Purification procedure and N-terminal amino acid sequence of yeast malate dehydrogenase isoenzymes

398

Biochimica et Bioph.vsica Acta 912 (1987) 398-403 Elsevier

BBA 32833

P u r i f i c a t i o n p r o c e d u r e and N - t e r m i n a l a m i n o acid s e q u e n c e of y e a s t m a l a t e dehydrogenase isoenzymes E r h a r d K o p e t z k i a, K a r l - D i e t e r E n t i a n a, F r i e d r i c h L o t t s p e i c h ~ and Dieter Mecke ~ Ph)'siologisch-Chemtsches lnstitut trn Medizinisch-Naturwissenschaftlichen-Forschungs lnstitut der (Jntversttiit Tiibtngen. T~bingen and t, Max Planck lnstitut fiir Biochemie. Mi~nchen (E R.G.) (Received 23 December 1986)

Key words: Malate dehydrogenase purification; Amino acid sequence, N-terminal: Glucose inactivation: Isoenzymes; ( S ceret'isiae )

A method has been devised for the rapid isolation of malate dehydrogenase isoenzymes. First, anionic proteins were precipitated with polyethyleneimine, whilst hydrophobic malate dehydrogenase remained in the supernatant fluid. Secondly, the supernatant was 30% saturated with ammonium sulfate and the two isoenzymes were separated by hydrophobic phenyI-Sepharose CL-4B chromatography. For further purification the enzymes were chromatofocused, and polybuffer was removed by hydrophobic chromatography. Affinity chromatography with blue Sepharose CL-6B [1] was used as final purification step. The purified isoenzymes were homogeneous as shown by isoelectric focusing and could be used for N-terminal sequencing. 34 amino acid residues could be identified for the cytoplasmic isoenzyme and 56 amino acid residues for the mitochondrial isoenzyme. Although there are regions of strong homology between both isoenzymes, the sequence differences clearly showed support that both isoenzymes are coded by different genes. Sequence comparison clearly indicated that the N-terminus of the cytoplasmic enzyme extended that of the mitochondrial enzyme by 12 amino acid residues. The amino acid sequence of the extending sequence resembled that of leading sequences known for enzymes which are transported into the mitochondria. The assumed leading sequence is discussed with respect to its possible role in glucose inactivation.

Introduction Yeast malate dehydrogenase (EC 1.1.1.37) is of particular interest because it is subject to glucose inactivation. Glucose inactivation refers to the marked reduction in activity of a number of enzymes (mostly gluconeogenic) within 1 h of adding glucose to derepressed cells [2-6] (see Ref. 7 for a review). The mechanism of irreversible glucose inactivation is still unknown. Since no degradation Correspondence: K.-D. Entian, Medizinisch-Naturwissenschaftlichen-Forschungs Institut, Ob dem Himmelreich 7, D-7400 Tiibingen, F.R.G.

products could be identified immunologically, a proteolytic mechanism has been proposed as being responsible for the glucose inactivation of malate dehydrogenase [1]. Such a mechanism was also consistent with findings for fructose-l,6-bisphosphatase [8] and phosphoenolpyruvate carboxykinase [9]. By contrast, a rapid reversible inactivation was observed for fructose-l,6-bisphosphatase in stationary cells [10]. Interconversion of the enzyme by phosphorylation was shown to be responsible for this type of inactivation [11,12]. Other gluconeogenic enzymes like malate dehydrogenase and phosphoenolpyruvate carboxykinase are only irreversibly degraded and no

0167-4838/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

399 phosphorylated enzymes were observed (Gancedo, J.M. and Holzer, H., personal communication). In mutants which cannot carry out irreversible inactivation of malate dehydrogenase, the inactivation of fructose-l,6-bisphosphatase remains reversible for at least 2 h [13]. Hence, the irreversible mechanism is probably the same for both enzymes. Towards elucidating the mechanism of irreversible inactivation of malate dehydrogenase we purified the isoenzymes to homogeneity and compared the N-terminal amino acid sequences. There are two malate dehydrogenase isoenzymes in yeast. One is cytoplasmic, whereas the other is in the mitochondrion [14]. Only the cytoplasmic isoenzyme is subject to glucose inactivation [15]. Comparison between the two enzymes should give further information for the understanding of glucose inactivation. Materials and Methods

Haploid yeast strain (catl. $3-14A) [23] was grown in 1% yeast extract, 2% bacto peptone (both Difco, U.S.A.) and 2% glucose until late logarithmic phase (5.108 cells/ml). For derepression of malate dehydrogenase cells were washed twice with sterile distilled water and resuspended in a 2-fold amount of derepression medium (0.2% ammonium sulfate, 0.069% diammonium hydrogene phosphate, 0.028% KCI, 0.015% magnesium sulfate heptahydrate, 0.14% calcium chloride dihydrate, 20 ppm myo-inositol, 10 ppm calcium pantothenate, 2 ppm thiamine, 0.5 ppm pyridoxamine hydrochloride, 0.01 ppm biotin, 20 ppm histidine, 0.78 ppm copper sulphate pentahydrate, 4.8 ppm iron(Ill) chloride hexahydrate, 3 ppm zinc sulphate heptahydrate, 3.5 ppm magnesium sulphate heptahydrate and 0.2% sodium acetate as carbon source). For derepression the cell suspension was incubated in a 20 1 fermenter for 6-8 h (aeration, 600 I/h). Cells were harvested by centrifugation, washed thrice with distilled water and frozen at - 2 0 ° C until used. For disruption, 250 g wet weight cells were suspended in 50 mM Tris-HCl buffer (pH 7), containing 1 mM EDTA and 1 mM mercaptoethanol and disintegrated using glass beads (Braun, Meisungen, F.R.C.) and a labora-

tory blender [20]. Cell debris was removed by centrifugation at 24000 × g (Beckman J-21C, 20 min) and the supernatant fluid was used as a crude extract. Polyethyleneimine (20% solution; trade name, Tydex 16) was a gift of Nordmann, Rassmann and Company (Hamburg, Kajen 2, F.R.G.). PhenylSepharose C L - 4 B (Pharmacia, Sweden) was equilibrated against 50 mM Tris-HCl (pH 7), 1 mM EDTA, 1 mM mercaptoethanol, saturated with 30% ammonium sulfate at 4°C. Regeneration was as in Ref. 25. Chromatofocusing gel PBE 94 (Pharmacia, Sweden) was equilibrated against 25 mM imidazole/CH3COOH (pH 7.4). Columns were packed as in Ref. 26. Dialysis tubes (Schleicher and Schiill, F.R.G.) were heated with 10 mM sodium hydrogene carbonate for 30 min followed by a wash with 50% ethanol analytical grade for 30 min. Malate dehydrogenase activity was assayed as in Ref. 27 with glycine/hydrazine buffer (pH 9.5). One unit of enzyme activity is defined as the conversion of 1 ~mol of substrate per min. Specific activity is defined in terms of 1 U / m g of protein [281. The isoelectric focusing was done as described in Ref. 29. The gel was composed of 3.6 ml acryl amide solution (22% acrylamide and 1.4% bisacrylamide), 15.4 ml 9 M urea and 1 ml ampholine (40%, LKB, Mtinchen, F.R.G.). The gel was polymerized on a glass plate which had been rubbed with a mixture of 20/~1 silan GF3 (Wacker Chemie, Mtinchen, F.R.G.) and 4 ~1 25% acetic acid; 0.5 mm gels were used. Polymerization was started with 40 ~mol ammonium peroxodisulfate (10% solution). Power was limited to 4 W. Initially voltage was 300 V and increased to a final level of about 1000 V within 6-8 h. Gels were fixed twice in 20% trichloroacetic acid for 30 min and stained according to the silver-stain method described in Refs. 30 and 31. Automated N-terminal amino acid sequence analysis was performed in a prototype spinningcup sequenator as described in detail previously [32], or in a gas-phase sequencer 470A from Applied Biosystems. The phenylthiohydantoin derivatives of the amino acids were analysed by a HPLC system which separates all components isocratically [33].

400

Results and Discussion

U/ml

. [mSl

mMDH

cMOH

A 280

Purification To 400 ml crude extract from derepressed yeast cells (see Materials and Methods) 40 ml of a 20% solution of polyethylenimine (Tydex 16) were added slowly with stirring. Polyethylenimine acts as a strong anionic exchanger and precipitates anionic proteins immediately. The suspension was centrifuged for 20 min at 12000 rpm (Beckman J 21 C). All the malate dehydrogenase activity remained in the supernatant fluid (see Table l), to which ammonium sulphate was added to 30% saturation and the solution was run through a 3 × 8-cm column of phenyl-Sepharose CL-4B (flow rate 120 mi/h). The column was equilibrated with 50 m M Tris-HC1, 1 mM E D T A . 1 mM mercaptoethanol (pH 7.0). Malate dehydrogenase was eluted with ammonium sulphate at decreasing concentrations (30-0%) and a simultaneously increasing gradient of polarity-reducing ethylene glycol (0--80%); (flow rate 60 m l / h , gradient volume 2 × 250 ml). Mitochondrial and cytoplasmic malate dehydrogenase isoenzymes were separated during this procedure at a conductivity of 5 and 8 mS, respectively (Fig. 1). Active fractions were pooled, dialysed and applied to chromatofocusing columns (1 × 40 cm) of polybuffer exchanger PBE 94 (flow rate 60 ml/h) which had been equilibrated against 25 mM imidazole/ CH~COOH (pH 7.4) and 350 ml polybuffer 96;

0.~

80'

gO" I ~ 20.

2

,~

~

"

-300

~oo

!roll Fig. 1. Hydrophobic chromatography of malate dehydrogenase. Enzyme activity (filled circles), conductivity (filled squares), cMDH, cytoplasmic malate dehydrogenase: mMDH, mitochondrial malate dehydrogenase.

elution was carried out, using polybuffer (diluted 1:13 from stock solution) adjusted to pH 6.0 (flow rate, 20 ml/h). Mitochondrial malate dehydrogenase was eluted at pH 6.35 and cytoplasmic malate dehydrogenase at pH 6.15 (Fig. 2). Polybuffer was removed by phenyI-Sepharose chromatography (see above) (1 × 10 cm column). Malate dehydrogenase was eluted in a single step with 50 mM Tris-HCl, 1 mM EDTA, 1 mM mercaptoethanol (pH 7) containing 70% ethyleneglycol. For final purification the enzymes were applied to a Blue Sepharose CL.-6B column (2 × 10 cm) and eluted by a KCI gradient (0-0.75 M,

]ABLE I PURIFICATION OF MALATE DEHYDROGENASE FROM YEAS]" (200 g WET W E I G H t ) cMDH. cytoplasmic malate dehydrogenase: mMDH. mitochondrial malate dehydrogenase. Purification stage

Total activity (U)

Specific activity (U/rag)

Crude extract Polyethylenimine precipitation Phenyl-Sepharose chromatography

14800 14100 3900 740O 2 860 3 700 2 560 3 600

0.6

Chromatofocusing Blue-Sepharose chromatograph>

cMDH mMDH cMDH mMDH cMDH mMDH

~o

Yield (~ )

95 16 5(1

76 44

40O 490

42

401 a

pH

U/ml

cMDM

A2BO

120 100 6.6 60'

b.(, 6.2

U/ml

pH

•7.2 7.0 200

• 6.8

160,

"6.6

t20,

'6.l,

BO

'6.2

z.O

' 6.0

~

m MDM

| O~

0.2

[mll

Fig. 2. Chromatofocusingof malate dehydrogenase. Enzyme activity(filledcircles), pH gradient(filled squares). (a) cMDH, cytoplasmicmalate dehydrogenase;(b) mMDH, mitochondrial malate dehydrogenase.

2 x 100 ml). The mitochondrial isoenzyme was eluted at 0.75 mS and the cytoplasmic isoenzyme at 2.15 mS. Finally, the enzymes were dialysed against 50 mM Tris-HCl, 1 mM EDTA, 1 mM mercaptoethanol (pH 7) and concentrated by centrifugation with concentrating patrons (Amicon, F.R.G.). The purified enzymes gave single bands after isoelectric focusing; the specific activity of the cytoplasmic isoenzymes was about 400 U / m g and that for the mitochondrial enzyme 490 U / m l (see also Table I). As previously found [1], the M,, as calculated by sodium dodecyl sulfate gel electrophoresis, was 40 000 for the cytoplasmic and 35 000 for the mitochondrial isoenzyme. The concentrated enzyme solutions were frozen at 20 o C after adding glycerol (final concentration, 50%) without any loss of activity.

Protein sequencing For N-terminal amino acid sequencing purified malate dehydrogenase was dialysed against 20 mM N H a H C O 3, freeze dried and washed twice with distilled water. 25 nmol mitochondrial malate dehydrogenase were subjected to N-terminal sequence analysis in a liquid-phase sequenator. Identification of 56 amino acid residues was possible. The repetitive yield was 97% (results are indicated in Fig. 3). To confirm these results, a second analysis was performed (20 nmol starting material, repetitive yield of 96%, 41 steps identified). Cytoplasmic malate dehydrogenase was analysed by gas-phase sequencing and liquid-phase sequencing. With the gas-phase sequenator using 300 pmol starting material, 21 residues could be identified (repetitive yield 92%). With the liquid-phase sequenator it was possible to determine 34 amino acid residues with a repetitive yield of 95%, using 15 nmol starting material (results are indicated in Fig. 3). The N-terminal sequences were arranged according to the principle of maximal homology and also compared with those of porcine mitochondrial malate dehydrogenase [16] (Fig. 3). There was strong homology between the three sequences compared with 64% homology between the two yeast isoenzymes, and 63% for the mitochondrial enzymes from yeast and pig. This indicated their genetical relationship. However, the differences between the yeast cytoplasmic and mitochondrial isoenzymes clearly showed that both proteins were coded by different genes. There is a strong conservative sequence of 12 residues for all three enzymes which may be important for the catalytic function or the tertiary structure. Furthermore, the cytoplasmic N-terminal sequence extended that of the mitochondrial enzymes by 12 amino acid residues, so the extending sequence may correspond with a leading sequence. Two types of leading sequence have been described for proteins. First, there are leading sequences of enzymes that are cotranslationally secreted into the endoplasmic reticulum according to the signal recognition hypothesis [17]. Characteristically, these sequences contain a central region of hydrophobic residues (reviewed in Ref. 18). Secondly, there are leading sequences of enzymes that are posttranslationally transported into mitochondria

402 I0

cMDH yeast:

Pro

His

Ser

Val

Thr

Pro

Ser

lie

Glu

Gln

Asp

Ser

mMDH yeast: mDMH pig:

Leu

Lys

Tyr Ala

~+

20

lie Val + 30

cMDH yeast:

Ala

lie

Leu

Gly

Ala

Ala

Gly

Gly

Ile

Gly

mMDH yeast: mMDH pig:

Thr +

Val +

+ +

+ t

+ +

Gly Ser

+ +

+ +

t +

+ +

cMDH yeast: mMDH yeast: mMDH pig:

Leu + +

Leu + +

Lys + +

Ala Leu Asn

Asn Ser

His Pro

Lys Leu

Val t

Thr Ser

Asp Arg

Leu +

Arg Thr

cMDH yeast mMDH yeast: mMDH pig:

Leu lie

Lys Ala

Gly His

Ala Thr

Lys Pro

Gly +

Val +

Ala +

Thr Ala

Asp +

Leu +

(Ser) +

cMDH yeast: mMDH yeast: mMDH pig:

Thr +

Asn Arg

Gly Ala

Val Thr

Val +

(Lys) Gly

Gin

(Pro)

+

+

+

+

Leu +

Set

Leu

+

+

4

+

Leu +

Tyr +

Asp +

His ~-

lie .-

Pro Glu

60

50

+

+

Phe Tyr

Fig. 3. N-terminal amino acid sequences of malate dehydrogenases. Amino acid residues determined with minor confidence are indicated by brackets. A plus sign indicates an amino acid identical to the one above it. [19-21]. These leading sequences c o n t a i n m o s t l y c h a r g e d a m i n o acids (reviewed in Ref. 22). The e x t e n d i n g sequence of c y t o p l a s m i c m a l a t e dehyd r o g e n a s e is strongly charged with eight h y d r o philic a m i n o acid residues, a n d hence resembles that of p o s t t r a n s l a t i o n a l l y t r a n s p o r t e d enzymes. This observation m a y b e c o m e i m p o r t a n t for und e r s t a n d i n g the m e c h a n i s m of glucose inactivation. H i t h e r t o our research on glucose inactivation was b l i n k e r e d by the idea that specific p r o t e i n a s e s m a y d e g r a d e the enzymes d u r i n g glucose inactivation (selective proteolysis). The evidence of a leading sequence justifies an alternative hypothesis, namely, that c y t o p l a s m i c m a l a t e d e h y d r o g e n a s e m a y be selectively t r a n s p o r t e d into the vacuole as well as unspecifically d e g r a d e d (selective transport). However, selective t r a n s p o r t requires that the leading sequence is m o d i f i e d u p o n a d d i t i o n of glucose to trigger the u p t a k e into the vacuole. E n z y m e m o d i f i c a t i o n by p h o s p h o r y l a t i o n at a serine residue has been shown for r a p i d reversible inactivation of f r u c t o s e - l , 6 - b i s p h o s p h a t a s e [10-12]. T h e e x t e n d i n g sequence of c y t o p l a s m i c m a l a t e d e h y d r o g e n a s e c o n t a i n s three serine a n d one t h r e o n i n e residues at which p h o s p h o r y l a t i o n m a y occur. So far no m a l a t e d e h y d r o g e n a s e p h o s p h o r y lation has been o b s e r v e d (Holzer, H. and G a n c e d o , J.M., personal c o m m u n i c a t i o n ) . Two reasons m a y

explain this failure: (1) D e g r a d a t i o n after the supp o s e d u p t a k e into the vacuole is too fast to detect a p h o s p h o r y l a t e d i n t e r m e d i a t e or (2) the s u p p o s e d p h o s p h o r y l a t e d leading sequence is cleaved from the enzyme d u r i n g its t r a n s p o r t into the vacuole. A l t h o u g h the h y p o t h e s i s of selective u p t a k e m a y be justified by the sequence data, it is speculative. A second alternative e x p l a n a t i o n of the e x t e n d i n g c y t o p l a s m i c sequence m a y be that both m a l a t e d e h y d r o g e n a s e isoenzymes were derived from a c o m m o n ancestral gene with the e x t e n d i n g sequence as a relict of their c o m m o n origin. H o w ever, the characteristic a m i n o acid c o m p o s i t i o n of the e x t e n d i n g sequence favours the hypothesis of selective t r a n s p o r t d u r i n g glucose inactivation.

Acknowledgements T h e authors thank Dr. H. T a b a k a n d Dr. K.-U. Fr~Shlich for m a n y s t i m u l a t i n g discussions a n d Dr. J.A. Barnett for reading the manuscript. This work was s u p p o r t e d by the Deutsche Forschungsgemeinschaft and the F o n d s d e r Chemie. It was p a r t i a l l y carried o u t in the M e d i z i n i s c h - N a t u r w i s senschaftliches F o r s c h u n g s i n s t i t u t of the University of Ttibingen. E.K. has been in receipt of a g r a n t of the S t u d i e n s t i f t u n g des Deutschen Volkes.

403

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