Advan. Enzyme Regul., Vol. 32, pp. 267-284, 1992 Printed in Great Britain. All rights reserved
0065-2571/92/$15.00 (~) 1992 Pergamon Press pie
PURIFICATION, CHARACTERIZATION, REGULATION AND MOLECULAR CLONING OF MITOCHONDRIAL PROTEIN KINASES ROBERT A. HARRIS, KIRILL M. POPOV, YOSHIHARU SHIMOMURA, YU Z H A O , J E R Z Y JASKIEWlCZ, NORIKO NANAUMI and MASASHIGE SUZUKI Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202
INTRODUCTION
Of the two protein kinases known to be present in the matrix space of mammalian mitochondria, the enzyme responsible for phosphorylation and inactivation of the pyruvate dehydrogenase complex (EC 1.2.4.1 + EC 2.3.1.12 + EC 1.8.1.4) is the most thoroughly studied (1, 2). Pyruvate dehydrogenase kinase (EC 2.7.1.99) renders the pyruvate dehydrogenase complex inactive by phosphorylation of specific sites on the Eltx subunit of the et-ketoacid dehydrogenase moiety of the complex. Short-term regulation of this kinase by allosteric effectors has been studied in considerable detail and is believed to be of physiological importance. However, much of the current interest in regulation of pyruvate dehydrogenase kinase stems from suggestive evidence for adaptive changes in activity of this enzyme in response to changes in nutritional and hormonal states of animals (3, 4). Although less well studied, the enzyme responsible for the phosphorylation and inactivation of the branched-chain ot-ketoacid dehydrogenase complex (EC 1.2.4.4 + EC no number + EC 1.8.1.4) is also of considerable interest (5-7). The activity of this enzyme, along with the opposing protein phosphatase, is believed responsible for regulation of the activity of the branched-chain a-ketoacid dehydrogenase complex and, therefore, the rate of disposal of the branched-chain amino acids (8, 9). Branched chain ot-ketoacid dehydrogenase kinase (EC 2.7.1.115) phosphorylates specific sites on the Elcx subunit of the branched-chain a-ketoacid dehydrogenase complex. Short-term regulation of this kinase seems to be confined simply to allosteric inhibitory effects of et-ketoisocaproate (transamination product of leucine). However, as with pyruvate dehydrogenase kinase, evidence has been presented for adaptive changes in the activity of this enzyme in response to nutritional and hormonal states of animals (6, 7, 9), but again like pyruvate dehydrogenase kinase, no information is available as to 267
268
R.A. HARRIS, et al.
the actual molecular mechanisms involved. Current efforts of this laboratory are directed toward obtaining the molecular tools necessary for a study of the regulation of gene expression of the mitochondrial protein kinases. We describe here the purification and characterization of these protein kinases from rat tissues, the cloning of cDNAs for branched-chain oL-ketoacid dehydrogenase kinase, and provide suggestive evidence that inhibition of branched-chain ot-ketoacid dehydrogenase kinase may be responsible for the development of myotonia in humans treated with clofibric acid. MATERIALS AND METHODS
Source of materials. Enzymes and biochemicals were obtained from Sigma Chemical Company; ot-chloroisocaproate was a gift from Dr. Ronald Simpson of Sandoz Pharmaceutical Corp., radiolabeled compounds from DuPont-New England Nuclear, molecular biology reagents from Bethesda Research Laboratories, protein sequencing reagents from Applied Biosystems, and chromatography supplies from Pharmacia. Purification of branched chain ot-ketoacid dehydrogenase and pyruvate dehydrogenase complexes from rat tissues. Recent modifications of purification procedures have led to greater retention of the protein kinase activities associated with the complexes (10, 11). In brief detail, our current purification procedure (12) for the rat heart complexes involves the following steps. Frozen rat hearts (200-300 g) are homogenized in a buffer containing Triton X-100, dithiothreitol (DTF) and a battery of protease inhibitors. The homogenate is centrifuged and the pellet homogenized again in the same buffer and centrifuged. The combined supernatants are made 1% in polyethylene glycol (PEG), the precipitate is discarded and the supernatant made 4.5% in PEG. The pellet obtained is suspended in a buffer containing Mg z+ and incubated at 37°C for 50 min, a treatment that activates the complexes by dephosphorylation. The suspension is made 3% in PEG, and the precipitate collected by centrifugation. The pellet is resuspended in 1 vol of Buffer A (50 mM potassium phosphate, pH 7.5, 0.1 mM EDTA, 3 mM DqT, 10 p,g/ml trypsin inhibitor, 0.1 /zM leupeptin) with 10% glycerol and 0.15 M KCI and applied to a 5 × 30 cm Phenyl Sepharose column equilibrated with the same buffer. The column is washed with Buffer A, Buffer A with 10% glycerol, and Buffer A containing 10% glycerol and 2% Tween 20. Both complexes are eluted from the column by the latter buffer prior to the major peak of absorbance. Fractions containing activity are pooled and applied to a hydroxylapatite column equilibrated with 5 vol of Buffer A with 0.1% Triton X-100. The column is washed with starting buffer and then developed stepwise with 100 and 200 mM potassium phosphate prepared in Buffer A with 0.1% Triton
MITOCHONDRIALPROTEIN KINASES
269
X-100. The pyruvate dehydrogenase complex elutes at 100 mM phosphate. Fractions containing pyruvate dehydrogenase activity are pooled and the enzyme is precipitated by centrifugation at 200,000 x g for 4 hr. The pellet is resuspended in Buffer A with 10% glycerol and stored at -70°C. Fractions containing branched-chain a-ketoacid dehydrogenase activity, eluted with 200 mM phosphate, are pooled and made 6.5% in PEG and centrifuged. Precipitated enzyme is suspended in a minimal vol of Buffer B (30 mM potassium phosphate, 0.1 mM EDTA, 1 mM MgSO4, 3 mM D'Iq', 0.05% Triton X-100, pH 7.5) and applied to a Q Sepharose column equilibrated with Buffer B. The column is washed with Buffer B and developed with a stepwise KCI gradient (300 and 600 mM). Fractions containing branched-chain ot-ketoacid dehydrogenase activity, eluted with about 600 mM KCI, are pooled and centrifuged at 200,000 x g for 4 hr. The pellet is resuspended in Buffer B with 10% glycerol and stored at -70°C. Both of the complexes isolated by this procedure possess high kinase activity (typical first order rate constants for ATP-dependent inactivation of 3.5 min -1 for pyruvate dehydrogenase kinase and 1.4 min -~ for branchedchain ot-ketoacid dehydrogenase kinase). The branched-chain ot-ketoacid dehydrogenase kinase complex and the pyruvate dehydrogenase kinase complex are separated into E1 and E2 subcomplexes by gel filtration in the presence of 1 M NaCI essentially as described by Cook et al. (13). A Superose 12 fast flow gel filtration column is used rather than Ultrogel AcA34. E2 elutes in the void vol, whereas El-kinase is retained by the matrix. Enzyme assays. The activity of the purified branched-chain ot-ketoacid dehydrogenase complex was assayed spectrophotometrically with 0.2 mM a-ketoisovalerate as substrate (9) but with 5 units of pig heart lipoamide dehydrogenase. Pyruvate dehydrogenase complex was assayed under the same conditions with 0.5 mM pyruvate as the substrate. One unit of enzyme activity catalyzed the formation of 1/xmole of NADH/min. Activities of the E1 components of the two complexes were assayed with the same assay conditions but in the presence of a molar excess of the corresponding E2 components. For the measurement of kinase activities, samples containing kinase-depleted pyruvate dehydrogenase complex or kinase-depleted branched-chain ot-ketoacid dehydrogenase complex were pre-incubated for 1 min at 32°C in 30 mM potassium phosphate, pH 7.5, 5 mM DTI', 0.1 mM EDTA, 2 mM MgCI 2, 0.05% Triton X-100, and 0.1 mg/ml bovine serum albumin. The reaction was initiated with the addition of 0.2 mM ATP. Aliquots of the mixture were removed as a function of time for the assay of pyruvate dehydrogenase and branched-chain et-ketoacid dehydrogenase residual activity. The procedures used for the assay of branched-chain et-ketoacid
270
R.A. HARRIS, et al.
dehydrogenase (15) and its kinase (9) in extracts of freeze-clamped tissue have been described previously.
Cloning strategy for cDNAs for branched-chain a-ketoacid dehydrogenase kinase. N-Terminal protein sequencing was performed on an Applied Biosystems Model 477A Pulse Liquid Protein Sequencer. The phenylthiobydantoin-derivatives were analyzed by reverse phase high pressure liquid chromatography with a Model 120A analyzer. Information obtained was used to design sense-antisense oligonucleotides as primers for generation of cDNA by the polymerase chain reaction (PCR). Total RNA, isolated from rat heart with RNAzol T M according to the instructions of Cinna/Biotecx Laboratories, was used for first strand cDNA synthesis by MMLV reverse transcriptase. PCR was performed in a programmable DNA thermal cycler (Perkin-Elemer Cetus) for 35 cycles. The PCR product was purified and sequenced. A non-degenerate oligonucleotide probe, synthesized according to the sequence of the PCR product, was used to screen rat liver and rat heart hgtl 1 libraries obtained from Clontech. Approximately 500,000 individual plaque-forming units were screened. Positive plaques were purified through four cycles of screening. Single-stranded DNA was prepared and sequenced by the dideoxy chain termination method (16). Western blot analysis. Proteins were electrophoresed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to polyvinyiidene difluoride membranes and analyzed immunochemically using antiserum raised in rabbits or guinea pigs against enzyme subunits or the kinases. In some cases, antibodies were purified from antiserum by epitope selection as described by Kelly et al. (17). Immunoblot analysis of the mass of branched-chain eL-ketoacid dehydrogenase. The branched-chain a-ketoacid dehydrogenase complex was quantitatively precipitated from crude tissue extracts as described by Goodwin et al. (15). The pellet was dissolved in a solution consisting of 4% (w/v) sodium dodecyl sulfate (SDS), 0.125 M-Tris, pH 6.8, 20% (v/v) glycerol, and 1.5 mg/ml DTT. The samples were boiled for 90 sec and applied to nitrocellulose paper in a dot blot apparatus in serial dilutions. The dot blotted paper was blocked with a solution containing bovine serum albumin, incubated with a mixture of polyclonal antibodies against the E l a and E2 subunits of branched-chain oL-ketoacid dehydrogenase complex (18), washed with bovine serum albumin, incubated with 125I-Protein A and washed again. Exposed autoradiograms were quantitated by densitometry. RNA isolation and Northern blot analysis. Total cellular RNA was
(1)
A
BCDE
60k50k- KINASE
30k-
FIG. 1. Purification of the rat heart branched-chain a-ketoacid dehydrogenase kinase. SDS-PAGE at steps of purification from the branched-chain a-ketoacid dehydrogenase kinase complex. (A) Branched chain a-ketoacid dehydrogenase kinase complex; (B) complex after ferricyanide treatment; (C) kinase released by ferricyanide treatment; (D) kinase purified by immunoadsorption chromatography; (E) kinase purified by DEAE-Sephacel chromatography. Reproduced with permission from Shimomura et al. (11)
MITOCHONDRIAL PROTEIN KINASES
271
extracted from freeze-clamped livers by the RNAzol TM method. 25/zg of total RNA of each sample was electrophoresed in a 1.5% agarose gel containing 2.2 M-formaldehyde and transferred to a NytranTM. The membrane was baked at 80°C in a vacuum oven for 2 hr. Rat branched-chain et-ketoacid dehydrogenase Elet, Ell3, E2 and tubulin cDNA were labelled by nick translation. Prehybridization and hybridization were carried out as described previously (18). Autoradiography was carried out with Kodak XAR-5 film and two intensifying screens at -70°C for 24-72 hr.
Isolation and incubation of hepatocytes. Hepatocytes were isolated from chow- and low-protein-fed rats as described previously (9). Hepatocytes were incubated in Krebs-Henseleit buffer supplemented with 1.25% (w/v) bovine serum albumin under an atmosphere of 95% 02/5% CO z. Clofibric acid, as the sodium salt, was added to the flasks at final concentrations indicated. After 15 min of incubation in a shaking water bath at 37°C, et-keto[1-14C]isovalerate (0.2 mM) was injected to initiate the reaction. The incubation was continued for an additional 15 min and then terminated. Carbon dioxide was collected in alkali and radioactivity determined by scintillation counting. RESULTS
AND DISCUSSION
Purification of the branched-chain ~-ketoacid dehydrogenase kinase and the pyruvate dehydrogenase kinase from rat liver and rat heart. Two procedures have now been developed by this laboratory for the isolation of the branched-chain ~-ketoacid dehydrogenase kinase from the branched-chain ~-ketoacid dehydrogenase kinase complex (11, 12). The key to the success of the first procedure was the finding that ferricyanide promotes dissociation of kinase activity from the branched-chain ~-ketoacid dehydrogenase complex (11). Thus, our first procedure involved an initial treatment of the complex with ferricyanide followed by high-speed centrifugation, immunoadsorption chromatography, and DEAE-Sephacel chromatography to purify the kinase. Both the rat heart and rat liver kinases were isolated by this procedure and gave one polypeptide band with a molecular weight of 44 kDa on SDS-PAGE (Fig. 1). Antibodies, monospecific for the branched-chain et-ketoacid dehydrogenase kinase by Western blot analysis of tissue extracts, inhibited kinase activity in a dose-dependent manner (Fig. 2) (19). In addition, chromatography of a mixture of the antibodies and the kinase on a protein A-Sepharose column resulted in the quantitative removal of kinase activity from the mixture (19). Since the latter column did not bind the kinase in the absence of antibodies, these observations confirm that the 44 kDa protein corresponds to the branched-chain et-ketoacid dehydrogenase kinase.
272
R.A. HARRIS, et al. i
1.0
0.2
,
i0 10
,
8'0
Antibody-eluUng buffer (,I) FIG. 2. Inhibition of branched-chain a-ketoacid dehydrogenase kinase by anti-kinasc antibodies. Rat liver branched-chain ~-ketoacid dehydrogenase kinase was mixed with different amounts of the purified antibodies. After preincubation, kinase-depleted branched-chain a-ketoacid dehydrogenase complex was added and ATP-dependent inactivation of dehydrogenase activity determined as an index of kinase activity. Reproduced with permission from Shimomura et aL (]9).
A new procedure for kinase isolation has been subsequently developed (12) based on the finding that ammonium chloride at a moderately high concentration will also promote dissociation of the kinase from the branched-chain a-ketoacid dehydrogenase complex. The developed procedure was also found applicable to the purification of pyruvate dehydrogenase kinase from its complex (12) as described below. The purified complex (i.e., either the branched-chain a-ketoacid dehydrogenase kinase complex or the pyruvate dehydrogenase kinase complex) is desalted on a PD-10 column equilibrated with Buffer B containing 0.2 mM oxidized glutathione. Desalted protein is applied at room temperature to a Q-Sepharose column (1.6 × 10 cm) equilibrated with the same buffer. The pyruvate dehydrogenase kinase elutes from this column with 0.18 M NHaCI added to the buffer; branched-chain ot-ketoacid dehydrogenase kinase with 0.25 M NHaCI. Kinase-depleted pyruvate dehydrogenase complex elutes with 0.4 M NHaCI; kinase-depleted branched-chain tx-ketoacid dehydrogenase with 0.6 M NHaCI. The kinase-depleted complexes are precipitated with PEG as described above, resuspended in Buffer A and dialyzed overnight against Buffer A with 10% (v/v) glycerol. Pooled kinase fractions obtained from either the branched-chain otketoacid dehydrogenase complex or the pyruvate dehydrogenase complex are supplemented with 5 mM DT-F and dialyzed against Buffer B with 5 mM DTT. The dialyzed preparation is applied to a Mono Q HR 5/5 column equilibrated with Buffer B plus 5 mM DTT (Figs. 3 and 4). The column is
A.
:i! i :'i:iii ~!!::~ i i~¸¸ ~i !¸!!!:!!!~i:iliii: i: ¸¸¸'~~~'':i!~¸:~¸!!i~¸~¸~¸i ........................ !~!~::i: :ill~ -3o ~ : ~:/ii! i~~ili: i!i~!i~i ::::i~ ...... Fraction numbers
15
le
lr
18
1~
20
21
100
11.0
B.
E o o
i
os T o
5O
0065-2571/92/$15.00 (~) 1992 Pergamon Press pie
PURIFICATION, CHARACTERIZATION, REGULATION AND MOLECULAR CLONING OF MITOCHONDRIAL PROTEIN KINASES ROBERT A. HARRIS, KIRILL M. POPOV, YOSHIHARU SHIMOMURA, YU Z H A O , J E R Z Y JASKIEWlCZ, NORIKO NANAUMI and MASASHIGE SUZUKI Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202
INTRODUCTION
Of the two protein kinases known to be present in the matrix space of mammalian mitochondria, the enzyme responsible for phosphorylation and inactivation of the pyruvate dehydrogenase complex (EC 1.2.4.1 + EC 2.3.1.12 + EC 1.8.1.4) is the most thoroughly studied (1, 2). Pyruvate dehydrogenase kinase (EC 2.7.1.99) renders the pyruvate dehydrogenase complex inactive by phosphorylation of specific sites on the Eltx subunit of the et-ketoacid dehydrogenase moiety of the complex. Short-term regulation of this kinase by allosteric effectors has been studied in considerable detail and is believed to be of physiological importance. However, much of the current interest in regulation of pyruvate dehydrogenase kinase stems from suggestive evidence for adaptive changes in activity of this enzyme in response to changes in nutritional and hormonal states of animals (3, 4). Although less well studied, the enzyme responsible for the phosphorylation and inactivation of the branched-chain ot-ketoacid dehydrogenase complex (EC 1.2.4.4 + EC no number + EC 1.8.1.4) is also of considerable interest (5-7). The activity of this enzyme, along with the opposing protein phosphatase, is believed responsible for regulation of the activity of the branched-chain a-ketoacid dehydrogenase complex and, therefore, the rate of disposal of the branched-chain amino acids (8, 9). Branched chain ot-ketoacid dehydrogenase kinase (EC 2.7.1.115) phosphorylates specific sites on the Elcx subunit of the branched-chain a-ketoacid dehydrogenase complex. Short-term regulation of this kinase seems to be confined simply to allosteric inhibitory effects of et-ketoisocaproate (transamination product of leucine). However, as with pyruvate dehydrogenase kinase, evidence has been presented for adaptive changes in the activity of this enzyme in response to nutritional and hormonal states of animals (6, 7, 9), but again like pyruvate dehydrogenase kinase, no information is available as to 267
268
R.A. HARRIS, et al.
the actual molecular mechanisms involved. Current efforts of this laboratory are directed toward obtaining the molecular tools necessary for a study of the regulation of gene expression of the mitochondrial protein kinases. We describe here the purification and characterization of these protein kinases from rat tissues, the cloning of cDNAs for branched-chain oL-ketoacid dehydrogenase kinase, and provide suggestive evidence that inhibition of branched-chain ot-ketoacid dehydrogenase kinase may be responsible for the development of myotonia in humans treated with clofibric acid. MATERIALS AND METHODS
Source of materials. Enzymes and biochemicals were obtained from Sigma Chemical Company; ot-chloroisocaproate was a gift from Dr. Ronald Simpson of Sandoz Pharmaceutical Corp., radiolabeled compounds from DuPont-New England Nuclear, molecular biology reagents from Bethesda Research Laboratories, protein sequencing reagents from Applied Biosystems, and chromatography supplies from Pharmacia. Purification of branched chain ot-ketoacid dehydrogenase and pyruvate dehydrogenase complexes from rat tissues. Recent modifications of purification procedures have led to greater retention of the protein kinase activities associated with the complexes (10, 11). In brief detail, our current purification procedure (12) for the rat heart complexes involves the following steps. Frozen rat hearts (200-300 g) are homogenized in a buffer containing Triton X-100, dithiothreitol (DTF) and a battery of protease inhibitors. The homogenate is centrifuged and the pellet homogenized again in the same buffer and centrifuged. The combined supernatants are made 1% in polyethylene glycol (PEG), the precipitate is discarded and the supernatant made 4.5% in PEG. The pellet obtained is suspended in a buffer containing Mg z+ and incubated at 37°C for 50 min, a treatment that activates the complexes by dephosphorylation. The suspension is made 3% in PEG, and the precipitate collected by centrifugation. The pellet is resuspended in 1 vol of Buffer A (50 mM potassium phosphate, pH 7.5, 0.1 mM EDTA, 3 mM DqT, 10 p,g/ml trypsin inhibitor, 0.1 /zM leupeptin) with 10% glycerol and 0.15 M KCI and applied to a 5 × 30 cm Phenyl Sepharose column equilibrated with the same buffer. The column is washed with Buffer A, Buffer A with 10% glycerol, and Buffer A containing 10% glycerol and 2% Tween 20. Both complexes are eluted from the column by the latter buffer prior to the major peak of absorbance. Fractions containing activity are pooled and applied to a hydroxylapatite column equilibrated with 5 vol of Buffer A with 0.1% Triton X-100. The column is washed with starting buffer and then developed stepwise with 100 and 200 mM potassium phosphate prepared in Buffer A with 0.1% Triton
MITOCHONDRIALPROTEIN KINASES
269
X-100. The pyruvate dehydrogenase complex elutes at 100 mM phosphate. Fractions containing pyruvate dehydrogenase activity are pooled and the enzyme is precipitated by centrifugation at 200,000 x g for 4 hr. The pellet is resuspended in Buffer A with 10% glycerol and stored at -70°C. Fractions containing branched-chain a-ketoacid dehydrogenase activity, eluted with 200 mM phosphate, are pooled and made 6.5% in PEG and centrifuged. Precipitated enzyme is suspended in a minimal vol of Buffer B (30 mM potassium phosphate, 0.1 mM EDTA, 1 mM MgSO4, 3 mM D'Iq', 0.05% Triton X-100, pH 7.5) and applied to a Q Sepharose column equilibrated with Buffer B. The column is washed with Buffer B and developed with a stepwise KCI gradient (300 and 600 mM). Fractions containing branched-chain ot-ketoacid dehydrogenase activity, eluted with about 600 mM KCI, are pooled and centrifuged at 200,000 x g for 4 hr. The pellet is resuspended in Buffer B with 10% glycerol and stored at -70°C. Both of the complexes isolated by this procedure possess high kinase activity (typical first order rate constants for ATP-dependent inactivation of 3.5 min -1 for pyruvate dehydrogenase kinase and 1.4 min -~ for branchedchain ot-ketoacid dehydrogenase kinase). The branched-chain ot-ketoacid dehydrogenase kinase complex and the pyruvate dehydrogenase kinase complex are separated into E1 and E2 subcomplexes by gel filtration in the presence of 1 M NaCI essentially as described by Cook et al. (13). A Superose 12 fast flow gel filtration column is used rather than Ultrogel AcA34. E2 elutes in the void vol, whereas El-kinase is retained by the matrix. Enzyme assays. The activity of the purified branched-chain ot-ketoacid dehydrogenase complex was assayed spectrophotometrically with 0.2 mM a-ketoisovalerate as substrate (9) but with 5 units of pig heart lipoamide dehydrogenase. Pyruvate dehydrogenase complex was assayed under the same conditions with 0.5 mM pyruvate as the substrate. One unit of enzyme activity catalyzed the formation of 1/xmole of NADH/min. Activities of the E1 components of the two complexes were assayed with the same assay conditions but in the presence of a molar excess of the corresponding E2 components. For the measurement of kinase activities, samples containing kinase-depleted pyruvate dehydrogenase complex or kinase-depleted branched-chain ot-ketoacid dehydrogenase complex were pre-incubated for 1 min at 32°C in 30 mM potassium phosphate, pH 7.5, 5 mM DTI', 0.1 mM EDTA, 2 mM MgCI 2, 0.05% Triton X-100, and 0.1 mg/ml bovine serum albumin. The reaction was initiated with the addition of 0.2 mM ATP. Aliquots of the mixture were removed as a function of time for the assay of pyruvate dehydrogenase and branched-chain et-ketoacid dehydrogenase residual activity. The procedures used for the assay of branched-chain et-ketoacid
270
R.A. HARRIS, et al.
dehydrogenase (15) and its kinase (9) in extracts of freeze-clamped tissue have been described previously.
Cloning strategy for cDNAs for branched-chain a-ketoacid dehydrogenase kinase. N-Terminal protein sequencing was performed on an Applied Biosystems Model 477A Pulse Liquid Protein Sequencer. The phenylthiobydantoin-derivatives were analyzed by reverse phase high pressure liquid chromatography with a Model 120A analyzer. Information obtained was used to design sense-antisense oligonucleotides as primers for generation of cDNA by the polymerase chain reaction (PCR). Total RNA, isolated from rat heart with RNAzol T M according to the instructions of Cinna/Biotecx Laboratories, was used for first strand cDNA synthesis by MMLV reverse transcriptase. PCR was performed in a programmable DNA thermal cycler (Perkin-Elemer Cetus) for 35 cycles. The PCR product was purified and sequenced. A non-degenerate oligonucleotide probe, synthesized according to the sequence of the PCR product, was used to screen rat liver and rat heart hgtl 1 libraries obtained from Clontech. Approximately 500,000 individual plaque-forming units were screened. Positive plaques were purified through four cycles of screening. Single-stranded DNA was prepared and sequenced by the dideoxy chain termination method (16). Western blot analysis. Proteins were electrophoresed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to polyvinyiidene difluoride membranes and analyzed immunochemically using antiserum raised in rabbits or guinea pigs against enzyme subunits or the kinases. In some cases, antibodies were purified from antiserum by epitope selection as described by Kelly et al. (17). Immunoblot analysis of the mass of branched-chain eL-ketoacid dehydrogenase. The branched-chain a-ketoacid dehydrogenase complex was quantitatively precipitated from crude tissue extracts as described by Goodwin et al. (15). The pellet was dissolved in a solution consisting of 4% (w/v) sodium dodecyl sulfate (SDS), 0.125 M-Tris, pH 6.8, 20% (v/v) glycerol, and 1.5 mg/ml DTT. The samples were boiled for 90 sec and applied to nitrocellulose paper in a dot blot apparatus in serial dilutions. The dot blotted paper was blocked with a solution containing bovine serum albumin, incubated with a mixture of polyclonal antibodies against the E l a and E2 subunits of branched-chain oL-ketoacid dehydrogenase complex (18), washed with bovine serum albumin, incubated with 125I-Protein A and washed again. Exposed autoradiograms were quantitated by densitometry. RNA isolation and Northern blot analysis. Total cellular RNA was
(1)
A
BCDE
60k50k- KINASE
30k-
FIG. 1. Purification of the rat heart branched-chain a-ketoacid dehydrogenase kinase. SDS-PAGE at steps of purification from the branched-chain a-ketoacid dehydrogenase kinase complex. (A) Branched chain a-ketoacid dehydrogenase kinase complex; (B) complex after ferricyanide treatment; (C) kinase released by ferricyanide treatment; (D) kinase purified by immunoadsorption chromatography; (E) kinase purified by DEAE-Sephacel chromatography. Reproduced with permission from Shimomura et al. (11)
MITOCHONDRIAL PROTEIN KINASES
271
extracted from freeze-clamped livers by the RNAzol TM method. 25/zg of total RNA of each sample was electrophoresed in a 1.5% agarose gel containing 2.2 M-formaldehyde and transferred to a NytranTM. The membrane was baked at 80°C in a vacuum oven for 2 hr. Rat branched-chain et-ketoacid dehydrogenase Elet, Ell3, E2 and tubulin cDNA were labelled by nick translation. Prehybridization and hybridization were carried out as described previously (18). Autoradiography was carried out with Kodak XAR-5 film and two intensifying screens at -70°C for 24-72 hr.
Isolation and incubation of hepatocytes. Hepatocytes were isolated from chow- and low-protein-fed rats as described previously (9). Hepatocytes were incubated in Krebs-Henseleit buffer supplemented with 1.25% (w/v) bovine serum albumin under an atmosphere of 95% 02/5% CO z. Clofibric acid, as the sodium salt, was added to the flasks at final concentrations indicated. After 15 min of incubation in a shaking water bath at 37°C, et-keto[1-14C]isovalerate (0.2 mM) was injected to initiate the reaction. The incubation was continued for an additional 15 min and then terminated. Carbon dioxide was collected in alkali and radioactivity determined by scintillation counting. RESULTS
AND DISCUSSION
Purification of the branched-chain ~-ketoacid dehydrogenase kinase and the pyruvate dehydrogenase kinase from rat liver and rat heart. Two procedures have now been developed by this laboratory for the isolation of the branched-chain ~-ketoacid dehydrogenase kinase from the branched-chain ~-ketoacid dehydrogenase kinase complex (11, 12). The key to the success of the first procedure was the finding that ferricyanide promotes dissociation of kinase activity from the branched-chain ~-ketoacid dehydrogenase complex (11). Thus, our first procedure involved an initial treatment of the complex with ferricyanide followed by high-speed centrifugation, immunoadsorption chromatography, and DEAE-Sephacel chromatography to purify the kinase. Both the rat heart and rat liver kinases were isolated by this procedure and gave one polypeptide band with a molecular weight of 44 kDa on SDS-PAGE (Fig. 1). Antibodies, monospecific for the branched-chain et-ketoacid dehydrogenase kinase by Western blot analysis of tissue extracts, inhibited kinase activity in a dose-dependent manner (Fig. 2) (19). In addition, chromatography of a mixture of the antibodies and the kinase on a protein A-Sepharose column resulted in the quantitative removal of kinase activity from the mixture (19). Since the latter column did not bind the kinase in the absence of antibodies, these observations confirm that the 44 kDa protein corresponds to the branched-chain et-ketoacid dehydrogenase kinase.
272
R.A. HARRIS, et al. i
1.0
0.2
,
i0 10
,
8'0
Antibody-eluUng buffer (,I) FIG. 2. Inhibition of branched-chain a-ketoacid dehydrogenase kinase by anti-kinasc antibodies. Rat liver branched-chain ~-ketoacid dehydrogenase kinase was mixed with different amounts of the purified antibodies. After preincubation, kinase-depleted branched-chain a-ketoacid dehydrogenase complex was added and ATP-dependent inactivation of dehydrogenase activity determined as an index of kinase activity. Reproduced with permission from Shimomura et aL (]9).
A new procedure for kinase isolation has been subsequently developed (12) based on the finding that ammonium chloride at a moderately high concentration will also promote dissociation of the kinase from the branched-chain a-ketoacid dehydrogenase complex. The developed procedure was also found applicable to the purification of pyruvate dehydrogenase kinase from its complex (12) as described below. The purified complex (i.e., either the branched-chain a-ketoacid dehydrogenase kinase complex or the pyruvate dehydrogenase kinase complex) is desalted on a PD-10 column equilibrated with Buffer B containing 0.2 mM oxidized glutathione. Desalted protein is applied at room temperature to a Q-Sepharose column (1.6 × 10 cm) equilibrated with the same buffer. The pyruvate dehydrogenase kinase elutes from this column with 0.18 M NHaCI added to the buffer; branched-chain ot-ketoacid dehydrogenase kinase with 0.25 M NHaCI. Kinase-depleted pyruvate dehydrogenase complex elutes with 0.4 M NHaCI; kinase-depleted branched-chain tx-ketoacid dehydrogenase with 0.6 M NHaCI. The kinase-depleted complexes are precipitated with PEG as described above, resuspended in Buffer A and dialyzed overnight against Buffer A with 10% (v/v) glycerol. Pooled kinase fractions obtained from either the branched-chain otketoacid dehydrogenase complex or the pyruvate dehydrogenase complex are supplemented with 5 mM DT-F and dialyzed against Buffer B with 5 mM DTT. The dialyzed preparation is applied to a Mono Q HR 5/5 column equilibrated with Buffer B plus 5 mM DTT (Figs. 3 and 4). The column is
A.
:i! i :'i:iii ~!!::~ i i~¸¸ ~i !¸!!!:!!!~i:iliii: i: ¸¸¸'~~~'':i!~¸:~¸!!i~¸~¸~¸i ........................ !~!~::i: :ill~ -3o ~ : ~:/ii! i~~ili: i!i~!i~i ::::i~ ...... Fraction numbers
15
le
lr
18
1~
20
21
100
11.0
B.
E o o
i
os T o
5O
