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Bioscience Reports i, 4'97-502 (1981) Printed in Great Britain 497 M a l a t e d e h y d r o g e n a s e : I s o l a t i o n f r o m E . c o l i and c o m p a r i s o n w i t h t h e e u k a r y o t i c m i t o c h o n d r i a l and c y t o p l a s m i c f o r m s Ross T. FERNLEY, ~ Steven R. LENTZ, and Ralph A. BRADSHAW Department of Biological Chemistry, Washington University School of Medicine, St. Louis, MO 63110, U.S.A. (Received 18 May 1981) Escherichia coli malate dehydrogenase has been isolated in homogeneous form by a procedure employing chromatography on DEAE-cellulose, 5'-AMP-Sepharose, and Sephacryl-200. It is c o m p o s e d of two identical polypeptide chains each of M r = 32 500. Like porcine mitochondrial malate dehydrogenase, it is devoid of tryptophan, but otherwise it is not particularly more similar in composition to one of the eukaryotic isozymes than to the other. However, amino-terminal sequence analysis of the first 36 residues shows r e m a r k a b l e s i m i l a r i t y of t h e b a c t e r i a l and mitochondrial enzymes (69% identical residues) in contrast to the cytoplasmic form (27%). The two p o r c i n e h e a r t e n z y m e s a r e identical in 2zt% of t h e p o s i t i o n s c o m p a r e d . These r e s u l t s clearly establish that all three forms of malate dehydrogenase have evolved from a c o m m o n p r e c u r s o r and t h a t t h e prokaryotic and mitochondrial forms have retained sequences that are much closer to the ancestral one than the cytoplasmic enzyme. These findings appear to further substantiate the endosymbiotic hypothesis for the origin of the mitochondrion. M a l a t e dehydrogenase ( M D H ) , which c a t a l y z e s the r e v e r s i b l e NAD+-dependent conversion of L-malate to oxaloacetate, occurs in a broad spectrum of living organisms. In e u k a r y o t e s , t h e r e are two d i s t i n c t f o r m s of the enzyme, one associated w i t h m i t o c h o n d r i a (mMDH) and the other found in the cytoplasm (sMDH) (Banaszak & Bradshaw, 1975). Prokaryotes possess only a single form. However, from one species of bacteria to a n o t h e r , t h e y appear to occur in either dimeric (Escherichia coli, Murphey eta]., 1967b; Pseudomonas testosteroni, You & Kaplan, 1979) or tetrameric (Bacillus subtills, B a c i l l u s stearothermophilus, Murphey eta]., 1967b) structures (Murphey et al., 1967a). Both e u k a r y o t i c isozymes are dimeric. The subunit size in all forms is 32 000-35 000 M r (Banaszak & Bradshaw, 1975). *Present address: Howard Florey Institute for Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia 9 1981 The Biochemical Society t498 FERNLEY ET A L . 0"8I '- ' ~ -I i ' ~ 'm 300 I I E ?~ 200 < 100 z >- ""% 0 300 FRACTION NUMBER I s o l a t i o n and c h a r a c t e r i z a t i o n of the two MDH isozymes of pig heart have revealed l i t t l e similarity except for molecular weight and subunit structure, suggesting that if they shared a common precursor, then the divergence must have occurred a l o n g time ago. It was of interest, therefore, to isolate a prokaryotic version of the enzyme and to compare it with its eukaryotic counterparts. E. coli w a s selected as th e s o u r c e b e c a u s e it had been established that its MDH had a dimeric s truct ur e (Murphey et al., 1967b). Methods Purification of E. coli malate dehydrogenase S t e p 1: Ten pounds of f r o z e n E. c o l i K12 c e l l s , grown in e n r i c h e d m e d i u m and h a r v e s t e d at t h r e e - f o u r t h s log phase, were thawed in 2.5 vol. of 0.02 M potassium phosphate, pH 7.0, containing 1 mM EDTA, 1 mM o - p h e n a n t h r o l i n e (O P), and 10 mM 2-m ercapt oethanol (2-ME) and were d i s r u p t e d by a single p a s s a g e t h r o u g h a M o u t o n - G a u l i n submicron disperser, chilled in ice, at 9000 psi. The homogenate was centrifuged for 60 rain at i0 000 g and t h e s u p e r natant brought to 45% saturation by the addition of solid (NHd)2SO 4. The precipitate was removed by centrifugation at 10 000 g for 30 rain a n d th e s u p e r n a t a n t r a i s e d to 7 5 % (NHd)~SO 4 s a t u r a t i o n . The precipitate was removed by centrifugation at 10 000 9' for t~5 rain and d i s s o l v e d in 10 mM T r i s , pH 7.5, 1 mM in EDTA, OP, 2-ME, and phenylmethylsulfonylfluoride. It was dialyzed against three changes of the same buffer. S t e p 2: The dialyzed sample was applied to a column (50 x 6 cm) of DEAE-cellulose (Whatman DE-52) equilibrated in 0.01 M Tris, pH 7.5, with i mM 2-ME and i mM EDTA. As shown in Fig. I, the column was washed with starting buffer until t h e - ~ 8 0 was < 0 . I and was then eluted with a 2-liter linear gradient from 0 - 0 . i M KCI in the same buffer. E. coli AND EUKARYOTIC MALATE DEHYDROGENASE lt99 Fig. i. Elution profile of the (NH4)2SO 4 precipitate of E. ooii malate dehydrogenase on a column (50 x 6 cm) of DE-52 cellulose. The columm, equilibrated in 0.01 M Tris, pH 7.5, c o n t a i n i n g 1 m M 2 - m e r c a p t o e t h a n o l and EDTA, was w a s h e d with starting buffer until the A280 was below 0.I (arrow). The enzyme was eluted with a 2-1iter linear gradient composed of the equilibrating buffer alone and with 0.i M KCI. The c o l u m n was d e v e l o p e d at ~50 m l / h and the active fractions pooled as indicated. Step 3: The a c t i v e f r a c t i o n s from the DE-52 column were pooled, dialyzed against l0 mM Tris, pH 8.0, containing 1 mM 2-ME, EDTA, a n d 0 . 0 5 % N a N a , and l o a d e d o n t o a c o l u m n (20 x 2.4 c m ) of 5 ! - A M P - S e p h a r o s e ( S i g m a ) e q u i l i b r a t e d in t h e s a m e b u f f e r . The column was washed with b u f f e r until the ~ o was <0.05. The e n z y m e w a s Muted as a sharp peak by the same b u f f e r containing 0.2 M NaCI (Fig. 2). ! I 400 08 T I I L O.6 ,i >F- 200 > U < 0.4 >N z 0.2 00 50 I00 FRACTION NUMBER 150 2000 Fig. 2. Elution profile of the fractionation of the DE-52 pool on a column (20 x 2.4 cm) of 5 ' - A M P Sepharose. The column was equilibrated with 0.01 M Tris, pH 8.0, containing i mM 2-mercaptoethanol and EDTA and 0.05% NaN 3. The column was washed until the A280 was below 0.05 (arrow) and then the enzyme was eluted directly with the same buffer containing 0.2 M NaCI. The active fractions were pooled as indicated. 500 FERNLEY ET AL. i I 0.6 I! I ....... I I II I! I I 1200 I I I ! 900 E o 6O0 ~ 03 3o0 z ~ 0 FRACTION NUMBER Fig. 3. Elution profile of the fractionation of E. coli malate dehydrogenase on a column (115 x 2.4 cm) of Sephacryl-200. The column was equilibrated in the same buffer as the 5'-AMP-Sepharose column (Fig. 2), and developed at 18 ml/h. The active fractions were pooled. Step 4: The e n z y m e eluted from the a f f i n i t y column was pooled and applied d i r e c t l y to a column (115 x 2.4 c m ) of S e p h a c r y l - 2 0 0 equilibrated in the same buffer as the 5 ' - A M P - S e p h a r o s e column. As shown in Fig. 3, all of the a c t i v i t y applied was found in the second (major) peak eluted. T h e s e f r a c t i o n s w e r e pooled and stored at -20oC. Enzyme activity and protein concentration E. coli m a l a t e d e h y d r o g e n a s e was assayed with 0.1 M m a l a t e and 2.5 mM NAD + as described previously ( G l a t t h a a r et al., 1974). Units were defined as pmol NADH/min a n d _ s p e c i f i c a c t i v i t y as units/mg of e n z y m e . An e x t i n c t i o n c o e f f i c i e n t , E ~ o n m = 1.73~ was d e t e r m i n e d and used for e s t i m a t i n g protein c o n c e n t r a t i o n s . Gel electrophoresis P u r i t y and subunit molecular weight d e t e r m i n a t i o n s were made on 15% p o l y a c r y l a m i d e gels in 0.1% sodium dodecyl s u l f a t e (SDS) run at 20 mA f o r 3 - 4 h as described by L a e m m l i (1970). ~Polyacrylamide e l e c t r o p h o r e s i s in 12.5% gels in the absence of SDS was also used to assay h o m o g e n e i t y . E. coli AND EUKARYOTIC MALATE DEHYDROGENASE 501 Amino acid analyses The amino acid composition was determined by aut om at i c analysis with a Durrum D-500 instrument in acid (6 N HCI) h y d r o l y s a t e s of 24, 4g, and 72 h p r e p a r e d a t 110~ under reduced pressure. Half-cystine was d e t e r m i n e d as c y s t e i c aci d a f t e r p e r f o r m i c aci d o x i d a t i o n ( M o o r e , 1963), and t r y p t o p h a n was quantitated spectrophotometrically (Edelhoch, 1967). Sequence analysis A m i n o - t e r m i n a l s e q u e n c e a n a l y s i s was performed in a Beckman g90c sequencer using the 0.33 M Quadrol program, a modification of t h e 0.1 M Quadrol program described by Brauer et al. (1975). All solvents and reagents w e r e obtained from Beckman. Polybrene, which was a d d e d to t h e spinning cup with t h e p r o t e i n sample to avoid e x t r a c t i v e losses, was a gift from Abbott Laboratories. The phenylthiazolinones obtained were converted to the corresponding hydantoins by heating at g 0 ~ in 1.0 N HC1 for 10 min and were identified by t h i n - l a y e r chromatography, gas chromatography, and high-performance liquid chromatography. The last two methods p r o v i d e d q u a n t i t a t i v e e s t i m a t e s (Thomas et al., 19gl). The enzyme was c a r b o x y m e t h y l a t e d a f t e r mild reduction (Angeletti et al., 1971) before being analyzed. Results The E. coli malate dehydrogenase obtained by the procedure described herein was judged to be homogeneous by gel etectrophoresis in the presence and in the absence of SDS. At a load sample of ,el00 IJg, only a very minor band comprising less than 1% of the t o t a l protein was evident in addition to the very intense main band, in the non-denaturing analysis. No contaminants were visible on the SDS gel at the same c o n c e n t r a t i o n . In the latter experiment, the E. c o l i protein migrated very slightly ahead of porcine heart mMDH (M r = 33 0g0; R. T. F e r n l e y , B. E. G l a t t h a a r , M. R. Sutton, and R. A. Bradshaw, manuscript in preparation) consistent with the Mr = 32 500 c a l c u l a t e d f r o m t he am i no aci d analyses (see below). The native protein has been reported by Murphey et al. (1967b) to be a dimer of i d e n t i c a l subunits with a Mr = 61 000. The elution position on gel filtration columns and the subunit molecular weight d e t e r m i n e d i r o m SDS-gel and a m i n o acid a n a l y s e s of t h e e n z y m e i s o l a t e d in the e x p e r i m e n t s r e p o r t e d h e r e s u g g e s t a M r -- 65 000, in e x c e l l e n t ag r eemen t with the earlier value. The amino acid composition of B. c o l i MDH is shown in Table 1. The values shown are for the subunit polypeptide. The 314 residues p r o d u c e a c a l c u l a t e d m o l e c u l a r w e i g h t of 32 500 and an average residue weight of 104, reflecting the r e l a t i v e e n r i c h m e n t of l o w e r molecular-weight amino acids, e.g. glycine, alanine, etc. The enzyme is also distinguished by t h e low t y r o s i n e (3) and t r y p t o p h a n (0) content which is r e f l e c t e d in the E~sZnm - 1.73 determined. 502 FERNLEY ET AL. Table i. Amino acid composition of E. co!i malate dehydrogenase a Porcine E. coli Aspartic acid Threonine Serine "Glutamic acid Proline Glycine Alanine Valine Half-cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Eistidine Lysine Arginine Tryptophan TOTAL 24 h 48 h 72 h 23.6 17.4 16.9 37.0 8.5 37.0 36.0 30.8 2.9 3.9 12.7 33.6 2.8 i0.0 2.1 20.7 8.6 0 24.3 24.2 18.0 17.7 16.1 16.6 37.5 37.8 i0.i 9.5 37.6 37.5 37.8 37.3 34.0 33.4 . . . . 3.8 3.2 14.7 14.5 34.2 34.2 3.5 3.4 I0.0 i0.0 2.2 2.2 21.8 21.9 8.0 7.9 . . . . Average Integer mMDH b sMDH c 24.0 17.7 17.2 37.4 9.4 37.4 37.0 33.7 2.9 3.6 14.6 34.0 3.2 I0.0 2.2 21.5 8.2 . . 24 18 17 37 9 37 37 34 3 4 15 34 3 I0 2 22 8 . . 24 21 18 24 21 28 32 28 8 6 23 27 5 ii 5 25 8 39 16 22 27 12 23 32 26 5 8 19 32 8 ii 4 31 i0 5 314 330 . 314 . aResidues/molecule of subunit bFrom complete amino acid sequence (R~ Fernley, B. E. glatthaar, M. R. Sutton, and R. A. Bradshaw, in preparation), mMDH~ mitochondrial malate dehydrogenase. CFrom acid hydrolysates (Banaszak & Bradshaw, 1975). sMDH, cytoplasmic malate dehydrogenase. The a m i n o - t e r m i n a l sequence of the first 36 residues of E. c o l i MDH, determined by automatic Edman degradation, is shown in Table 2. Unambiguous assignments were made for all positions except 26, 28, and 31, which were preliminarily determined to be serine residues by gas-liquid c h r o m a t o g r a p h y . H o w e v e r , the very low levels of dehydroserine observed did not allow more than t e n t a t i v e i d e n t i f i c a tion. Discussion The protocol for the purification of E. c o l i MDH is similar to that d e s c r i b e d by Murphey et el. (1967b) in the use oi ( N H 4 ) 2 5 0 4 p r e c i p i t a t i o n , DEAE c e l l u l o s e c h r o m a t o g r a p h y , and gel filtration. However, the use of affinity chromatography on 5'-AMP-Sepharose, as applied by Weininger and Banaszak (197g) to the isolation of porcine h e a r t s- and mMDH, and the s u b s t i t u t i o n of S e p h a c r y l - 2 0 0 f o r 5 e p h a d e x G-100 e l i m i n a t e d the need ior crystallization as the final purification step. The same affinity c h r o m a t o g r a p h y s t e p has also been e f f e c t i v e l y applied by Wright and S u n d a r a m (1979) to the purification of malate dehydrogenases from a number of thermophilic and mesophilic bacteria. The overall yield of enzyme from 10 lb of E. coZi AND Table 2. 503 Amino terminal sequence of E. co2i malate dehydrogenase Cycle Residue 1 2 3 4 5 6 7 8 9 i0 ii 12 13 14 15 16 17 18 E U K A R Y O T I C MALATE DEHYDROGENASE Method of identification a Methionine Lysine Valine Alanine Valine Leucine Glycine Alanine Alanine Glycine Glycine Isoleucine Glycine Glutamine Alanine Leucine Alanine Leucine T/G T/G T/G T/G T/G T/G/H T/G T/G T/G T/G T/G T/G/H T/G T/G T/G T/G/H T/G T/G/H Cycle 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Residue Method of identification a Leucine Leucine Lysine Threonine Glutamine Leucine Proline (Serine) Glyeine (Serine) Glutamic acid Leucine (Serine) Leucine Tyrosine Aspartic acid Isoleucine Alanine T/G/H T/G/H T/G T/G T/G T/G/H T/G T/G T/G G T/G T/G/H G T/G/H T/G G G/H G aAbbreviations: T~ thin-layer (silica-gel) chromatography; G, gas-liquid chromatography; H~ high-performance liquid chromatography. f r o z e n cells was ~'100 mg (sp. act. = 350 units/rag), which r e p r e s e n t s about 5% of t h e a c t i v i t y r e c o v e r e d f r o m t h e a m m o n i u m s u l f a t e precipitation. This p e r c e n t yield is c o m p a r a b l e to the value of 6% r e p o r t e d by Murphey et al. (1967b). However~ these workers s t a r t e d w i t h t w i c e as m a n y c e l l s and r e c o v e r e d o n l y o n e - f o u r t h as much enzyme. This c a n be e x p l a i n e d ~ in p a r t y by t h e f ~ c t t h a t t h e y o v e r e s t i m a t e d the e x t i n c t i o n coefficient~ r e p o r t e d as E28n = 3.39~ by a f a c t o r of 2. This may have been due to some c o n t a m i n a t i n g protein in t h e i r preparation s i n c e t h e y f o u n d 1.75 r e s i d u e s of t r y p t o phan/subunit and the MDH~ as isolated in this study~ was d e v o i d of this residue. This may also r e f l e c t the i n a c c u r a c i e s in the methods available for d e t e r m i n i n g both the t r y p t o p h a n c o n t e n t and e x t i n c t i o n c o e f f i c i e n t s in the earlier studies. The r e m a i n d e r of their composition is in reasonable a g r e e m e n t with t h a t r e p o r t e d here. The comparison of the E. c o Z i MDH composition with t h a t of the two e u k a r y o t i c MDHs of p o r c i n e h e a r t is g i v e n in T a b l e 1. The composition of the mitochondrial isozyme~ c a l c u l a t e d from the p r i m a r y st[ucture9 is also c h a r a c t e r i z e d by t h e a b s e n c e of t r y p t o p h a n b u t d i f f e r s m a r k e d l y in g l u t a m i c acid~ prolin% cysteine~ and isoleucine. Similar deviations with the c y t o p l a s m i c f o r m a r e also e v i d e n t . In fact~ t h e r e is l i t t l e compelling reason~ on the basis of this evidence~ to e x p e c t t h a t the p r o k a r y o t i c e n z y m e would be more similar to one or the other of the e u k a r y o t i c MDHs~ if in f a c t it is similar to e i t h e r . However~ as shown in Fig. tt~ an e n t i r e l y d i f f e r e n t p i c t u r e e m e r g e s f r o m a c o m p a r i s o n of t h e a m i n o - t e r m i n a l sequences of these t h r e e MDHs. Clearly~ E. c o 2 i MDH shares an e x t e n s i v e number of identical residues with the mitochondrial i s o z y m e t h a t is considerably reduced in 50~ FERNLEY ET AL. E. coli I.iDH NE 2-M[K-V-A-V-L-G-A-A-G-G- porcine mMDH NH2-At K-V-A-V-L-G-A~G-G- porcine sMDH Ac-(S ,Z, P)- I - - ~ ~ G - A - A - G ~ E. coli }[DH porcine mMDH porcine sMDH A-Y.-s>_b->s->c->G E. coli }UJH porcine mMD.H porcine sMDH Fig. 4. Comparison of the amino-terminal sequences of E. co2i porcine heart mitochondrial (mMDH) and p o r c i n e h e a r t cytoplasmic malate dehydrogenase (sMDH). Residues identical in at least two of the segments are enclosed in boxes. Data taken from (mMDH) R. T. Fernley, B. E. Glatthaar, M. R. Sutton, and R. A. Bradshaw (manuscript in preparation) and (sMDH) R. A. Bradshaw, M. J. Wade, B. E. Glatthaar~ G. R. Barbarash, and M~ R. Sutton (unpublished observations). a comparison with the cytoplasmic form. F u r t h e r m o r e , both the p r o k a r y o t i c and mitochondrial MDHs c o m m e n c e at the s a m e p o s i t i o n and possess f r e e m-amino groups whereas the c y t o p l a s m i c e n z y m e has t h r e e additional residues and an NCZ-acetyl group. As s u m m a r i z e d in Table 3, E. c o l i and porcine mMDH share 25/36 positions ( 6 9 % ) in c o n t r a s t to the 9/33 i d e n t i t i e s c h a r a c t e r i z i n g t h e E. c o i l ~ p o r c i n e sMDH comparison. Interestingly, the two e u k a r y o t i c isozymes show a similar r e l a t e d n e s s (8/33, or 2Lt%). Although the q u a n t i t a t i v e a s p e c t s shown in Table 3 a r e derived from s e g m e n t s t h a t r e p r e s e n t only about I0% of each polypeptide and t h e r e f o r e c o u l d v a r y s o m e w h a t f r o m t h o s e c a l c u l a t e d f r o m a comparison of the c o m p l e t e sequences, the two main conclusions, i.e. 1) t h a t all t h r e e proteins s h o w s u f f i c i e n t r e l a t e d n e s s to suggest a c o m m o n a n c e s t r a l precursor and 2) t h a t the p r o k a r y o t i c form of the e n z y m e is much more s i m i l a r t o t h e m i t o c h o n d r i a l i s o z y m e t h a n to t h e c y t o p l a s m i c one, are unlikely to be materially altered. E. coli AND EUKARYOTIC MALATE DEHYDROGENASE 505 Table 3. Comparison of the amino terminal sequences of the malate dehydrogenases of E. c o l i and porcine heart The lower left side of the matrix shows the number of identical residues per total positions compared. The extra three amino terminal residues of sMDH have not been included. The deletion introduced in the mMDH sequence corresponding to residue 22 in the E. c o l i protein has been treated as a non-identity. The values listed in the upper right side express the ratios as percentages. E. E. Porcine coli Mitochondrial Cytoplasmic -- 69 27 Mitochondrial 25/36 -- 24 Cytoplasmic 9/33 8/33 -- coli Porcine The relationship ~ observed between the bacterial and animal MDHs is very similar to that previously reported for superoxide d i s m u t a s e s ( S t e i n m a n & Hill, 1973). In that study, comparison of the aminoterminal structures of two forms (Fe and Mn) from E. c o l i with the c h i c k e n l i v e r m i t o c h o n d r i a l e n z y m e (Mn) showed t h a t 20 of 27 residues of the mitochondrial e nz ym e segment were identical to one or another of the two bacterial dismutases. The two E. c o l i enzymes were also r e l a t e d to e a c h o t h e r to a b o u t t h e s a m e e x t e n t . In contrast, the bovine e r y t h r o c y t e superoxide dismutase, which contains Cu and Zn ions, did not show significant homology with any of t h e b a c t e r i a l or c h i c k e n e n z y m e s . B r i d g e n et al. (1975), from their sequence determination of the first 60 residues of B. s e e a r o t h e r m o p h i l u s superoxide dismutase (Mn), found a similar relationship to the same eukaryotic enzymes. As noted by Steinman and Hill (1973), the striking homology of the prokaryotic and m i t o c h o n d r i a l f o r m s of t h e s a m e e n z y m e c l e a r l y s u g g e s t s a c o m m o n a n c e s t o r and appears to substantiate the endosymbiotic theory for the origin of mitochondria and chloroplasts. This theory proposes that these organelles arose from the internalization of specific prokaryotic ceils by p r o t o e u k a r y o t e s , s u r v i v i n g i n i t i a l l y as i n t r a c e l l u l a r s y m b i o n t s and ultimately, through evolutionary change, adopting their present-day s t r uc t ur e and function (Margulis, 1970). An important aspect of this hypothesis requires that much of the genome of the i n t e r n a l i z e d p r o k a r y o t e be t r a n s f e r r e d t h r o u g h s u b s e q u e n t evolutionary events to the nucleus, which now directs the synthesis of most mitochondrial proteins. The principal opposing t h e o r y s u g g e s t s that th er e was a continuous development of eukaryotes from prokaryores with the autogenic production of all intracellular o r g a n d i e s (Raff & M a h l e r , 1972; U z z e l l & Spolsky, 197#). Support for the endosymbiotic hypothesis has been drawn from morphological and functional s i m i l a r i t i e s b e t w e e n m i t o c h o n d r i a / c h l o r o p l a s t s and prokaryotes and, more r e c e n t l y , f r o m t h e e x t r a o r d i n a r y s i m i l a r i t y in t h e p r i m a r y s t r u c t u r e s of n u c l e i c acid e l e m e n t s of t h e o r g a n i s m s / o r g a n e l l e s 506 FERNLEY ET AL, (Schwarz & KBssel, 1980; Phillips & Carr, 19gl). However, Anderson et al. (1981), who have determined t h e c o m p l e t e s e q u e n c e of t h e h u man m i t o c h o n d r i a l g e n o m e , h a v e suggested that 'the mammalian mitochondrial genetic system cannot generally be classified as e i t h e r p r o k a r y o t e - l i k e or eukaryote.like' because of the distinct differences f o u n d in this g e n o m e (and its t r a n s l a t i o n ) and all o t h e r l i v i n g organisms studied. Also Uzzell and Spolsky (19gl) have argued that much of the structural data, used to c o n s t r u c t p h y l o g e n e t i c t r e e s ( S c h w a r t z & D a y h o f f , 1978), that appear to strongly support endosymbiosis can be used equally well to support autogenesis. The protein sequence data for the MDHs presented here do not, as with the results of Steinman and Hill (1973), resolve the mitochondrial origin controversy. However, they extend that study in that sMDH, as an evolutionary homolog of both the p r o k a r y o t i c and m i t o c h o n d r i a l f o r m s , p r o v i d e s an ' i n t e r n a l evolutionary control' that is not complicated by either species or tissue variation. Except for the unlikely possibility that E. co2~" and porcine mitochondrial MDH evolved in a parallel fashion a f t e r t h e f o r m a t i o n of e u k a r y o t i c cel l s and in a d i s t i n c t l y d i f f e r e n t manner than the porcine cytoplasmic enzyme, it must be concluded that the bacterial and m i t o c h o n d r i a l f o r m s m o r e c l o s e l y r e s e m b l e t he a n c e s t r a l precursor, and that the cytoplasmic enzyme, for whatever reason, has u n d e r g o n e much m o r e e x t e n s i v e mutational change. Thus, t h e mitochondrial and bacterial enzymes have retained their high d e g r e e of s i m i l a r i t y o v e r t he s a m e t i m e p e r i o d t h a t a n o t h e r ' s i b l i n g ' of the original ancestor gene has not. These d e v e l o p m e n t s s e e m m o r e c o m p a t i b l e with an e n d o s y m b i o t i c pathway than an autogenic one. Acknowledgements This work was supported by U.S.P.H.S. research grant AM 13362. 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