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Bioscience Reports i, 4'97-502 (1981)
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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
'
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300
I
I
E
?~
200
<
100 z
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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.
S.R.L. was supported by National Research Service Award GM 07200,
Medical Scientist.
The authors wish to thank Dr. R. Ray Fall, Dept.
of Chemistry, University of Colorado, Boulder, Colorado, for supplying
a p o r t i o n of t he c e l l s used and Waiter Nulty for his assistance in
lysing them. The amino acid composition and sequence analyses were
p e r f o r m e d in t h e P r o t e i n C h e m i s t r y F a c i l i t y , D e p t . of Biological
Chemistry, Washington University, established in p a r t by a g r a n t to
R.A.B. f r o m t he National Science Foundation.
The authors wish to
thank Ms. Karen DeVries for her help in preparing the enzyme and Ms.
Solveig Storvick-Pollei for her advice and assistance in the preparation
of this manuscript.
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