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A spin label EPR study of tobacco mosaic virus protein
Volume 85, number 1
FEBS LETTERS
A SPIN LABEL
EPR STUD~f OF TOBACCO
January 1978
MOSAIC Vt[RUS PROTEIN
M. A. HEMM[NGA, T. V A N DEN B 0 0 M G A A R D and J. L. DE W[7? Department o f Molecular Physics, Agricultural University, De Drei]en 6, PO Box 8091, 6 700 F P Wagenbzgen, The Netherlands
Received 31 October 1977
1. [ritroduefion 0 T o b a c c o Mosaic Virus (TMV) is a rodlike particle c o m p o s e d o f coat protein and RNA. The coat protein consists o f 2130 identical subunRs with tool. wt 17 5130. These subunits itselves are able to forl~ welldefined oligomers, such as trimers, double disks and helices [11. d e p e n d e n t on protein c o n c e n t r a t i o n , o H . lo~c
s~-en~--~til
~ d
I
II 0
tempera_ure_
R e c e n t l y a 0 . 5 n m resolution three-dimens~nai
model o f the double disk i~ published, based on an X-ray diffraction study [ 2 ] . F r o m this model .it is seen that the sin~e SH-group, which is present in the protein subunit [ 3 ] , is located at the side o f ~le subunit that has interaction with adjacent subuni~_s in the olioomers. In a previous paper spin labeled TMV protein with maleinfide sp/n label I attached to this SH-group is the subject o f a saturation transfer EPR study [4~. In this c o m m u n i c a t i o n convenfionM E P R spin label results are presented with TMV-proteht that i; labeled with maleirrdde spin labels with different c h i n length (I and H), given in fig. 1. It is demonstrated tkat these spin labels provide i n f o r m a t i o n a b o u t the diss ~ciationassociation equilibrium o f the spin labeled p~otein subunits as a function o f p H and m o n i t o r a c~nformafional change in the protein subunits above pH 9.5.
rr
o
Fig.1. The structure o_*t_h_emaleimide spin labels I and II.
EPR measurements were carried out on a Vafian E-6 spectrometer equipped with variable temperature controller. Protein samples were contained in quartz capillaries [ 4 ] . The temperature was measured with a copper-cor,~!.~:~_tan thermoconp!e and is accurate within -+ I°C. For the E P R measurements the spin labeled protein samples were dialysed at 4°C against water ( p H 5.5) or appropriate buffers: 12 mM Tris--HCi (pH 8.8), 12 mM NaHCOa--NaOH (pH 9.5) and 10 mM NaOH (pH 11.7).
2. ~d[aeer~a~s and rr~e~:hods 3. Results
TMV-protein was labeled with spin labels I and [[ as described previously [ 4 ] . The labeling was carried out h~. the presence o f 4 M urea and was a b o u t t 0 0 % for spin labels I and IL The spin labels were obtained from S ~ , a (Calif. USA). Elsevier/Worth-Holland Biomedical Press
Typical E P R spectra o f TMV-protein labeled with spin labels [ and U are shown ha fig.2. The spectra contain t w o c o m p o n e n t s : A sharp three-Hne spectrum arising from mobile spin labels o n the protein, super171
Volume gS, number 1
FEBS LETTERS
/
p~t g.:3
/
3
Z. G C
/~"
January 1 9 7 8
s p i n I¢.~el t
/
hl
/
L/
~ /
pin I~bel ff
2-
.'-oGAuss
Fig.2. EP_Rspectra of TMV protein labeled with spin labels i and II. Protein concentration approx. 6 g/liter, in 12 mbl NaHCO3--NaOH buffer, pH 9.5. h~ and h= are the heights of the low-field line~ of the gharp and broad spectral components, respectively. The samples were contained in small capil!arids. EPR conditions: frequency 9.12 GHz, microwave power 50 roW, modulation amplitude 1.6 G, temp. 4°C.
'i 0
i p N----~
imposed on a broad spectrum arising f r o m immobiiized sp-n labels. The sharp spectral c o m p o n e n t o f tkm longest ~pin label Ii has a larger amplitude with respect to the broad c o m p o n e n t than this c o m p o n e n t o f spin label I. Below pH 9.5 the widths o f the low and high-field lines o f the sharp spectral c o m p o n e n t are approximately equal for spin labels I en II and do n o t change with pH. This also applies to the b r o a d spectral comp o n e n t . This means that the mobility o f the mobile and immobile spin labels remains c o n s t a n t and that
the amplitudes of the sharp and broad spectral components represent approximately the c o n c e n t r a t i o n o f mobile and immobile spin labels, respectively. Thus
Fig.3. Varialion ofh,/h., with pH for TMV protein labeled with spin lat~els i and H. The error lirn~ts m h~/h 2 are about -+ 0.I.
pH 11.7 a :~harp three-line spectrum is present only and h:/h2 goes to infinity. At p H 5.5 file sharp spectral c o m p o n e n t is almost absent for spin label L The curves in fig.3 are reversible. At p H l 1.7 the lhae width o f the l o w and higJafield lines o f the three-line spectrum, which is similar for spin labels [ and I[, are a b o u t 15% smaller than for the corresponding lines at p H 5 . 5 - 9 . 5 . This indicates m~t increase o f m o t i o n o f the mobile spin labels whef~ the p H is raised f r o m 9.5--11.7.
the ratio h~/h2, where h~ and h2 are the heights of the low-field lines o f the sharp and broad c o m p o n e n t s , respectively (see fig.2), is a suitable parameter to express the relative n u m b e r o f mobile spin labels. When the p_H o f a spin labeled T_MV-protein sample is raised from 5.5--9.5 at equal settings o f the E P R spectrometer and equal protein concentrations, the amplitude o f the sharp c o m p o n e n t increases at the cost o f the broad c o m p o n e n t . This indicates that at increasing pH spin labels go over from the immobile state to the mobile state. A plot ofh~/h2 against pH is given in fig.3 for TMV protein labeled with spin labels [ and I!. Note that h i/h2 increases when the p H is raised and that the longer the .~p~ label, the hio~qer the value ofh~/h2. At 172
4. Discussbm The E P R spectra in fig.2 indicate the presence o f t w o states .af the spin labels I and [I o n the surface o f spin labeled TMV protein. In one state the spin label is very mabiie with a rotational correlation time Te ~,~ 1{}-9 L;, :ghereas ha hhe other state the spin label is immobflr_zed with r c ~ 5 × 10 -s s [ 5 ] . The resldl~.s o f fig.2 and 3 have led t o the m o d e l given in f i g 4 , to explain the effect o f p H and differenl chain lengths o f spin labels [ and 1[. In a single subunit o f spht labeled TMV protein the spin label extends in aolution and is mobile. When a subunlt
Volume 85, number 1
FEBS LETTERS
Fig.4. S c h e m a t i c m o d e l t o e x p l a i n t h e p r e s e n c e o f t h e s h a r p a n d b r o a d s p e c t r a / c o m p o n e n t s in t h e E P R s p e c t r a in fig.2. T h e shape o f the T M V protein s u b u n i t and the position o f t h e s p i n label ( i n d i c a t e d b y a b l a c k d o t ) , w h i c h is a t t a c h e d t o
the SH-group, are derived from [ 1[,2]. The spin label at the outside of the two subunits is mobile, whereas the spL-~label that is squeezed between the subunits is LrnmobRe. This spin label h a s a r o t a t i o n a l c o r r e l a t i o n t i m e c o r r e s p o n d i n g t o t h e
overall rotational motion ef the two subun.ils. interacts w i t h a n o t h e r one, the m o b i l i t y o f one spin label will be reduced b y the presence o f the adjacc::t p r o t e i n surface (see fig.4). An increase o f p H will dissociate oligomers o f spin labeled T M V protein and increase the m o b i l e c o m p o n e n t at the cost o f tim i m m o b i l e c o m p o n e n t . This gives an increase o f t'. ~/h 2 in accordance w i t h fig.3. An increase o f the length o f the spin label will reduce the interaction b e t w e e n the subunits and enhance dissociation, also leading to an increase o f h ~/h2. A t p H values above 9.5 an increase in m o t i o n o f the spin labels o n the p r o t e i n surface is detected. This can n o t arise f r o m dissociation o f the oligomers, b u t results f r o m a change in t_he e n v i r o n m e n t o f the spin labels. This d e m o n s t r a t e s that in the p H region 9 . 5 - - 1 1 . 7 a c o n f o r m a t i o n a l change takes place in the spin labeled protein subunits. F r o m c o m p u t e r simulations o f superpositions o f m o b i l e a n d i m m o b i l e E P R spectra b y [ 6 ] , it is possible t o express h~/h2 in t e r m s o f f , the fraction o f m o b i l e sphn labels. Because the line widths in the c o m p u t e r simulated [6] and e x p e r i m e n t a l spectra are different,
January ~978
t h e y m u s t be t a k e n into a c c o u n t b y the relation h!A~ = c o n s t a n t [ 7 ] , where ~ is the w~dth o f the low-field line o f the sharp c o m p o n e n t in the spectra. Tiffs gives hJh~ ~ 16.3 f / l - - f , forhJh2 <~ 5. It is calculated f r o m fig.3 that at p H 9.5 the vah:es f o r f are a p p r o x . 7 and 17% for T M V p r o t e i n labeled with spin labels I and H, respectively. When it is assumed t h a t the protein oligomers consist o f h n e a r chains o f n subunits, it is easily seen that n = l / f , o y extending the m o d e l in rigA. This yields values for n o f a b o u t 14 and 6, respectively, in the case t h a t the subunits p o l y m e r i z e in t w o or m o r e layers [ 1 ] , the a p p r o x i m a t e values o f n are larger, so t h a t the calcula ted n u m b e r s are m i n i m u m values o f n . The molecular weight o f these oligomers at pH 9.5 is sufficiently high to explain the presenct; o f the b r o a d spectral cor_lponent in fig.2 arising f r o m spin labels squeezed b e t w e e n the surface ~, o f the subunits. It is interesting to m a k e a c o m p a r i s o n b e t w e e n the b e h a v i o u r o f native T M V protein as a function o f pH [ 1 ] and the spin labeled protein. At tow ionic strength and a t e m p e r a t u r e 0t" 5°C native TMV protein forms helical oligomers at p H ,~ 5, which dissociate into m a i n l y trimers at p H -~, 9 [ 1 ] . The spin label results also indicate a dissociation o f o h g o m e r s o f spin labeled T M V protein at increasing pH, in a g r e e m e n t with the b e h a v i o u r o f ,lative T M V protein. However, since the smallest n u m b e r o f subunits o f spin labeled T M V p r o t e i n in oligomers at p H 9.5 is a b o u t 6, the spin labeled protein has a t e n d e n c y to associate to larger oligomers t h a n native protein. Tiffs is in a g r e e m e n t w i t h previous e x p e r i m e n t s , using s a t u r a t i o p transfer EPR, where the same effect is f o u n d at p H 7 [ 4 ] . It is obvious f r o m the present e x p e r i m e n t s that association o f T~IV protein is very- sensitive to changes at the surface o f the protein. C h e , d c a ! m o d i f i c a t i o n o f the T M V protein, b y attaching a maleimide spin label at the SH-group, changes it: association properties, b u t also increasing the length o f the spin label at the SH-group has large effects on the p r o t e i n - - p r o t e i n interaction. Acknowledgements The excellent technical assistence o f Miss M. Boerjan with the p r e p a r a t i o n o f T M V protein is gratefully acknowledged. We are i n d e b t e d to the D e p a r t m e n t o f Virology for providing l a b o r a t o r y facilities. i73
Volume 85, number I
FEBS LETTERS
References [11 D u r h a m , A- C. H. ( 1 9 7 2 ) J. Mol. Biol. 67, 2 8 9 - - 3 0 5 . {2] C h a m p u e s s , J. N.. B l o o m e r , A. C., Bricogne, G., Butler, P. J. G. a n d Klug, A. 0 9 7 6 ) N a t u r e 259, 2 0 - 2 4 . [3] A n d e r e r , F. H., W i t t m a n n - L e i b o ! d , B. a n d W i t t m a n n , I4 G. (19653 Z. Naturforsc!h. 20b. I 2 0 3 - - 1 2 1 3 .
174
January 1978
[ 4 ] H e m m i n g a , M. A., De Iager, P. A. a n d De Wit, 1. L. ( 1 9 7 7 ) B i o c h e m . Biophys. Res. C o m m u n . in press. [ 5 ] Hsia, J. C. a n d P~ette, L. H. ( 1 9 6 9 ) A r c h . B f o c h e m . Biophys. L29, 2 9 6 - - 3 0 7 . [ 6 ] l o s t , P., G r i f f i t h , O. H., Capaldi, R. N. a n d V a n d e r k o o i , G. ( 1 9 7 3 ) Proc. Natl. &cad. Sci. U S A 7 0 , 4 8 0 - 4 8 4 . [7J H e m m i n ~ a , M. A. a n d Post, $. F. M. ( 1 9 7 7 ) Bfoehim. Biophys_ &eta 4 3 6 , 2 2 2 - - 2 3 4 .
FEBS LETTERS
A SPIN LABEL
EPR STUD~f OF TOBACCO
January 1978
MOSAIC Vt[RUS PROTEIN
M. A. HEMM[NGA, T. V A N DEN B 0 0 M G A A R D and J. L. DE W[7? Department o f Molecular Physics, Agricultural University, De Drei]en 6, PO Box 8091, 6 700 F P Wagenbzgen, The Netherlands
Received 31 October 1977
1. [ritroduefion 0 T o b a c c o Mosaic Virus (TMV) is a rodlike particle c o m p o s e d o f coat protein and RNA. The coat protein consists o f 2130 identical subunRs with tool. wt 17 5130. These subunits itselves are able to forl~ welldefined oligomers, such as trimers, double disks and helices [11. d e p e n d e n t on protein c o n c e n t r a t i o n , o H . lo~c
s~-en~--~til
~ d
I
II 0
tempera_ure_
R e c e n t l y a 0 . 5 n m resolution three-dimens~nai
model o f the double disk i~ published, based on an X-ray diffraction study [ 2 ] . F r o m this model .it is seen that the sin~e SH-group, which is present in the protein subunit [ 3 ] , is located at the side o f ~le subunit that has interaction with adjacent subuni~_s in the olioomers. In a previous paper spin labeled TMV protein with maleinfide sp/n label I attached to this SH-group is the subject o f a saturation transfer EPR study [4~. In this c o m m u n i c a t i o n convenfionM E P R spin label results are presented with TMV-proteht that i; labeled with maleirrdde spin labels with different c h i n length (I and H), given in fig. 1. It is demonstrated tkat these spin labels provide i n f o r m a t i o n a b o u t the diss ~ciationassociation equilibrium o f the spin labeled p~otein subunits as a function o f p H and m o n i t o r a c~nformafional change in the protein subunits above pH 9.5.
rr
o
Fig.1. The structure o_*t_h_emaleimide spin labels I and II.
EPR measurements were carried out on a Vafian E-6 spectrometer equipped with variable temperature controller. Protein samples were contained in quartz capillaries [ 4 ] . The temperature was measured with a copper-cor,~!.~:~_tan thermoconp!e and is accurate within -+ I°C. For the E P R measurements the spin labeled protein samples were dialysed at 4°C against water ( p H 5.5) or appropriate buffers: 12 mM Tris--HCi (pH 8.8), 12 mM NaHCOa--NaOH (pH 9.5) and 10 mM NaOH (pH 11.7).
2. ~d[aeer~a~s and rr~e~:hods 3. Results
TMV-protein was labeled with spin labels I and [[ as described previously [ 4 ] . The labeling was carried out h~. the presence o f 4 M urea and was a b o u t t 0 0 % for spin labels I and IL The spin labels were obtained from S ~ , a (Calif. USA). Elsevier/Worth-Holland Biomedical Press
Typical E P R spectra o f TMV-protein labeled with spin labels [ and U are shown ha fig.2. The spectra contain t w o c o m p o n e n t s : A sharp three-Hne spectrum arising from mobile spin labels o n the protein, super171
Volume gS, number 1
FEBS LETTERS
/
p~t g.:3
/
3
Z. G C
/~"
January 1 9 7 8
s p i n I¢.~el t
/
hl
/
L/
~ /
pin I~bel ff
2-
.'-oGAuss
Fig.2. EP_Rspectra of TMV protein labeled with spin labels i and II. Protein concentration approx. 6 g/liter, in 12 mbl NaHCO3--NaOH buffer, pH 9.5. h~ and h= are the heights of the low-field line~ of the gharp and broad spectral components, respectively. The samples were contained in small capil!arids. EPR conditions: frequency 9.12 GHz, microwave power 50 roW, modulation amplitude 1.6 G, temp. 4°C.
'i 0
i p N----~
imposed on a broad spectrum arising f r o m immobiiized sp-n labels. The sharp spectral c o m p o n e n t o f tkm longest ~pin label Ii has a larger amplitude with respect to the broad c o m p o n e n t than this c o m p o n e n t o f spin label I. Below pH 9.5 the widths o f the low and high-field lines o f the sharp spectral c o m p o n e n t are approximately equal for spin labels I en II and do n o t change with pH. This also applies to the b r o a d spectral comp o n e n t . This means that the mobility o f the mobile and immobile spin labels remains c o n s t a n t and that
the amplitudes of the sharp and broad spectral components represent approximately the c o n c e n t r a t i o n o f mobile and immobile spin labels, respectively. Thus
Fig.3. Varialion ofh,/h., with pH for TMV protein labeled with spin lat~els i and H. The error lirn~ts m h~/h 2 are about -+ 0.I.
pH 11.7 a :~harp three-line spectrum is present only and h:/h2 goes to infinity. At p H 5.5 file sharp spectral c o m p o n e n t is almost absent for spin label L The curves in fig.3 are reversible. At p H l 1.7 the lhae width o f the l o w and higJafield lines o f the three-line spectrum, which is similar for spin labels [ and I[, are a b o u t 15% smaller than for the corresponding lines at p H 5 . 5 - 9 . 5 . This indicates m~t increase o f m o t i o n o f the mobile spin labels whef~ the p H is raised f r o m 9.5--11.7.
the ratio h~/h2, where h~ and h2 are the heights of the low-field lines o f the sharp and broad c o m p o n e n t s , respectively (see fig.2), is a suitable parameter to express the relative n u m b e r o f mobile spin labels. When the p_H o f a spin labeled T_MV-protein sample is raised from 5.5--9.5 at equal settings o f the E P R spectrometer and equal protein concentrations, the amplitude o f the sharp c o m p o n e n t increases at the cost o f the broad c o m p o n e n t . This indicates that at increasing pH spin labels go over from the immobile state to the mobile state. A plot ofh~/h2 against pH is given in fig.3 for TMV protein labeled with spin labels [ and I!. Note that h i/h2 increases when the p H is raised and that the longer the .~p~ label, the hio~qer the value ofh~/h2. At 172
4. Discussbm The E P R spectra in fig.2 indicate the presence o f t w o states .af the spin labels I and [I o n the surface o f spin labeled TMV protein. In one state the spin label is very mabiie with a rotational correlation time Te ~,~ 1{}-9 L;, :ghereas ha hhe other state the spin label is immobflr_zed with r c ~ 5 × 10 -s s [ 5 ] . The resldl~.s o f fig.2 and 3 have led t o the m o d e l given in f i g 4 , to explain the effect o f p H and differenl chain lengths o f spin labels [ and 1[. In a single subunit o f spht labeled TMV protein the spin label extends in aolution and is mobile. When a subunlt
Volume 85, number 1
FEBS LETTERS
Fig.4. S c h e m a t i c m o d e l t o e x p l a i n t h e p r e s e n c e o f t h e s h a r p a n d b r o a d s p e c t r a / c o m p o n e n t s in t h e E P R s p e c t r a in fig.2. T h e shape o f the T M V protein s u b u n i t and the position o f t h e s p i n label ( i n d i c a t e d b y a b l a c k d o t ) , w h i c h is a t t a c h e d t o
the SH-group, are derived from [ 1[,2]. The spin label at the outside of the two subunits is mobile, whereas the spL-~label that is squeezed between the subunits is LrnmobRe. This spin label h a s a r o t a t i o n a l c o r r e l a t i o n t i m e c o r r e s p o n d i n g t o t h e
overall rotational motion ef the two subun.ils. interacts w i t h a n o t h e r one, the m o b i l i t y o f one spin label will be reduced b y the presence o f the adjacc::t p r o t e i n surface (see fig.4). An increase o f p H will dissociate oligomers o f spin labeled T M V protein and increase the m o b i l e c o m p o n e n t at the cost o f tim i m m o b i l e c o m p o n e n t . This gives an increase o f t'. ~/h 2 in accordance w i t h fig.3. An increase o f the length o f the spin label will reduce the interaction b e t w e e n the subunits and enhance dissociation, also leading to an increase o f h ~/h2. A t p H values above 9.5 an increase in m o t i o n o f the spin labels o n the p r o t e i n surface is detected. This can n o t arise f r o m dissociation o f the oligomers, b u t results f r o m a change in t_he e n v i r o n m e n t o f the spin labels. This d e m o n s t r a t e s that in the p H region 9 . 5 - - 1 1 . 7 a c o n f o r m a t i o n a l change takes place in the spin labeled protein subunits. F r o m c o m p u t e r simulations o f superpositions o f m o b i l e a n d i m m o b i l e E P R spectra b y [ 6 ] , it is possible t o express h~/h2 in t e r m s o f f , the fraction o f m o b i l e sphn labels. Because the line widths in the c o m p u t e r simulated [6] and e x p e r i m e n t a l spectra are different,
January ~978
t h e y m u s t be t a k e n into a c c o u n t b y the relation h!A~ = c o n s t a n t [ 7 ] , where ~ is the w~dth o f the low-field line o f the sharp c o m p o n e n t in the spectra. Tiffs gives hJh~ ~ 16.3 f / l - - f , forhJh2 <~ 5. It is calculated f r o m fig.3 that at p H 9.5 the vah:es f o r f are a p p r o x . 7 and 17% for T M V p r o t e i n labeled with spin labels I and H, respectively. When it is assumed t h a t the protein oligomers consist o f h n e a r chains o f n subunits, it is easily seen that n = l / f , o y extending the m o d e l in rigA. This yields values for n o f a b o u t 14 and 6, respectively, in the case t h a t the subunits p o l y m e r i z e in t w o or m o r e layers [ 1 ] , the a p p r o x i m a t e values o f n are larger, so t h a t the calcula ted n u m b e r s are m i n i m u m values o f n . The molecular weight o f these oligomers at pH 9.5 is sufficiently high to explain the presenct; o f the b r o a d spectral cor_lponent in fig.2 arising f r o m spin labels squeezed b e t w e e n the surface ~, o f the subunits. It is interesting to m a k e a c o m p a r i s o n b e t w e e n the b e h a v i o u r o f native T M V protein as a function o f pH [ 1 ] and the spin labeled protein. At tow ionic strength and a t e m p e r a t u r e 0t" 5°C native TMV protein forms helical oligomers at p H ,~ 5, which dissociate into m a i n l y trimers at p H -~, 9 [ 1 ] . The spin label results also indicate a dissociation o f o h g o m e r s o f spin labeled T M V protein at increasing pH, in a g r e e m e n t with the b e h a v i o u r o f ,lative T M V protein. However, since the smallest n u m b e r o f subunits o f spin labeled T M V p r o t e i n in oligomers at p H 9.5 is a b o u t 6, the spin labeled protein has a t e n d e n c y to associate to larger oligomers t h a n native protein. Tiffs is in a g r e e m e n t w i t h previous e x p e r i m e n t s , using s a t u r a t i o p transfer EPR, where the same effect is f o u n d at p H 7 [ 4 ] . It is obvious f r o m the present e x p e r i m e n t s that association o f T~IV protein is very- sensitive to changes at the surface o f the protein. C h e , d c a ! m o d i f i c a t i o n o f the T M V protein, b y attaching a maleimide spin label at the SH-group, changes it: association properties, b u t also increasing the length o f the spin label at the SH-group has large effects on the p r o t e i n - - p r o t e i n interaction. Acknowledgements The excellent technical assistence o f Miss M. Boerjan with the p r e p a r a t i o n o f T M V protein is gratefully acknowledged. We are i n d e b t e d to the D e p a r t m e n t o f Virology for providing l a b o r a t o r y facilities. i73
Volume 85, number I
FEBS LETTERS
References [11 D u r h a m , A- C. H. ( 1 9 7 2 ) J. Mol. Biol. 67, 2 8 9 - - 3 0 5 . {2] C h a m p u e s s , J. N.. B l o o m e r , A. C., Bricogne, G., Butler, P. J. G. a n d Klug, A. 0 9 7 6 ) N a t u r e 259, 2 0 - 2 4 . [3] A n d e r e r , F. H., W i t t m a n n - L e i b o ! d , B. a n d W i t t m a n n , I4 G. (19653 Z. Naturforsc!h. 20b. I 2 0 3 - - 1 2 1 3 .
174
January 1978
[ 4 ] H e m m i n g a , M. A., De Iager, P. A. a n d De Wit, 1. L. ( 1 9 7 7 ) B i o c h e m . Biophys. Res. C o m m u n . in press. [ 5 ] Hsia, J. C. a n d P~ette, L. H. ( 1 9 6 9 ) A r c h . B f o c h e m . Biophys. L29, 2 9 6 - - 3 0 7 . [ 6 ] l o s t , P., G r i f f i t h , O. H., Capaldi, R. N. a n d V a n d e r k o o i , G. ( 1 9 7 3 ) Proc. Natl. &cad. Sci. U S A 7 0 , 4 8 0 - 4 8 4 . [7J H e m m i n ~ a , M. A. a n d Post, $. F. M. ( 1 9 7 7 ) Bfoehim. Biophys_ &eta 4 3 6 , 2 2 2 - - 2 3 4 .