High resolution electron microscopy of Fraction I protein from plant leave

Fernandez-Moran, H.

Haselkorn, R.

Kieras, F. J.

Van Bruggen, E. F. J.

English

NASA Technical Reports Server

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. -. <-ELECTRON MICROSCOPIC AND BIOCHEMICAL 
CHARACTERIZATION OF./PRACTION I PROTEIN .-- 
R.  Haselkorn 
H ,  Fernandez-Moran 
F. J. Kieras 
E. F. J. van Bruggen 
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https://ntrs.nasa.gov/search.jsp?R=19660002909 2020-03-16T22:16:49+00:00Z
Electron Microscopic and Biochemical 
Characterization of Fraction I Protein 
ABSTRACT. High resolution electron micrographs of 
Fraction I protein from Chinese cabbage leaves have been 
obtained. The protein, which has ribulose 1, 5-diphos- 
phate carboxylase activity, appears to be a cube with edge 
of about 120 8 .  Substructure can be seen in individual 
particles, consistent with a model having 24 subunits, the 
number prescribed by the available physical and chemical 
data. 
When crude extracts of tobacco leaves were examined 
in the analytical ultracentrifuge over a decade ago, a 
major soluble protein fraction, termed the Fraction I 
protein, was found t o  have a sedimentation coefficient of 
about 18s (1). Subsequent work showed that the Fraction 
I protein is located predominantly, if not exclusively, 
in the chloroplast (2). There, it comprises by weight 
at least 50 percent of the soluble proteins ( 2 , 3 ) .  
Fraction I protein has been reported to have the following 
biological activities: (i) protochlorophyll holochrome: 
the protein to which protochlorophyll is attached, and 
which catalyzes the light-induced conversion of protochlor- 
ophyll to chlorophyll (3,4);  (Ti) ribose-1-phosphate iso- 
merase (5); (iii) ribulose-1-phosphate kinase (5); and 
(iv) ribulose 1,5-diphosphate carboxylase (carboxydismutase) 
(6). Trown has recently shown that the,,activities under 
(ii) and (iii) are readily separated from Fraction I 
protein by gel filtration on Sephadex G-200, whereas car- 
boxydismutase activity (iv) is superimposable on the 
elution profile of Fraction I protein (7). He concluded 
that Fraction I protein and carboxydismutase were one and 
the same. 
-1- 
Trown found the molecular weight of carboxydismutase 
to be 515,000 by sedimentation equilibrium (7). He, as 
well as Markham and coworkers, and Wildman and coworkers, I 
found the 18s species prone to form linear aggregates, of 
which the dimer and trimer have sedimentation coefficients 
of 25s and 32s respectively (7,8,1). The molecular weight, 
sedimentation coefficient , and aggregation characteristics 
of carboxydismutase are very similar to those of the E. 
coli RNA polymerase (9). 
-
A molecular weight of 515,000 is very high F o r  a 
single polypeptide chain. Simple general considerations 
of the type that led Watson and Crick to the subunit model 
for the construction of virus protein shells suggest that 
carboxydismutase must be made up of subunits (10). If 
this is so, it becomes germane to inquire whether the 
rules governing the interaction of subunits are the same 
as those that apply in the case of small viruses (10). 
This is a realistic question in the case of carboxydis- 
mutase, since large amounts of purified material are 
readily available. 
We therefore undertook a combined electron microscopic 
and biochemical investigation of Fraction I protein, w i t n  
the following specific aims: 
(5) Is carboxydismutase composed of subunits? 
(ii) If so, what rules govern their interaction? 
(iii) Do morphological and/or functional relation- 
ships exist between carboxydismutase and RNA 
polymerase? 
only carboxydismutase? 
(iv) Is Fraction I protein carboxydismutase, and 
In this communication we provide an affirmative 
answer to (i), a tentative answer to (ii), a negative 
answer to (iii), and no answer to (iv). 
-2- 
Fresh ,  re-ribbed Chinese cabbage leaves were ground 
i n  the  cold and f i l t e r e d  through seve ra l  l a y e r s  of  cheese 
c l o t h .  T h i s  e x t r a c t  was then cent r i fuged  f o r  t e n  minutes 
a t  12,000 rpm i n  a Serval1 c e n t r i f u g e ,  and t h e  supernatant  
made 50 percent  s a t u r a t e d  w i t h  ammonium s u l f a t e .  The 
p r e c i p i t a t e  was co l l ec t ed  by c e n t r i f u g a t i o n ,  dissolved i n  
a small volume o f  0.01 M t r i s ,  p H  7.5,  and d ia lysed  i n  t h e  
cold aga ins t  t h e  same buffer .  One m l  o f  t h e  d ia lysed  
prepara t ion  was then  subjected t o  zone c e n t r i f u g a t i o n  on 
a glycerol grad ien t .  Carboxydismutase a c t i v i t y  i s  seen 
(Figure 1) t o  be associated s o l e l y  w i t h  t h e  peak o f  u l t r a -  
v i o l e t  absorbing ma te r i a l  a t  18s. T h i s  r e s u l t  i s  cons i s t -  
en t  w i t h  two i n t e r p r e t a t i o n s .  The one favored by Trown 
i s  t h a t  t h e  18s p r o t e i n  i s  i n  f a c t  carboxydismutase. 
A l t e r n a t i v e l y ,  i t  might  be argued t h a t  casboxydismutase 
i s  a small  molecule a c t i v e  only when a s soc ia t ed  w i t h  t h e  
18s p r o t e i n .  
Each F rac t ion  i n  Figure 1 was assayed f o r  RNA poly- 
merase a c t i v i t y  using ca l f  thymus DNA a s  template under 
condi t ions  found t o  be optimal f o r  t h e  E. c o l i  enzyme. _.- 
( 9 ) .  No s i g n i f i c a n t  a c t i v i t y  was found. 
I n  some p repa ra t ions ,  m a t e r i a l  iiaviiig aii a b s ~ r p t l s n  
spectrum c h a r a c t e r i s t i c  of reduced pyr id ine  nuc leo t ide  
was observed s p e c i f i c a l l y  a s soc ia t ed  w i t h  t h e  18s peak. 
These f r a c t i o n s  were tes ted  f o r  t r i o s e  phosphate dehydro- 
genase a c t i v i t y ,  b u t  none was found. The s ign i f i cance  o f  
t h e  bound pyr id ine  nucleot ide remains unknown. 
Mater ia l  f r o m  t h e  peak o f  absorbancy i n  a grad ien t  
l i k e  t h a t  of Figure 1 was then  examined i n  t h e  e l e c t r o n  
microscope. 
-3- 
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Fraction Number 
The specimens were prepared f o r  e l e c t r o n  microscopy 
using shadow cas t ing  and negative s t a i n i n g  techniques.  
The specimens were examined i n  t h e  n a t i v e  s t a t e  
s h o r t l y  a f t e r  prepara t ion .  The f i n a l  c o n c e n t r a t i m  of 
t h e  specimens was genera l ly  3.1 mg. o f  p r o t e i n  p e r  m l .  
i n  0.01 t r i s  or TMA M t r i s ,  M magnesium 
a c e t a t e  and 2 . 2  x lo-* M ammonium c h l o r i d e )  a t  pH 7.15. 
Ammonium a c e t a t e  bu f fe r  was used f o r  t h e  shadowing 
experiments. The specimens were mounted on extremely 
t h i n  carbon f i l m s  (10 A t o  50 A t h i c k ) ,  prepared by 
evaporat ion on f r e s h l y  cleaved mica i n  a Varian Vac-ion 
pump 921 u n i t  a t  a vacuum o f  
u l t r a t h i n ,  p r a c t i c a l l y  s t r u c t u r e l e s s  carbon f i l m s  a r e  
supported on s p e c i a l  f enes t r a t ed  s u b s t r a t e s  (11). 
t o  lo-' mm Hg. These 
The microdroplet  cross-spraying technique (11,12) 
and a d rop le t  technique (13) were used for negat ive  
s t a i n i n g  (Figure 3) .  By means o f  a s p e c i a l  mul t ip le -  
spraying device microdroplets o f  t he  specimen and o f  1 
t o  2 percent potassium phosphotungstate a t  pH 7.2 t o  7.4 
c o l l i d e ,  and i n t e r a c t  very r ap id ly  s h o r t l y  before  i m -  
p inging on the  specimen g r id .  Pos i t i ve  s t a i n i n g  w i t h  
1 or 2 percent  uranyl  a c e t a t e  was a l s o  obttalned with 
t h i s  technique. 
A drople t  o f  t he  specimen d i l u t e d  w i t h  t r i s  o r  TMA 
b u f f e r  was placed on the  carbon coated f e n e s t r a t e d  f i l m s .  
A f t e r  1 t o  2 minutes excess enxyme s o l u t i o n  was removed 
by f l o a t i n g  t h e  g r i d  upside down on TMA b u f f e r  f o r  about 
15 seconds. Then the  specimen was s t a ined  by f l o a t i n g  
on 0 .5  percent  t o  1 percent neu t r a l i zed  potassium phos- 
photungstate  (PTA) or on 1 percent  t o  2 percent  unbuffered 
u rany l  a c e t a t e  ( U A ) .  After  1 t o  2 minutes t h e  specimen 
was dr ied  by  b l o t t i n g  the  edge-\of t he  g r i d  on f i l t e r  
paper .  
-4- 

The electron microscopic studies were carried out in 
a facility designed to provide optimum conditions for 
consistent attainment of high resolution. Electron micro- 
graphs were recorded with Siemens Elmiskops I and Ia, at 
electron optical magnifications of 40,000 and 46,000 at 
83 kV. These microscopes were operated with a highly 
regulated power supply, and used pointed filaments of 
single-crystal tungsten (14,15) to provide microbeam 
illumination of high coherence yielding enhanced contrast. 
For critical high resolution analysis through-focus series 
were taken in steps of 80 A to 200 h,:-ani3 the measured 
astigmatism of the objective lens was 0.05 CL. or less. A 
liquid nitrogen anti-contamination device was used while 
recording most of the micrographs. Calibrations were 
carried out using a diffraction grating replica of 54,864 
lines per inch, and checked by using combined electron 
microscopy and electron diffraction of selected planes 
from copper phthalocyanine and chrysotile crystals. 
The analytical data obtained by Rees (17) established 
that the bulk of Fraction I protein consists of 24 identi- 
- cal subunits, each of molecular weight 22,500. Twenty-four 
subunits can be fitted into equivalent positions by placing 
them in groups of three on the vertices of a cu-be. Sucn a 
model for the enzyme can be ruled out on several grounds. 
First, the electron micrographs of concentrated droplets 
show no tendency toward aggregation in regular arrays. 
Secondly, attempts at crystallization from ammonium sulfate 
solutions have produced fibrous precipitates instead. 
Thirdly, when aggregates do occur, they are seen to be 
linear. Hence, the c o r r e c t  molecular model must be one 
which contains a unique two-fold axis. 
The shadowed preparations (Figure 2) show uniform 
cubical particles often with rounded edges and a central 
depression which may be partly due to preparative (de- 
hydration) effects. A model consistent with the views 
shown i n  e l e c t r o n  micrographs (F igures  3 and 4 ) ,  a s  wel l  
a s  w i t h  macroscopic p rope r t i e s  of t h e  p r o t e i n ,  i s  t h a t  of 
a cube w i t h  t h r e e  subuni t s  along each edge, and one i n  
t h e  cen te r  of each o f  fou r  f aces .  P a r t i c l e s  r e s t i n g  on 
a s i d e  face w i l l  show three rows o f  t h r e e  subuni t s  (F ig-  
u r e  4 a ) .  P a r t i c l e s  r e s t i n g  on a top  or bot tom face w i l l  
show a r i n g  of e igh t  subunits (F igure  4b )  corresponding 
to t h e  s i d e  view w i t h  t he  c e n t r a l  subunit  missing. The 
subuni ts  have a diameter of approximately 20 A t o  30 A .  
P a r t i c l e s  r e s t i n g  on an edge should show t h r e e  rows ,  
w i t h  t h e  p a r t i c l e  length i n  t he  d i r e c t i o n  of  t h e  r3ws up 
t o  40 percent  g r e a t e r  than i t s  w i d t h  perpendicular  to 
t h e  rows. P a r t i c l e s  r e s t i n g  on a ver tex  should have a 
hexagonal appearance,  w i t h  a c e n t r a l  subuni t  and twelve 
subuni t s  around t h e  perimeter.  Although s e v e r a l  p a r t i c l e s  
have been observed corresponding t o  t h e  l a t t e r  two views, 
t h e s e  a r e  r a r e ,  and could be due t o  d i s t o r t i o n  o r  assoc ia-  
t i o n  of p a r t i c l e s  on t h e  subs t r a t e .  P a r t i c l e s  w i t h  hexa- 
gonal o u t l i n e  a r e  seen occas iona l ly ,  but t hese  usua l ly  
have one r igh t -angle  corner,  and probably a r i s e  from 
p a r t i a l  embedding o f  t h e  square face .  
The diameter of a Stokes sphere o f  molecular weight 
500,000 and sedimentation c o e f f i c i e n t  18s (7) i s  c a i c u i a t -  
e d  t o  be 120 A .  T h i s  value i s  i n  exce l l en t  agreement w i t h  
t h e  measured dimensions of t h e  cubica l  p a r t i c l e s  ( 1 2 0  A 
edge) a s  shown by e l ec t ron  microscopy using seve ra l  pre-  
p a r a t i o n  techniques.  
Subunits i n  t h e  model proposed a r e  not  r e l a t e d  by t h e  
p r i n c i p l e  of quasi-equivalence t h a t  governs t h e  s t r u c t u r e  
of many small  v i ruses  ( 1 0 ) .  I n  t h e  model proposed, sub- 
u n i t s  a r e  found i n  t h r e e  p o s i t i 9 n s :  on f a c e s ,  edges,  or 
v e r t i c e s .  We assume t h a t  the plane n e t ,  f m m  which t h e  
s t r u c t u r e  may formally o r i g i n a t e ,  c o n s i s t s  of a square 
l a t t i c e .  Then t h e  d i s t o r t i o n  required t o  p l ace  a subunit  
r 
-b- 
I .  

I .  
on a ve r t ex ,  maintaining t h e  same bonding r u l e ,  becomes ~ 
unacceptable ( 1 0 ) .  We a r e  therefore  obliged t o  propose t h a t  
e i t h e r  subuni t s  a r e  capable o f  making seve ra l  kinds o f  bond, 
o r  t h a t  subuni t s  undergo considerable  conformational changes 
a s  a r e s u l t  o f  t h e i r  s t a t i s t i c a l  l o c a t i o n  i n  t h e  completed 
p a r t i c l e .  
These f ind ings  may be summarized: F rac t ion  I p r o t e i n  
c o n s i s t s  of a cube, about 120 A along each edge, containing 
24 subuni ts  v i sua l i zed  i n  high r e s o l u t i o n  e l e c t r o n  micrographs. 
The p r o t e i n  contains  a l l  t h e  carboxydismutase a c t i v i t y  recover- 
a b l e  from g lyce ro l  g rad ien t s .  It has t h e  same sedimentation 
c o e f f i c i e n t  and molecular weight a s  b a c t e r i a l  RNA polymerase, 
but lacks  the  l a t t e r  a c t i v i t y .  The p a r t i c l e  s t r u c t u r e  does 
not  appear t o  be governed b y  t h e  same r u l e s  t h a t  apply i n  t h e  
case o f  small v i r u s  p r o t e i n  s h e l l s .  We be l i eve  t h i s  t o  be 
t h e  f i r s t  occasion upon which  t h e  subs t ruc tu re  o f  a p r o t e i n  
enzyme of about 120 A diameter has been resolved i n  e l e c t r o n  
micrographs. 
R .  Haselkorn 
H .  Fernandez-Moran 
F . J .  Kieras 
E . F . J .  van Bruggen ( 2 0 )  
/ / 
Department o f  Biophysics 
T h e  Univers i ty  of  Chicago 
-7- 
LEGENDS TO FIGURES 
fi'j pure 1. Glycerol pradient centrifugation of Fraction I 
protein. @ne ml of a dialysed 3 percent to 50 percent saturat- 
ed amrnonj-urn sulfate fraction was layered on a 5 percent to 30 
percent glycerol gradient containing 0.01 M tris sulfate, 
0.031. M clutathione, pH 7.7, and centrifuged for 24 hours at 
4'C at 23,000 rpm in the SW25 rotor of the Model L Spinco. 
After centrifugation, one ml fractions were collected in the 
following system: 0.07 ml glycerol fraction, 1.5 p mole 
NaHC 0 (5.1 mC/m mole), 2 p moles MgC12, 15 p moles tris, 
pH 7.7, and 0.06 p mole ribulose 1,5-diphosphate in a final 
volume of 0.20 ml were incubated at 25OC for 15 minutes. 
Glacial acetic acid (0.05 ml) was then added, and 0.05 ml 
aliquots were dried on planchets and counted (16). 
14 
3 
Figure 2. Fraction I protein from Chinese cabbage leaves 
shadowed with platinum on thin carbon substrate(X"160,OOO). 
Figure 3. Fraction I protein from Chinese cabbage leaves 
negatively stained with 2 percent phosphotungstate (pH 7.4) 
by microdroplet cross-spraying technique ( X 360,000). 
Figure 4 (a,b). Particles ol '  Fraction I proieirl i i ega t ivz l j ;  
stained with 2 percent phosphotungstate (pH 7.4) in differ- 
ent orientations ( X 800,000). 
Text of (possible) Cover Picture: Electron micrograph of 
Fraction I protein, the major soluble protein component in 
green leaf chloroplasts, showing subunit organization of these 
enzyme protein particles of about 120 A ( X 360,000 - micro- 
graph A; or X 640,000-micrograph B) . 
REFERENCES AND NOTES 
1. 
2. 
3. 
4. 
5. 
6. 
7. 
8. 
9. 
S.J. Singer, L. Eggman, J.M. Campbell, and S . G .  Wildman, 
J. Biol. Chem. - 197, 233 (1952). 
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- 205 , 969 ( 1953) 
J.W. Lyttleton and P.O.P. T'so, Arch. Biochem. Biophys. 
- 73, 120 (1958). 
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N.K. Boardman, Biochem. Biophys, Acta - 62, 63 (1962). 
, Biochem. Biophys. Acta - 64, 279 (1962). 
G .  van Noort, W. Hudson and S . G .  Wildman, Plant Physiol. 
36, Suppl XIX (1961). 
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A. Weissbach, B.L. Horecker and J. Hurwitz, J. Biol. 
Chem. - 218, 795 (1956). 
R.W. Dorner, A. Kahn and S . G .  Wildman, J. Biol. Chem. - 229, 
R.B. Park and N . G .  Pon, J. Mol. B i o l .  3, 1 (1961). 
945 (1957). 
P.W. Trown, Biochemistry - 4, 908 (1965). 
R. Markham, Adv. in Virus Research - 9 ,  241 (1962). 
E. Fuchs, W. Zillig, P.H.  Hofschneider and A. Preuss, 
J. Mol. Biol. - 10, 546 (1964). 
A.J.E. Colvill, E.F.J. van Bmggen and H. Fernandez-Moran, 
to be submitted to Science. 
REFERENCES AND NOTES CONT. 
10. F.H.C. Crick and J.D. Watson, Nature 177, 473 (1956). 
D.L.D. Caspar and A. Klug, Cold Spring. Harbor Symp. 
Quant Biol. 27, 1 (1962). - 
/ / 
11. H. Fernandez-Moran, Circulation - 26, 1039 (1962). 
12. , L.J. Reed, M. Koike, and C.R.Willms, 
Science 145, 3635 (1964). 
13. H.E. Huxley, J. Mol. Biol. - 1, 281 (1963). 
1 f 
14. H. Fernandez-Moran, J. Appl. Phys. - 31, 1840 (1960). 
15. , T. Oda, P.V. Blair, and D.E. Green, 
J. Cell Biol. - 22, 63 (1964). 
16. B.R. Rabin and P.W. Trown, Proc. Natl. Acad. Sci. U.S. 
- 51, 497 (1964) 
17. M.W. Rees, Personal Communication. 
18. We are indebted to Misses C.M. Kunner ana D.A. iviecidofi' 
for their technical assistance in specimen preparation 
and t o  Mrs. L. Deloria for her technical assistance, 
and to Mr. C. L. Hough for expert assistance with photo- 
graphic reproductions. 
19. This work was supported by the following grants: 
AI-14448 and AI-06279, NB-04267 and GM-13243 of the 
United States Public Health Service; by NsG 441-63 
of the National Aeronautics and Space Administration; 
by U.S. Atomic Energy Commission Contract AT-(11-1)- 
1344; by the L. Block Fund; and by the Otho Sprague 
Memorial Fund of The University of Chicago. 
c 
REFERENCES AND NOTES CONT. 
23. RH i s  a r e c i p i e n t  of a Research Career Development 
Award f rom t h e  United S t a t e s  Public Health Service; 
FJK i s  a predoctora l  t r a i n e e  o f  the  United S t a t e s  
Public Health Service;  EFJvB was an  I n t e r n a t i o n a l  
Postdoctoral  Research Fellow o f  t h e  United S t a t e s  
Public Health Serv ice ,  present  address  Laboratorium 
v o o r  Structuurchemie,  Bloemsingel 10 ,  Groningen, 
The Netherlands.  


Electron microscopic and biochemical characterization of Fraction 1 protein

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