymes from bovine mammary gland [9], human heart [23] and pig heart [25], respectively. Interactions of electrostatic or hydrophobic nature between the protein and the gel can also affect the elution of a protein during chromatography [26]. We showed the Kav of native isocitrate dehydrogenase did change when gel filtration was performed under conditions which would alter possible electrostatic interactions (e.g., lowering the ionic strength and increasing the p H of the elution buffers). We suggest, therefore, that these factors can explain the difference in M r between the native and denatured ovarian isocitrate dehydrogenase and may contribute to some of the discrepancies of the native M r reported in the literature [7,23,25]. There is disagreement in the literature concerning the quaternary structure of NADP+-dependent isocitrate dehydrogenase. Structures proposed for the enzyme isolated from the heart have included a monomer (a single polypeptide chain) or multiples of this structural unit to form dimer or tetramer structures [3,5,25,27]. The native enzymes isolated from the liver and adrenal gland have been reported to be dimers [7,8]. Several groups have reported that dimerisation of NADP+-dependent isocitrate dehydrogenase is dependent upon the presence of Mg 2+ and DL-isocitrate [7,27,28]. Kelly and Plaut [25] reported that the purified porcine heart enzyme

226 eluted from a gel filtration column as a dimer in the presence of Mg 2+ and DL-isocitrate, and as a monomer in their absence. We found the elution position of the ovarian enzyme did not alter in the absence of isocitrate and Mg 2+ nor in the presence of EDTA, however the recovery of protein and enzyme activity did. A similar observation was made by McFarlane and co-workers for the enzyme from the ox heart [5]. When gel filtration was performed in the presence of 6 M guanidine hydrochloride, to disrupt putative dimerisation, the M r of isocitrate dehydrogenase was apparently reduced, but the change was too small to represent conclusive proof of subunit structure. A lag period in attaining a steady state reaction velocity during the assay of isocitrate dehydrogenase, similar to the one reported here, has also been reported for the enzyme from the porcine heart [25,27] and bovine liver [7]. The lag phase for the heart and liver enzymes has been attributed to the time necessary for the transition of the inactive monomer to the active dimer form of the enzyme [7,25,27,28]. The conditions which influence the lag of the enzymes from these tissues are similar to those which bring about the lag during the assay of the ovarian enzyme, i.e., low activating ion concentrations and high dilution of the enzyme. Both the discrepency in the apparent M r between the native and denatured enzyme, and the presence of a lag phase are suggestive of dimeric structure. Although it is difficult to compare the kinetic properties of isocitrate dehydrogenase isolated from different tissue sources, due to differing assay conditions and levels of purity, the specific activity of 57.8 units/mg for the purified enzyme from the porcine corpus luteum is one of the highest found to date. Comparable specific activities have been obtained for the purified enzyme from the liver (46.5 units/mg), mammary gland (52.5 units/mg) and adrenal glands (60.3 units/mg) [6-9]. The enzyme isolated from heart tissue generally has a lower specific activity [3,5,25] with the exception of the human heart enzyme (63,6 units/mg) [4]. The K m for substrate and cofactor for the ovarian enzyme did not differ markedly from those reported for the purified mitochondrial and cytoplasmic isoenzymes from other tissues [7,9,11,23]. The pH profile and optimum observed for the ovarian enzyme is similar to those reported for the enzyme from the liver, adrenal and mammary glands [6,8,9,11]. The ovarian enzyme is also similar to that from other tissues in that it requires the divalent cations Mg 2+ and Mn 2+ for activity and is inhibited by Ca 2+. Of interest was the influence the relative concentrations of C a 2 + and Mg 2+ had on the activity of the purified ovarian enzyme. In the presence of 0.1 mM Mg 2+, enzyme activity was significantly inhibited by 0.1 mM Ca 2+ and the lag phase greatly increased by Ca 2+ concentrations down to 1.0 /~M. The intracellular con-

centration of free Ca e+ is 0.1-1.0/~M. Changes in the concentration are highly localised and controlled by intricate buffering systems [29]. With the intracellular concentration of Mg 2+ in the vicinity of 1.0 mM it is reasonable to speculate that an acute in vivo control of ovarian isocitrate dehydrogenase activity by the relative levels of free Ca 2+ and Mg 2+ might operate. Comparisons of N A D P + dependent isocitrate dehydrogenase between tissues is complicated by the various schemes of purification and different methods used to characterise its properties in different laboratories. The physical and kinetic properties of the cytoplasmic enzyme purified from the porcine corpus luteum were similar to those reported in the literature for the cytoplasmic enzymes of the liver and adrenal gland. Many similarites also exist between the ovarian and heart enzyme although the mitochondrial enzyme which predominates in the heart is a different isoenzyme. Therefore, it is difficult to judge whether ovarian isocitrate dehydrogenase is a similar or different isoenzyme to the enzyme isolated from other tissues. Protein blotting enabled us to show a clear distinction between the cytoplasmic enzyme from the ovary and the heart mitochondrial enzyme. An immunological relationship was demonstrated between the cytoplasmic enzyme from the superovulated ovary, liver, adrenal gland and heart. Based on the similarity of the substrate turnover numbers and M r we conclude that the cytoplasmic isocitrate dehydrogenases from the ovary, liver and adrenal gland may be the same isoenzyme. Our evidence also suggests the heart may possess a similar cytoplasmic isoenzyme. The ovarian isocitrate dehydrogenase differs from the other cytoplasmic isocitrate dehydrogenases in two ways. First, the enzyme concentration and specific activity are much higher in the corpus luteum than in the other tissues examined and second, there is a marked induction of enzyme activity. The increase in enzyme activity is induced by the gonadotropins and shows the same developmental pattern as steroidogenesis [1,2]. Our thesis is the enzyme plays an important role in lipid metabolism and the maintenance of the steroidogenic apparatus of ovarian cells.

Acknowledgements We wish to acknowledge grants from A R G C (No. D281/15245), Medical School Fund of Western Australia and the University of Western Australia. We are grateful to Watsonia Abbatoirs, Spearwood W.A. and would like to thank Dr. R.C. Tuckey.

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