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Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Isolation and Partial Characterization of a Cytochrome b Complex and Cytochrome Oxidase from Yeast Mitochondria Leo A. Marjanen
a
a
Department of Developmental Biology , Research School of Biological Sciences, Australian National University , Canberra, A. C. T., 2601, Australia Published online: 05 Dec 2006.
To cite this article: Leo A. Marjanen (1976) Isolation and Partial Characterization of a Cytochrome b Complex and Cytochrome Oxidase from Yeast Mitochondria, Preparative Biochemistry, 6:2-3, 153-175, DOI: 10.1080/00327487608061610 To link to this article: http://dx.doi.org/10.1080/00327487608061610
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PREPARATIVE BIOCHEMISTRY, 6 ( 2 & 3 ) , 153-175 (1976)
ISOLATION AND PARTIAL CHARACTERIZATION OF A CYTOCHROME b COMPLEX AND CYTOCHROME OXIDASE FROM YEAST MITOCHONDRIA
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Leo A. Marjanen Department o f Developmental B i o l o g y , Research School o f B i o l o g i c a l S c i e n c e s , A u s t r a l i a n N a t i o n a l U n i v e r s i t y , Canberra, A.C.T. 2601, A u s t r a l i a ABSTRACT A cytochrome b complex and cytochrome o x i d a s e
have been p u r i f i e d 1 4 - and 20-fold r e s p e c t i v e l y from y e a s t s u b m i t o c h o n d r i a l p a r t i c l e s by a s i m p l e p r o c e d u r e i n v o l v i n g t h e i r s p o n t a n e o u s p r e c i p i t a t i o n from a deoxycholate e x t r a c t .
The r e c o v e r y o f b o t h p r o t e i n s
was a l m o s t q u a n t i t a t i v e .
The s p e c i f i c heme c o n t e n t s
were 11 and 8 nmoles/mg p r o t e i n f o r t h e cytochrome b complex and cytochrome o x i d a s e r e s p e c t i v e l y and b o t h
were s p e c t r a l l y pure.
Sodium dodecyl s u l f a t e g e l
e l e c t r o p h o r e s i s r e s o l v e d t h e cytochrome b complex i n t o seven d i s t i n c t s u b u n i t s w i t h m o l e c u l a r w e i g h t s 42,000, 3 3 , 0 0 0 , 27,500,
23,000, 1 5 , 5 0 0 , 13,000 a n d 10,500.
Cytochrome o x i d a s e c o n t a i n e d f i v e bands w i t h m o l e c u l a r w e i g h t s 4 2 , 0 0 0 , 26,500, 21,000, 14,000 and 10,500.
153 Copyright 0 1976 hy Marel Dekker, Inc All Rights Reserved Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical. including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher
154
MARJANEN
Much of t h e cytochrome b complex (and a l l o f t h e cytochrome o x i d a s e ) could be r e s o l u b i l i z e d i n aqueous b u f f e r f o l l o w i n g p r e c i p i t a t i o n from t h e deoxycholate extract.
The f r a c t i o n o f t h e cytochrome b p r e p a r a t i o n
which remained i n s o l u b l e appeared i d e n t i c a l t o t h e Downloaded by [University of Waterloo] at 06:07 14 January 2015
s o l u b l e p r o t e i n i n terms o f p o l y p e p t i d e composition b u t c o n t a i n e d less phospholipid and bound d e t e r g e n t , s u g g e s t i n g t h a t i n s o l u b i l i t y may r e s u l t from i n t e r a c t i o n between hydrophobic r e g i o n s o t h e r w i s e occupied by amphiphiles. The s o l u b l e cytochrome b complex migrated a s a s i n g l e species upon a n a l y t i c a l u l t r a c e n t r i f u g a t i o n and column chromatography, and d u r i n g e l e c t r o p h o r e s i s on polyacrylamide g e l s .
T r i t o n X-100, urea, o r b i l e
s a l t s , f a i l e d t o d i s s o c i a t e t h e complex.
These
findings suggest t h a t t h e subunits a r e t i g h t l y associated i n s i t u . INTRODUCTION
Considerable i n t e r e s t i s c u r r e n t l y c e n t e r e d on t h e mechanism of m i t o c h o n d r i a l b i o g e n e s i s , p a r t i c u -
l a r l y t h e i d e n t i t y of p o l y p e p t i d e s s y n t h e s i z e d
by
t h e g e n e t i c system w i t h i n t h e o r g a n e l l e i t s e l f ( 1 , 2 ) . Where m i t o c h o n d r i a l DNA is a b s e n t , a s i n some cyto-
plasmic p e t i t e mutants of y e a s t , o r where t r a n s l a t i o n on mitochondrial ribosomes i s p r e v e n t e d w i t h chloram-
CYTOCHROME b FROM YEAST MITOCHONDRIA
155
phenicol, cytochrome b, cytochrome oxidase and the oligomycin-sensitive ATPase are no longer formed indicating that their biogenesis requires products of intramitochondrial protein synthesis.
To date, this
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has been clearly confirmed by labelling studies only in the case of cytochrome oxidase ( 3 , 4 ) and the oligomycin-sensitive ATPase (1). Mitochondria1 cytochrome b has not so far been obtained in a well characterized form from yeast, although the heme-containing polypeptide has recently been isolated from Neurospora and shown to be synthesized on mitochondria1 ribosomes ( 5 , 6 ) .
It is the
purpose of this paper to describe a method by which both a cytochrome b complex and cytochrome oxidase can be purified in high yields from yeast mitochondria, and to define the properties and subunit structure of the former sufficiently to make it a useful starting point for studies of cytochrome b biogenesis. MATERIALS AND METHODS Preparation of Submitochondrial Particles Saccharomyces cerevisiae (a locally isolated diploid wild type) was grown at 28OC in an aerobic medium containing 1.5% (w/v) galactose, 0 . 7 5 % yeast extract, 0.12% (NHI,)~SO,+,0.05% NaC1, 0.01% CaC12, 0.2% MgS04.7H20, 0.1% KH2P04 and 3 x
FeC13.
156
WANEN
Cells in the early stationary phase of growth were harvested by centrifugation, washed twice in cold water and once in 0.5 M sorbitol, 1mM EDTA and l O m M Tris-HC1, pH 7.4, then broken in the latter medium by
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treatment in a Braun homogenizer for 30 sec ( 7 ) .
The
homogenate was centrifuged three times at 1,000 xg for 5 min, then mitochondria were collected at 15,000 xg
for 10 min and washed once in 0.25 M sorbitol and lOmM Tris-HC1, pH 7 . 4 .
The pellet was resuspended in
the same medium at 10 mg protein/ml and sonicated for 30 sec using a Branson sonifier at an output of 7 amp.
Submitochondrial particles were sedimented at 100,000 xg for 30 min and washed once. Cytochrome Isolation All steps were carried out at OOC.
Submito-
chondrial particles were resuspended in 0.25
M
sorbitol and lOmM Tris-HC1, pH 7.4, at 20 mg protein/ml, then solid K C 1 followed by sodium deoxycholate (10% "/v, pH 7 - 8 ) were added slowly to the stirred solution to concentrations of 1 M and 0.15 mg detergent/mg protein respectively.
After stirring for
30 min the suspension was centrifuged at 100,000 xg
for 45 min and the supernatant, which contained a l l of the cytochrome c, was carefully removed. pellet was resuspended in 0 . 2 5
M
The
sorbitol and l O m M
15 7
CYTOCHROME b FROM YEAST MITOCHONDRIA Tris-HC1,
pH 7 . 0 ,
t h e n s o l i d K C 1 and 1 0 % sodium
deoxycholate were slowly added t o c o n c e n t r a t i o n s of 1 M and 0 . 5 mg detergent/mg p r o t e i n r e s p e c t i v e l y .
The
s o l u t i o n w a s s t i r r e d f o r 30 min t h e n c e n t r i f u g e d a t
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1 0 0 , 0 0 0 xg f o r 30 min t o o b t a i n a clear brown super-
n a t a n t which c o n t a i n e d almost a l l of t h e remaining cytochromes. The c l e a r s u p e r n a t a n t w a s l e f t t o s t a n d overnight. T u r b i d i t y developed w i t h i n t h a t time and was removed by c e n t r i f u g a t i o n a t 4 0 , 0 0 0 xg f o r 1 5 min.
The f i r s t
p e l l e t was normally d i s c a r d e d s i n c e it c o n t a i n e d deoxycholic a c i d w i t h l i t t l e cytochrome.
Centrifug-
a t i o n was r e p e a t e d t h e r e a f t e r whenever s l i g h t t u r b i d i t y developed (ncrmally every 2-4 h ) .
The p e l l e t s ,
which c o n t a i n e d s p e c t r a l l y p u r e cytochrome b t o g e t h e r w i t h v a r y i n g amounts of deoxycholic a c i d , were r e s u s pended i n 1 0 mM T r i s - H C 1 ,
pH 7 . 4 .
The cytochrome
should be removed f r e q u e n t l y as a g g r e g a t i o n c o n t i n u e s s i n c e an i n c r e a s e d p r o p o r t i o n could n o t be resolubili z e d i n aqueous b u f f e r s (see below) when t h e p r e c i p i t a t e d p r o t e i n was l e f t i n c o n t a c t w i t h t h e deoxycholate e x t r a c t . P r e c i p i t a t i o n o f t h e cytochrome b complex w a s complete w i t h i n 4 8 h and was followed by a p r o g r e s s i v e a g g r e g a t i o n of cytochrome o x i d a s e .
The f i r s t c y t o -
chrome o x i d a s e f r a c t i o n ( s ) may show some c o n t a m i n a t i o n
158
W A N E N
by cytochrome b and can b e d i s c a r d e d .
Much of t h e c y t o -
chrome o x i d a s e (60-80%) w a s c o l l e c t e d w i t h i n t h e following 48 h b u t up t o 5 days w a s r e q u i r e d f o r complete p r e c i p i t a t i o n .
The f i n a l s u p e r n a t a n t c o n t a i n e d
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s p e c t r a l l y pure cytochrome
c1.
S p e c t r a 1 Ana 1y sis S p e c t r a were o b t a i n e d e i t h e r a t room o r l i q u i d n i t r o g e n temperature u s i n g a Cary 1 4 r e c o r d i n g s p e c t r o photometer.
Absolute s p e c t r a were recorded a g a i n s t
e q u a l c o n c e n t r a t i o n s of bovine serum albumin.
Heme
c o n t e n t s were c a l c u l a t e d from t h e a b s o r p t i o n d i f f e r e n c e
a t t h e a-peak maximum between a reduced and an o x i d i z e d sample.
The following m i l l i m o l a r e x t i n c t i o n coef f i -
c i e n t s were used: chrome a , 1 0 . 4 10).
cytochrome b , 13.2 ( r e f . 8 1 , cyto-
( r e f . 9) and cytochrome
cl,
17.1 ( r e f .
The p y r i d i n e hemochromogen spectrum was o b t a i n e d
as d e s c r i b e d by Basford
et &.
(11).
Other methods Methods f o r t h e a s s a y of cytochrome o x i d a s e ( 1 2 1 , NADH
-
and s u c c i n a t e cytochrome c r e d u c t a s e ( 1 2 ) and
ATPase (13) have been d e s c r i b e d elsewhere.
The a s s a y s
were c a r r i e d o u t a t t h e f o u r t h day o f cytochrome precipitation. aa3
The p r e c i p i t a t e s o f cytochromes b and
were d i s s o l v e d immediately b e f o r e t h e a s s a y s were
performed.
Polyacrylamide g e l e l e c t r o p h o r e s i s i n t h e
presence of sodium dodecyl s u l f a t e h a s been p r e v i o u s l y
CYTOCHROME b FROM YEAST MITOCHONDRIA
described ( 1 4 ) .
159
Densitometer t r a c i n g s o f t h e s t a i n e d
g e l s were o b t a i n e d using t h e scanning a t t a c h m e n t o f a G i l f o r d r e c o r d i n g spectrophotometer. P r o t e i n w a s e s t i m a t e d according t o Lowry
et &.
(151, using bovine serum albumin a s t h e s t a n d a r d .
Phos-
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p h o l i p i d w a s measured a f t e r a s h i n g o f t h e samples by t h e method o f Chen e t a l .
(16).
Materials ' C h o l i c and deoxycholic a c i d (Sigma) were r e c r y s t a l l i z e d from 5 0 % e t h a n o l b e f o r e use.
[14C]deoxycholic
a c i d ( s p e c i f i c a c t i v i t y 2 . 9 8 mCi/mmole) w a s o b t a i n e d from M a l l i n c k r o d t . RESULTS S o l u b i l i z a t i o n and P u r i f i c a t i o n Deoxycholate a t a c o n c e n t r a t i o n of 0.5 mg d e t e r gent/mg mitochondria1 p r o t e i n s o l u b i l i z e d a major propo r t i o n (>80%) of t h e cytochromes a a 3 , b , and c
but a
c o n s i d e r a b l y smaller amount ( ~ 3 0 % )o f t h e t o t a l p r o t e i n (Table 1 ) . The r e s u l t i n g p u r i f i c a t i o n , measured as t h e s p e c i f i c heme c o n t e n t , was 3- t o 4-fold f o r each cytochrome.
The slow, spontaneous p r e c i p i t a t i o n o f t h e
cytochrome b complex, followed by cytochrome aa3, y i e l d e d very high r e c o v e r i e s and f i n a l heme p u r i f i c a t i o n s of 1 4 - and 20-fold r e s p e c t i v e l y compared t o t h e p a r e n t membranes.
Autoxidation of t h e p r o t e i n s w a s
a p p a r e n t l y i n s i g n i f i c a n t s i n c e p r e p a r a t i o n s under a i r
200
280
640
490
310
130
320
440*
Total heme a c1 nmoles
700
b
11.2
2.8
0.8
8.1
1.3
0.4
2.3
1.4
0.5*
Specific heme b a C1 nmo 1e s/mg protein
30.6
0.04
1.2
pmo les/mi n/mg protein
Specific activity**
**Cytochrome c oxidase activity; soybean phospholipids.
assayed in the presence of 2 mg/ml of sonicated
*These values are only approximate owing to spectral interference by cytochrome c.
Purification of the cytochromes from yeast mitochondria. Details of the method of purification are given in the text. The values for cytochrome b and cytochrome oxidase are the sum of the spectrally pure cytochrome precipitates which were collected in this experiment. Following complete precipitation of these cytochromes, the cytochrome c 1 supernatant was dialysed against 0.1 M then 0.01 M NaPi, pH 7.4, cleared by centrifugation at 40,000 xg for 15 min and concentrated by Amicon pressure filtration using a PM 10 membrane. The concentrate was again centrifuged before the spectrum was recorded.
Total protein mg Submitochondrial particles 860 Solubilized cytochromes 225 Cytochrome b precipitats 44 Cytochrome aa precipitate 25 Cytochrome c 1 supernatant 57
Fraction
TABLE 1
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rr:
% M
161
CYTOCHROME b FROM YEAST MITOCHONDRLA
o r n i t r o g e n had i d e n t i c a l s u b u n i t p a t t e r n s on sodium dodecyl s u l f a t e g e l s .
Cytochrome c o x i d a s e a c t i v i t y
i n c r e a s e d 25-fold d u r i n g t h e p u r i f i c a t i o n o f cytochrome a a 3 whereas t h e cytochrome b complex c o n t a i n e d a l m o s t no cytochrome c o x i d a s e a c t i v i t y (Table 1);
these values
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may be underestimated s i n c e t h e a c t i v i t y ( b u t n o t t h e s p e c i f i c heme c o n t e n t ) d e c r e a s e d i n t h e d e o x y c h o l a t e e x t r a c t w i t h a h a l f - t i m e o f 50 h.
Neither preparation
c o n t a i n e d any measurable A T P a s e o r NADH- o r s u c c i n a t e cytochrome c r e d u c t a s e a c t i v i t i e s . Cytochromes b ,
c1
and a were p r e s e n t i n t h e deoxy-
c h o l a t e e x t r a c t a t a r a t i o of approximately 2:l:l (Table 1 ) . The s o l u b i l i z a t i o n o f
cytochrome b and
c1
may occur a s a b i n a r y complex (as a p p a r e n t l y o c c u r s i n
bovine mitochondria (8,17,19)) s i n c e t h e r a t i o remained near 2 : l even a t deoxycholate c o n c e n t r a t i o n s where o n l y p a r t i a l s o l u b i l i z a t i o n of t h e s e cytochromes o c c u r r e d . The h i g h e r p r o p o r t i o n of cytochrome b i s n o t a b l e s i n c e
a t l e a s t two f u n c t i o n a l forms a r e now r e c o g n i s e d i n mitochondria1 membranes (20). The " s o l u b l e " form o f t h e cytochrome b complex (see below) could n o t be f u r t h e r p u r i f i e d e i t h e r by
g l y c e r o l d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o r column chromatography on Sepharose 4B ( b o t h i n t h e p r e s e n c e of cholate).
A s i n g l e major peak
was observed by t h e s e
methods, and by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n ( F i g u r e
MARJANEN
162
lA), i n d i c a t i n g t h a t the s o l u b l e p r e p a r a t i o n may e x i s t as a s i n g l e p r o t e i n complex.
Cytochrome o x i d a s e l i k e -
w i s e migrated as a s i n g l e peak upon u l t r a c e n t r i f u g a t i o n (Figure 1 B ) .
As shown by polyacrylamide g e l e l e c t r o -
p h o r e s i s , the cytochrome b complex c o u l d n o t be f u r t h e r Downloaded by [University of Waterloo] at 06:07 14 January 2015
d i s a g g r e g a t e d w i t h e i t h e r 1%T r i t o n X-100 or 8 M u r e a ,
A
FIGURE 1 A n a l y t i c a l u l t r a c e n t r i f u g a t i o n of t h e cytochrome b complex and cytochrome o x i d a s e . The cytochromes were prepared as d e s c r i b e d i n t h e t e x t t h e n c e n t r i f u g e d a t 1 0 0 , 0 0 0 xg f o r 6 0 min t o remove small amounts of aggregated material and a d j u s t e d t o 4 mg protein/rnl Sedimentation a n a l y s i s i n 1 0 0 mM T r i s - H C 1 , pH 7 . 4 . was performed a t 22OC and a t 60,000 rpm u s i n g t h e s i n g l e s e c t o r c e l l of a Beckman model E a n a l y t i c a l u l t r a c e n t r i f u g e . Photographs were t a k e n a t 1 9 6 , 2 6 0 , 3 2 4 and 4 5 2 min (cytochrome b complex ( A ) ) o r 13, 3 0 , 94 and 313 min (cytochrome o x i d a s e (B)) a f t e r r e a c h i n g maximum speed. The d i r e c t i o n o f s e d i m e n t a t i o n i s from r i g h t to l e f t .
CYTOCHROME b FROM YEAST MITOCHONDRIA
163
o r w i t h 1%sodium d e o x y c h o l a t e o r 2 % sodium c h o l a t e . Phenolacetic acid-urea
( 2 1 1 , sodium dodecyl s u l f a t e and
t h e c a t i o n i c d e t e r g e n t cetyltrimethylammonium bromide caused d i s s o c i a t i o n b u t w i t h a l o s s of t h e heme absorption. Downloaded by [University of Waterloo] at 06:07 14 January 2015
Spectral Analysis The reduced cytochrome b complex e x h i b i t e d a , B and
y
bands a t 562, 532 and 430 nm i n b o t h d i f f e r e n c e
( F i g u r e 2 ) and a b s o l u t e ( F i g u r e 3A) s p e c t r a .
A t -196°C
t h e peaks s h i f t e d t o 559, 529 and 429 nm ( F i g u r e 3 B ) . A s e x p e c t e d , t h e p y r i d i n e hemochromogen spectrum corres-
ponded t o t h e p y r i d i n e d e r i v a t i v e of f e r r o p r o t o p h o r p h y r i n I X ( F i g u r e 3C).
No c o n t a m i n a t i o n by t h e o t h e r c y t o -
chromes c o u l d b e d e t e c t e d e i t h e r a t 20°C o r -196"C,
or
by a p p l i c a t i o n of t h e d i f f e r e n t i a l heme method ( 9 ) . Cytochrome a a 3 e x h i b i t e d a and
nm r e s p e c t i v e l y ( F i g u r e 2 ) .
y
bands a t 6 0 4 and 445
The cytochrome c1 which
remained i n t h e s u p e r n a t a n t a f t e r p r e c i p i t a t i o n o f t h e o t h e r cytochromes was s p e c t r a l l y p u r e ( F i g u r e 2 ) b u t of low s p e c i f i c heme c o n t e n t ( T a b l e 1 ) . Sodium Dodecyl S u l f a t e G e l E l e c t r o p h o r e s i s D i s s o c i a t i o n and e l e c t r o p h o r e s i s o f the cytochrome b complex on 1 0 % p o l y a c r y l a m i d e g e l s i n t h e p r e s e n c e of sodium dodecyl s u l f a t e r e v e a l e d s e v e n d i s t i n c t bands ( F i g u r e 4 ) two of which (V and V I ) were i n c o m p l e t e l y separated.
Molecular w e i g h t s of t h e bands, o b t a i n e d
MARJANEN
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164
CYTOCHROME b
CYTOCHROME c,
553
A
FIGURE 2 Room temperature difference spectra (dithionite reduced minus ferricyanide oxidized) of yeast submitochondria-ticles and purified cytochromes. The spectra were recorded in 100 mM Tris-HC1, pH 7.4, either at 2 mg protein/ml (submitochondrial particles) or 0.13-0.20 mg protein/ml (cytochromes) The baselines (i.e. oxidized minus oxidized spectra) have been omitted from the figure.
.
by calibrating the gels with standard proteins were: I, 42,000;
11, 33,000;
111, 27,500;
V, 15,500; VII 13,000; VII, 10,500.
IV, 23,000;
CYTOCHROME b FROM YEAST MITOCHONDRIA
165
A
CYTOCHROME b COMPLEX
A
I
I
I
I I
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I
I I I I I I I I I I
[!
0
A BSORPT 1 0N SPECTRA
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Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Isolation and Partial Characterization of a Cytochrome b Complex and Cytochrome Oxidase from Yeast Mitochondria Leo A. Marjanen
a
a
Department of Developmental Biology , Research School of Biological Sciences, Australian National University , Canberra, A. C. T., 2601, Australia Published online: 05 Dec 2006.
To cite this article: Leo A. Marjanen (1976) Isolation and Partial Characterization of a Cytochrome b Complex and Cytochrome Oxidase from Yeast Mitochondria, Preparative Biochemistry, 6:2-3, 153-175, DOI: 10.1080/00327487608061610 To link to this article: http://dx.doi.org/10.1080/00327487608061610
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PREPARATIVE BIOCHEMISTRY, 6 ( 2 & 3 ) , 153-175 (1976)
ISOLATION AND PARTIAL CHARACTERIZATION OF A CYTOCHROME b COMPLEX AND CYTOCHROME OXIDASE FROM YEAST MITOCHONDRIA
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Leo A. Marjanen Department o f Developmental B i o l o g y , Research School o f B i o l o g i c a l S c i e n c e s , A u s t r a l i a n N a t i o n a l U n i v e r s i t y , Canberra, A.C.T. 2601, A u s t r a l i a ABSTRACT A cytochrome b complex and cytochrome o x i d a s e
have been p u r i f i e d 1 4 - and 20-fold r e s p e c t i v e l y from y e a s t s u b m i t o c h o n d r i a l p a r t i c l e s by a s i m p l e p r o c e d u r e i n v o l v i n g t h e i r s p o n t a n e o u s p r e c i p i t a t i o n from a deoxycholate e x t r a c t .
The r e c o v e r y o f b o t h p r o t e i n s
was a l m o s t q u a n t i t a t i v e .
The s p e c i f i c heme c o n t e n t s
were 11 and 8 nmoles/mg p r o t e i n f o r t h e cytochrome b complex and cytochrome o x i d a s e r e s p e c t i v e l y and b o t h
were s p e c t r a l l y pure.
Sodium dodecyl s u l f a t e g e l
e l e c t r o p h o r e s i s r e s o l v e d t h e cytochrome b complex i n t o seven d i s t i n c t s u b u n i t s w i t h m o l e c u l a r w e i g h t s 42,000, 3 3 , 0 0 0 , 27,500,
23,000, 1 5 , 5 0 0 , 13,000 a n d 10,500.
Cytochrome o x i d a s e c o n t a i n e d f i v e bands w i t h m o l e c u l a r w e i g h t s 4 2 , 0 0 0 , 26,500, 21,000, 14,000 and 10,500.
153 Copyright 0 1976 hy Marel Dekker, Inc All Rights Reserved Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical. including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher
154
MARJANEN
Much of t h e cytochrome b complex (and a l l o f t h e cytochrome o x i d a s e ) could be r e s o l u b i l i z e d i n aqueous b u f f e r f o l l o w i n g p r e c i p i t a t i o n from t h e deoxycholate extract.
The f r a c t i o n o f t h e cytochrome b p r e p a r a t i o n
which remained i n s o l u b l e appeared i d e n t i c a l t o t h e Downloaded by [University of Waterloo] at 06:07 14 January 2015
s o l u b l e p r o t e i n i n terms o f p o l y p e p t i d e composition b u t c o n t a i n e d less phospholipid and bound d e t e r g e n t , s u g g e s t i n g t h a t i n s o l u b i l i t y may r e s u l t from i n t e r a c t i o n between hydrophobic r e g i o n s o t h e r w i s e occupied by amphiphiles. The s o l u b l e cytochrome b complex migrated a s a s i n g l e species upon a n a l y t i c a l u l t r a c e n t r i f u g a t i o n and column chromatography, and d u r i n g e l e c t r o p h o r e s i s on polyacrylamide g e l s .
T r i t o n X-100, urea, o r b i l e
s a l t s , f a i l e d t o d i s s o c i a t e t h e complex.
These
findings suggest t h a t t h e subunits a r e t i g h t l y associated i n s i t u . INTRODUCTION
Considerable i n t e r e s t i s c u r r e n t l y c e n t e r e d on t h e mechanism of m i t o c h o n d r i a l b i o g e n e s i s , p a r t i c u -
l a r l y t h e i d e n t i t y of p o l y p e p t i d e s s y n t h e s i z e d
by
t h e g e n e t i c system w i t h i n t h e o r g a n e l l e i t s e l f ( 1 , 2 ) . Where m i t o c h o n d r i a l DNA is a b s e n t , a s i n some cyto-
plasmic p e t i t e mutants of y e a s t , o r where t r a n s l a t i o n on mitochondrial ribosomes i s p r e v e n t e d w i t h chloram-
CYTOCHROME b FROM YEAST MITOCHONDRIA
155
phenicol, cytochrome b, cytochrome oxidase and the oligomycin-sensitive ATPase are no longer formed indicating that their biogenesis requires products of intramitochondrial protein synthesis.
To date, this
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has been clearly confirmed by labelling studies only in the case of cytochrome oxidase ( 3 , 4 ) and the oligomycin-sensitive ATPase (1). Mitochondria1 cytochrome b has not so far been obtained in a well characterized form from yeast, although the heme-containing polypeptide has recently been isolated from Neurospora and shown to be synthesized on mitochondria1 ribosomes ( 5 , 6 ) .
It is the
purpose of this paper to describe a method by which both a cytochrome b complex and cytochrome oxidase can be purified in high yields from yeast mitochondria, and to define the properties and subunit structure of the former sufficiently to make it a useful starting point for studies of cytochrome b biogenesis. MATERIALS AND METHODS Preparation of Submitochondrial Particles Saccharomyces cerevisiae (a locally isolated diploid wild type) was grown at 28OC in an aerobic medium containing 1.5% (w/v) galactose, 0 . 7 5 % yeast extract, 0.12% (NHI,)~SO,+,0.05% NaC1, 0.01% CaC12, 0.2% MgS04.7H20, 0.1% KH2P04 and 3 x
FeC13.
156
WANEN
Cells in the early stationary phase of growth were harvested by centrifugation, washed twice in cold water and once in 0.5 M sorbitol, 1mM EDTA and l O m M Tris-HC1, pH 7.4, then broken in the latter medium by
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treatment in a Braun homogenizer for 30 sec ( 7 ) .
The
homogenate was centrifuged three times at 1,000 xg for 5 min, then mitochondria were collected at 15,000 xg
for 10 min and washed once in 0.25 M sorbitol and lOmM Tris-HC1, pH 7 . 4 .
The pellet was resuspended in
the same medium at 10 mg protein/ml and sonicated for 30 sec using a Branson sonifier at an output of 7 amp.
Submitochondrial particles were sedimented at 100,000 xg for 30 min and washed once. Cytochrome Isolation All steps were carried out at OOC.
Submito-
chondrial particles were resuspended in 0.25
M
sorbitol and lOmM Tris-HC1, pH 7.4, at 20 mg protein/ml, then solid K C 1 followed by sodium deoxycholate (10% "/v, pH 7 - 8 ) were added slowly to the stirred solution to concentrations of 1 M and 0.15 mg detergent/mg protein respectively.
After stirring for
30 min the suspension was centrifuged at 100,000 xg
for 45 min and the supernatant, which contained a l l of the cytochrome c, was carefully removed. pellet was resuspended in 0 . 2 5
M
The
sorbitol and l O m M
15 7
CYTOCHROME b FROM YEAST MITOCHONDRIA Tris-HC1,
pH 7 . 0 ,
t h e n s o l i d K C 1 and 1 0 % sodium
deoxycholate were slowly added t o c o n c e n t r a t i o n s of 1 M and 0 . 5 mg detergent/mg p r o t e i n r e s p e c t i v e l y .
The
s o l u t i o n w a s s t i r r e d f o r 30 min t h e n c e n t r i f u g e d a t
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1 0 0 , 0 0 0 xg f o r 30 min t o o b t a i n a clear brown super-
n a t a n t which c o n t a i n e d almost a l l of t h e remaining cytochromes. The c l e a r s u p e r n a t a n t w a s l e f t t o s t a n d overnight. T u r b i d i t y developed w i t h i n t h a t time and was removed by c e n t r i f u g a t i o n a t 4 0 , 0 0 0 xg f o r 1 5 min.
The f i r s t
p e l l e t was normally d i s c a r d e d s i n c e it c o n t a i n e d deoxycholic a c i d w i t h l i t t l e cytochrome.
Centrifug-
a t i o n was r e p e a t e d t h e r e a f t e r whenever s l i g h t t u r b i d i t y developed (ncrmally every 2-4 h ) .
The p e l l e t s ,
which c o n t a i n e d s p e c t r a l l y p u r e cytochrome b t o g e t h e r w i t h v a r y i n g amounts of deoxycholic a c i d , were r e s u s pended i n 1 0 mM T r i s - H C 1 ,
pH 7 . 4 .
The cytochrome
should be removed f r e q u e n t l y as a g g r e g a t i o n c o n t i n u e s s i n c e an i n c r e a s e d p r o p o r t i o n could n o t be resolubili z e d i n aqueous b u f f e r s (see below) when t h e p r e c i p i t a t e d p r o t e i n was l e f t i n c o n t a c t w i t h t h e deoxycholate e x t r a c t . P r e c i p i t a t i o n o f t h e cytochrome b complex w a s complete w i t h i n 4 8 h and was followed by a p r o g r e s s i v e a g g r e g a t i o n of cytochrome o x i d a s e .
The f i r s t c y t o -
chrome o x i d a s e f r a c t i o n ( s ) may show some c o n t a m i n a t i o n
158
W A N E N
by cytochrome b and can b e d i s c a r d e d .
Much of t h e c y t o -
chrome o x i d a s e (60-80%) w a s c o l l e c t e d w i t h i n t h e following 48 h b u t up t o 5 days w a s r e q u i r e d f o r complete p r e c i p i t a t i o n .
The f i n a l s u p e r n a t a n t c o n t a i n e d
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s p e c t r a l l y pure cytochrome
c1.
S p e c t r a 1 Ana 1y sis S p e c t r a were o b t a i n e d e i t h e r a t room o r l i q u i d n i t r o g e n temperature u s i n g a Cary 1 4 r e c o r d i n g s p e c t r o photometer.
Absolute s p e c t r a were recorded a g a i n s t
e q u a l c o n c e n t r a t i o n s of bovine serum albumin.
Heme
c o n t e n t s were c a l c u l a t e d from t h e a b s o r p t i o n d i f f e r e n c e
a t t h e a-peak maximum between a reduced and an o x i d i z e d sample.
The following m i l l i m o l a r e x t i n c t i o n coef f i -
c i e n t s were used: chrome a , 1 0 . 4 10).
cytochrome b , 13.2 ( r e f . 8 1 , cyto-
( r e f . 9) and cytochrome
cl,
17.1 ( r e f .
The p y r i d i n e hemochromogen spectrum was o b t a i n e d
as d e s c r i b e d by Basford
et &.
(11).
Other methods Methods f o r t h e a s s a y of cytochrome o x i d a s e ( 1 2 1 , NADH
-
and s u c c i n a t e cytochrome c r e d u c t a s e ( 1 2 ) and
ATPase (13) have been d e s c r i b e d elsewhere.
The a s s a y s
were c a r r i e d o u t a t t h e f o u r t h day o f cytochrome precipitation. aa3
The p r e c i p i t a t e s o f cytochromes b and
were d i s s o l v e d immediately b e f o r e t h e a s s a y s were
performed.
Polyacrylamide g e l e l e c t r o p h o r e s i s i n t h e
presence of sodium dodecyl s u l f a t e h a s been p r e v i o u s l y
CYTOCHROME b FROM YEAST MITOCHONDRIA
described ( 1 4 ) .
159
Densitometer t r a c i n g s o f t h e s t a i n e d
g e l s were o b t a i n e d using t h e scanning a t t a c h m e n t o f a G i l f o r d r e c o r d i n g spectrophotometer. P r o t e i n w a s e s t i m a t e d according t o Lowry
et &.
(151, using bovine serum albumin a s t h e s t a n d a r d .
Phos-
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p h o l i p i d w a s measured a f t e r a s h i n g o f t h e samples by t h e method o f Chen e t a l .
(16).
Materials ' C h o l i c and deoxycholic a c i d (Sigma) were r e c r y s t a l l i z e d from 5 0 % e t h a n o l b e f o r e use.
[14C]deoxycholic
a c i d ( s p e c i f i c a c t i v i t y 2 . 9 8 mCi/mmole) w a s o b t a i n e d from M a l l i n c k r o d t . RESULTS S o l u b i l i z a t i o n and P u r i f i c a t i o n Deoxycholate a t a c o n c e n t r a t i o n of 0.5 mg d e t e r gent/mg mitochondria1 p r o t e i n s o l u b i l i z e d a major propo r t i o n (>80%) of t h e cytochromes a a 3 , b , and c
but a
c o n s i d e r a b l y smaller amount ( ~ 3 0 % )o f t h e t o t a l p r o t e i n (Table 1 ) . The r e s u l t i n g p u r i f i c a t i o n , measured as t h e s p e c i f i c heme c o n t e n t , was 3- t o 4-fold f o r each cytochrome.
The slow, spontaneous p r e c i p i t a t i o n o f t h e
cytochrome b complex, followed by cytochrome aa3, y i e l d e d very high r e c o v e r i e s and f i n a l heme p u r i f i c a t i o n s of 1 4 - and 20-fold r e s p e c t i v e l y compared t o t h e p a r e n t membranes.
Autoxidation of t h e p r o t e i n s w a s
a p p a r e n t l y i n s i g n i f i c a n t s i n c e p r e p a r a t i o n s under a i r
200
280
640
490
310
130
320
440*
Total heme a c1 nmoles
700
b
11.2
2.8
0.8
8.1
1.3
0.4
2.3
1.4
0.5*
Specific heme b a C1 nmo 1e s/mg protein
30.6
0.04
1.2
pmo les/mi n/mg protein
Specific activity**
**Cytochrome c oxidase activity; soybean phospholipids.
assayed in the presence of 2 mg/ml of sonicated
*These values are only approximate owing to spectral interference by cytochrome c.
Purification of the cytochromes from yeast mitochondria. Details of the method of purification are given in the text. The values for cytochrome b and cytochrome oxidase are the sum of the spectrally pure cytochrome precipitates which were collected in this experiment. Following complete precipitation of these cytochromes, the cytochrome c 1 supernatant was dialysed against 0.1 M then 0.01 M NaPi, pH 7.4, cleared by centrifugation at 40,000 xg for 15 min and concentrated by Amicon pressure filtration using a PM 10 membrane. The concentrate was again centrifuged before the spectrum was recorded.
Total protein mg Submitochondrial particles 860 Solubilized cytochromes 225 Cytochrome b precipitats 44 Cytochrome aa precipitate 25 Cytochrome c 1 supernatant 57
Fraction
TABLE 1
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rr:
% M
161
CYTOCHROME b FROM YEAST MITOCHONDRLA
o r n i t r o g e n had i d e n t i c a l s u b u n i t p a t t e r n s on sodium dodecyl s u l f a t e g e l s .
Cytochrome c o x i d a s e a c t i v i t y
i n c r e a s e d 25-fold d u r i n g t h e p u r i f i c a t i o n o f cytochrome a a 3 whereas t h e cytochrome b complex c o n t a i n e d a l m o s t no cytochrome c o x i d a s e a c t i v i t y (Table 1);
these values
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may be underestimated s i n c e t h e a c t i v i t y ( b u t n o t t h e s p e c i f i c heme c o n t e n t ) d e c r e a s e d i n t h e d e o x y c h o l a t e e x t r a c t w i t h a h a l f - t i m e o f 50 h.
Neither preparation
c o n t a i n e d any measurable A T P a s e o r NADH- o r s u c c i n a t e cytochrome c r e d u c t a s e a c t i v i t i e s . Cytochromes b ,
c1
and a were p r e s e n t i n t h e deoxy-
c h o l a t e e x t r a c t a t a r a t i o of approximately 2:l:l (Table 1 ) . The s o l u b i l i z a t i o n o f
cytochrome b and
c1
may occur a s a b i n a r y complex (as a p p a r e n t l y o c c u r s i n
bovine mitochondria (8,17,19)) s i n c e t h e r a t i o remained near 2 : l even a t deoxycholate c o n c e n t r a t i o n s where o n l y p a r t i a l s o l u b i l i z a t i o n of t h e s e cytochromes o c c u r r e d . The h i g h e r p r o p o r t i o n of cytochrome b i s n o t a b l e s i n c e
a t l e a s t two f u n c t i o n a l forms a r e now r e c o g n i s e d i n mitochondria1 membranes (20). The " s o l u b l e " form o f t h e cytochrome b complex (see below) could n o t be f u r t h e r p u r i f i e d e i t h e r by
g l y c e r o l d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o r column chromatography on Sepharose 4B ( b o t h i n t h e p r e s e n c e of cholate).
A s i n g l e major peak
was observed by t h e s e
methods, and by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n ( F i g u r e
MARJANEN
162
lA), i n d i c a t i n g t h a t the s o l u b l e p r e p a r a t i o n may e x i s t as a s i n g l e p r o t e i n complex.
Cytochrome o x i d a s e l i k e -
w i s e migrated as a s i n g l e peak upon u l t r a c e n t r i f u g a t i o n (Figure 1 B ) .
As shown by polyacrylamide g e l e l e c t r o -
p h o r e s i s , the cytochrome b complex c o u l d n o t be f u r t h e r Downloaded by [University of Waterloo] at 06:07 14 January 2015
d i s a g g r e g a t e d w i t h e i t h e r 1%T r i t o n X-100 or 8 M u r e a ,
A
FIGURE 1 A n a l y t i c a l u l t r a c e n t r i f u g a t i o n of t h e cytochrome b complex and cytochrome o x i d a s e . The cytochromes were prepared as d e s c r i b e d i n t h e t e x t t h e n c e n t r i f u g e d a t 1 0 0 , 0 0 0 xg f o r 6 0 min t o remove small amounts of aggregated material and a d j u s t e d t o 4 mg protein/rnl Sedimentation a n a l y s i s i n 1 0 0 mM T r i s - H C 1 , pH 7 . 4 . was performed a t 22OC and a t 60,000 rpm u s i n g t h e s i n g l e s e c t o r c e l l of a Beckman model E a n a l y t i c a l u l t r a c e n t r i f u g e . Photographs were t a k e n a t 1 9 6 , 2 6 0 , 3 2 4 and 4 5 2 min (cytochrome b complex ( A ) ) o r 13, 3 0 , 94 and 313 min (cytochrome o x i d a s e (B)) a f t e r r e a c h i n g maximum speed. The d i r e c t i o n o f s e d i m e n t a t i o n i s from r i g h t to l e f t .
CYTOCHROME b FROM YEAST MITOCHONDRIA
163
o r w i t h 1%sodium d e o x y c h o l a t e o r 2 % sodium c h o l a t e . Phenolacetic acid-urea
( 2 1 1 , sodium dodecyl s u l f a t e and
t h e c a t i o n i c d e t e r g e n t cetyltrimethylammonium bromide caused d i s s o c i a t i o n b u t w i t h a l o s s of t h e heme absorption. Downloaded by [University of Waterloo] at 06:07 14 January 2015
Spectral Analysis The reduced cytochrome b complex e x h i b i t e d a , B and
y
bands a t 562, 532 and 430 nm i n b o t h d i f f e r e n c e
( F i g u r e 2 ) and a b s o l u t e ( F i g u r e 3A) s p e c t r a .
A t -196°C
t h e peaks s h i f t e d t o 559, 529 and 429 nm ( F i g u r e 3 B ) . A s e x p e c t e d , t h e p y r i d i n e hemochromogen spectrum corres-
ponded t o t h e p y r i d i n e d e r i v a t i v e of f e r r o p r o t o p h o r p h y r i n I X ( F i g u r e 3C).
No c o n t a m i n a t i o n by t h e o t h e r c y t o -
chromes c o u l d b e d e t e c t e d e i t h e r a t 20°C o r -196"C,
or
by a p p l i c a t i o n of t h e d i f f e r e n t i a l heme method ( 9 ) . Cytochrome a a 3 e x h i b i t e d a and
nm r e s p e c t i v e l y ( F i g u r e 2 ) .
y
bands a t 6 0 4 and 445
The cytochrome c1 which
remained i n t h e s u p e r n a t a n t a f t e r p r e c i p i t a t i o n o f t h e o t h e r cytochromes was s p e c t r a l l y p u r e ( F i g u r e 2 ) b u t of low s p e c i f i c heme c o n t e n t ( T a b l e 1 ) . Sodium Dodecyl S u l f a t e G e l E l e c t r o p h o r e s i s D i s s o c i a t i o n and e l e c t r o p h o r e s i s o f the cytochrome b complex on 1 0 % p o l y a c r y l a m i d e g e l s i n t h e p r e s e n c e of sodium dodecyl s u l f a t e r e v e a l e d s e v e n d i s t i n c t bands ( F i g u r e 4 ) two of which (V and V I ) were i n c o m p l e t e l y separated.
Molecular w e i g h t s of t h e bands, o b t a i n e d
MARJANEN
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164
CYTOCHROME b
CYTOCHROME c,
553
A
FIGURE 2 Room temperature difference spectra (dithionite reduced minus ferricyanide oxidized) of yeast submitochondria-ticles and purified cytochromes. The spectra were recorded in 100 mM Tris-HC1, pH 7.4, either at 2 mg protein/ml (submitochondrial particles) or 0.13-0.20 mg protein/ml (cytochromes) The baselines (i.e. oxidized minus oxidized spectra) have been omitted from the figure.
.
by calibrating the gels with standard proteins were: I, 42,000;
11, 33,000;
111, 27,500;
V, 15,500; VII 13,000; VII, 10,500.
IV, 23,000;
CYTOCHROME b FROM YEAST MITOCHONDRIA
165
A
CYTOCHROME b COMPLEX
A
I
I
I
I I
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I I
I
I I I I I I I I I I
[!
0
A BSORPT 1 0N SPECTRA
r
,.o1
rn
n
