Planta (Berl.) 79, 254--267 (1968)

Isolation, Properties, and Structure of Fraction I Protein from Avena sativa L. M. W . STE]~$, B. E. S. GU~ING~ T. A. GRAHAM, a n d D. J. CAR~ Department of Botany, Queen's University of Belfast, Belfast 9, ~orthern Ireland ]~eceived ~,Tovember 15, 1967 Summary. 1. A method is described for the extraction and purification of :Fraction I protein from Avena sativa L. leaves. 2. The protein possesses ribulose diphosphate carboxylase activity. Chromatography on gels of Sephadex G-200 separates phosphoribulokinase and ribose phosphate isomerase from the carboxylase. 3. The S~ was calculated to be 18.2, the Stokes radius (determined by gel filtration on a cabibrated column) 74 A, the molecular weight 5.7 • l0 B, and the frictional ratio 1.35. 4. An amino acid analysis is presented. 5. Electron microscope observations of negatively-stained Avena Fraction I protein molecules are compatible with the suggestion that they consist of 24 protomers disposed on the smfface of an octahedral shell with 4:3:2 symmetry, and of diameter approximately 105 A.

Introduction

The isolation a n d p a r t i a l c h a r a c t e r i s a t i o n of F r a c t i o n I p r o t e i n from Avena 8ativa L. was u n d e r t a k e n as p a r t of a p r o g r a m m e of i n v e s t i g a t i o n s of t h e c o m p o n e n t s of t h e s t r o m a of chloroplasts. I n p a r t i c u l a r , we wished to discover if t h e r e were a n y r e l a t i o n s h i p s b e t w e e n t h e a b u n d a n c e of this p a r t i c u l a r p r o t e i n a n d t h e presence in t h e s t r o m a of Avena p l a s t i d s of a fibrillar spherulite t e r m e d for convenience t h e " s t r o m a c e n t r e " ( G u y i n G , 1965) a n d considered to be proteinaceous. A p a r t from this a s p e c t of t h e work, which is d e a l t w i t h in a s e p a r a t e p a p e r ( G u ~ I ~ G , STEER a n d COCHRA~E, in press), t h e i n f o r m a t i o n p r e s e n t e d here is of c o m p a r a t i v e v a l u e : so far n e a r l y all of t h e p u b l i s h e d w o r k on F r a c t i o n I p r o t e i n has been carried o u t using Dicotyledons, a n d i n d e e d one p a r t i c u l a r species - - spinach - - has been t h e source of p r o t e i n in t h e m a j o r i t y of investigations. The p r e s e n t c o m m u n i c a t i o n is concerned w i t h t h e isolation, e n z y m i c c h a r a c t e r i s a t i o n , properties, a n d s u b - s t r u c t u r e of F r a c t i o n I p r o t e i n from Avena 8ativa. * Present address : Department of Botany, University of Wisconsin, Madison, Wisconsin, U.S.A.

Fraction I Protein from Avena

255

Materials and Methods

a) Plant Materials Avena sativa, var Victory, seeds were germinated and grown for 7--9 days in trays of sand under Philips VHO fluorescent lamps providing 750--1,000 ft. candles. The first leaves were harvested when 5 - - 8 cm long.

b) Negative Staining Solutions of pure protein, impure fractions, and crude leaf homogenates were mixed with sodium phosphotungstate solution, p H 7.4, in small beakers. Films of curbon were t h e n floated on the surface of the mixture and samples t a k e n b y iramersing 400-mesh grids and picking up areas of the film from below. After blotting a n d airdrying, the grids were examined at 60 k v using a n A.E.I. E M 6 B fitted with half micron thick 30 or 50 ~ objective apertures (C. French & Co.). Direct magnifications of • 80,000--• 2OO,0O0 were used.

c) Isolation o/Avena Fraction I Protein Preliminary experiments (STEER, 1966; STILES, G U ~ I ~ G and CARE, 1966) demonstrated the value of a two-stage molecular sieve chromatography procedure. The first stage was the removal of m a n y of the low molecular weight contaminants present in the supernatant from 100,000 X G centrifugation of homogenised leaves. Such contaminants were retarded relative to Fraction I protein during passage through a column of Sephadex G-100. The second stage was the purification of selected fractions using Sephadex G-200. The first stage of purification yielded a slightly green solution capable of fixing C1402 into 3-phosphoglycerate when supplied with ribose-5-phosphate and ATP. Electron microscopy of this eluate after negative staining with phosphotungstic acid showed t h a t some fragments of membranous materiM were present in addition to recognisable Fraction I protein molecules and occasional stromacentre fibrils. The second stage resolved two main protein-containing fractions. The first to be Muted was usually slightly green and contained some nucleic acid. The second was colourless, contained less t h a t 0.5% RNA, was free of ribose phosphate isomerase and phosphoribulo-kinase activity, and O.D.2s 0 measurements across the fraction paralleled the activity of ribulose diphosphate carboxylase. Much cleaner preparations were obtained if the P~NA and membrane fragments, normally Muted in the first, peak to come off the G-20O column, were removed prior to the first stage of purification. This was achieved b y incorporating magnesium ions into the homogenisation medium and by centrifuging the homogenate at a higher speed. Peak (a) in Figs. I and 2 represents all t h a t remains of the first peak of RNA-containing material. Presumably the leaf ribosomes retained their structural integrity in the presence of Mg ++ so t h a t 16 S RNA, known to be present in ribosomes, including chloroplast ribosomes (e.g. S~nNCER and W~ITFIELn, 1966) was not released. Ribulose diphosphate carboxylase has been shown to be sensitive to chloride ions (TRows, 1965) and to phosphate (WEIsSBAC~, HORECK~ and HVRWITZ, 1956; PAVLSE~ and LA~E, 1966). The Avena enzyme was found to be similarly inhibited by these ions. These observations, plus previous experience of the protective effect of cysteine and glutathione on the activity of the Avena enzyme (STEER et al., 1966), led to the selection of the following media and procedures:

256

M . W . S~EnR, B. E. S. GV~NING, T. A. GRAI~AM, and D. J. CARR:

Grinding Medium. 0.2 M tris-sulphate, p H 7.4, containing 10-3 M cysteine a n d 3.10 -2 M MgS04. 40--50 gm fresh weight batches of leaves were ground b y pestle and m o r t a r in 25--30 ml of this medium and t h e n strained through muslin before centrffugation a t 157,000 X G (average) for 40 rain. A 10 x 10 ml angle rotor was used on an M.S.E. SS50TC centrifuge. Isolation, Stage 1. Sephadex G-100: Aliquots of the s u p e r n a t a n t were passed into a 2 • 26 cm column of Sephadex G-100 prepared in and Muted with 0.1 M trissulphate, p H 8.0, containing 10-4 M cysteine. The low molecular weight material darkened rapidly upon exposure to air, b u t b y the time this became noticeable, the Fraction I protein had become separated from it. Isolation, Stage 2. Sephadex G-200: Ribulose diphosphate carboxylase was satisfactorily separated from the isomerase and kinase enzymes in either of two ways. A small sample (1.0 ml) of stage 1 eluate was passed into a small column (2.5 x 40 cm) of Sephadex G-200; alternatively, a large sample (up to 40 ml) was passed into a large column (4.8 • 185 cm) of G-200. I n b o t h cases the eluent was 0.03 M tris-sulphate, p H 8.0, containing 10 -4 M cysteine. All of the above operations were carried out a t 2~ Examples of the elution patterns are shown in Figs. 1 a n d 2. d) Enzyme Assays ~) Ribulose Diphosphate Carboxylase (EC. 4.1.1.39). 0.2 ix moles ribulose 1.5-diphosphate (Sigma), 5 ix moles MgSO 4 a n d 3 ~z moles glutathione in 0.4 ml 0.I M tris-sulphate buffer, p H 8.0 were mixed with 4.0 ix moles (0.1 m], activity 1.2 IXC) :Na214COa, a n d the reaction started b y adding 0.1 ml of test enzyme. The reaction mixture was incubated at 25~ for 30 rain. This method is based on the systems used by WEISSBACH, HORECKE~ and HUI~WITZ (1956) and b y RABI~r and T ~ o w ~ (1964); the carbon dioxide concentration is considerably sub-optimM (P~VLSE~ and LA~E, 1966). Other details m a y be found in STnER et al. (1966). fi) RibosephosphateIsomerase (EC. 5.3.1.6). 0.5 ix moles ribose-5~phosphate a n d 2.0 ix moles cysteine in 0.1 ml of 0.2 M tris-sulphate buffer, p H 7.0, were reacted with 0.1 ml of test enzyme at 37~ for 10 rain. The carbazole method of AXEL~On a n d JA~C (1954) was used to assay the ribulose-5-phosphate formed in the reaction. ~) Phosphoribulokinase (EC. 2.7.1.19). 0.4 ml of test enzyme was added to a reaction mixture and incubated a t 37~ for 30 min. The mixture contained 3.0 ix moles adenosine triphosphate, 2.0 ix moles cysteine, 5.0 ix moles MgS04, 1.2 ix moles phospho-enol pyruvate, 0.12 ix moles reduced nicotinamide adenine dinucleotide, 2.0 units of phospho-enol p y r u v a t e kinase (Sigma), 4.0 units of lactic dehydrogenase (Sigma) a n d 2.0 ix moles ribulose-5-phosphate in 2.1 ml of 0.04 M potassium phosp h a t e buffer p H 7.9. The change in O.D.3~0 measured the formation of adenosine diphosphate and hence of ribulose 1.5-diphosphate formation. This method was based on the system used b y Hu~w~Tz, WEISSBAC]I,HORECKER and SMYRN~OT~S (1956).

e) Other Properties o/ Avena Fraction I Protein ct) Stokes Radius. The elution volume of Fraction I protein samples passed through a 2.5 X 45 cm column of Sephadex G-200 already calibrated using blue dextran, alcohol dehydrogenase, thyroglobulin a n d catalase was determined in order to estimate the Stokes radius (SIEGEL and MONTY, 1966). The parameters Kd89 (GELOTTE, 1960) ~nd (--Kay) 89 (LAURENT and KILLANDEI~, 1964) gave essentially identical results when plotted against the Stokes radius of the standards. fi) Amino Acid Analysis. The protein used was t a k e n from the centre of the final Sephadex G-200 peak, corresponding to ribulose diphosphate carboxylase freed from the other two enzymes. The selected samples were concentrated and

Fraction I Protein from Avena

257

again chromatographed on Sephadex G-200. The samples were filtered through cellulose acetate at all possible stages of the preparation, and the final material was essentially bacteria-free. The protein was collected by centrifugation following prolonged (and monitored) dialysis against distilled water. The pellet was washed with water, ethanol, ethanol-ether, and ether, yielding dry protein that could easily be weighed on a Cahn microbalanee and that was h'ee of fortuitously associated lipid (RmLEY, T~OR~BE~ and BAILEY, 1967). Weighed samples were dissolved in 6 N. HC1 and aliquots taken to provide duplicated hydrolysates. Hydrolysis proceeded in 6 N. HC1 at 110~ under nitrogen in sealed tubes. A Technicon 3 column Autoanalyser was calibrated in all columns and used according to standard procedures. Cysteine content was not corrected for losses; methionine was calculated as methionine plus methionine sulphone; tryptophane was estimated by calculating the molar ratio tyrosine: tryptophane from optical density measurements on the protein in alkaline solution (BEAVEN and I-Ior~n)Au 1952) and using the tyrosine value provided by the Autoanalyser; all other residues were estimated from maximum values, or by first-order extrapolations to zero time. Amide nitrogen was not determined. Duplicate samples of 11, 22, 46, 73 and 96 hour hydrolysates were analysed. y) Molecular Weight. Pure protein, at 0.75 mg/ml in 0.03 IV[ tris-HC1 buffer, pH 7.8, of measured density and viscosity, was examined in a Spinco analytical ultracentrifuge. The $20w value obtained was corrected to S~ w using data published by RIDLEu et al. (1967) and this, together with the Stokes radius and partial specific volume (calculated by summation of the contributions of the individual amino acids) provided an estimate of the molecular weight. Results

a) Enzymatic and Physical Properties o/Avena Fraction I Protein The two-stage molecular sieve c h r o m a t o g r a p h y p r o c e d u r e p r o v i d e d a fraction which c o n t a i n e d only one m a j o r p r o t e i n species, as j u d g e d b y a c r y l a m i d e gel electrophoresis. The r a t i o of t h e optical densities a t 280 a n d 260 millimicrons (DE Moss a n d BARD, 1957) was u p to 1.8. This fact, plus n e g a t i v e Orcinol reactions a n d tests for d e o x y r i b o s e all i n d i c a t e d freedom from c o n t a m i n a t i o n b y nucleic acids. The s e d i m e n t a t i o n coefficient (S20w) of a 0 . 7 5 m g m / m l p r e p a r a t i o n was 17.7; t h e Schlieren p a t t e r n was a n o t h e r i n d i c a t i o n of t h e p u r i t y of t h e sample. E n z y m a t i c analyses showed t h a t a t least two enzymes which c o m m o n l y c o n t a m i n a t e F r a c t i o n I p r o t e i n (e.g. M~,~Dmr,A a n d AKAZAWA, 1964) were r e m o v e d (Figs. 1 a n d 2). A t t h e c o n s i d e r a b l y s u b - o p t i m M CO~ concentrations used in t h e c a r b o x y l a s e assay, t h e p r e p a r a t i o n s h a d specific activities of u p to 0.08 ~ moles s u b s t r a t e fixed p e r m g m p r o t e i n p e r m i n u t e a t 25~ The process of molecular sieve c h r o m a t o g r a p h y , used in t h e purificat i o n of t h e Avena ribulose d i p h o s p h a t e carboxylase, also p r o v i d e d a m e a s u r e of its Stokes radius. A linear relationship b e t w e e n a p a r a m e t e r of t h e elution volume a n d the Stokes r a d i u s was e s t a b l i s h e d for one p a r t i c u l a r column a n d for t h r e e p r o t e i n s t a n d a r d s . Using this c a l i b r a t e d column, a n d assuming t h a t F r a c t i o n I p r o t e i n does n o t b e h a v e anomalously, its Stokes r a d i u s was e s t i m a t e d to be 74 A.

258

3I. W. STEEg, B. E. S. GTJ~gr~G, T. A. GgAm~, and D. J. CARt:

~ (b)

I

0.12 0.10 0.08

c~J 30

3_

20

~2~

0.06 0.04

X3

(a)

lO

2.5 x Z~Ocm/ o/

0.02

/

T

25 -o 0.25 -5

.~

/,

/ ~ -o--o ~~

1

50

75

100

150

125

2

m

.u

~z o 0.20

I

a0:--_E 30

0.15

lsomerase +

+

Kinase

20

0.10,

x o

0.05[

•

Corb0xyiase

&8x185cm

r

10

-...--o~ o o~.%2-- . . . .

o

011280 0D260

I

1200

,

A/I

p

I

I

1 600 2 000 Volume (mls)

Fig. 1. Elution profile of ribulose diphosphate earboxylase, phosphoribulokinase, and ribose phosphate isomerase from a 2.5 • 40 cm column of Sephadex G-200 Fig. 2. As Fig. l, but from a 4.8 • ]85 cm column. Initial sample volume 40 m]. PhosphoribuloMnase omitted. In both Figs. peak (a) represents a fraction that is contaminated with RNA (note the low 0D2s0/~60 ratio, Fig. 2), peak (b) is ribulose diphosphate carboxylase, and peak (e) contains the other two enzymes A m i n o acid anMysis of a s a m p l e f r o m which b a c t e r i a h a d been carefully e x c l u d e d gave t h e results shown in t h e table. B y s u m m a t i o n of t h e p a r t i a l specific volumes of t h e c o n s t i t u e n t amino acids t h e p a r t i a l specific v o l u m e of t h e p r o t e i n was c a l c u l a t e d to be 0.73. This value c o m p a r e s well with d a t a for t h e spinach a n d spinach b e e t e n z y m e s ( P A ~ L S ~ a n d LANE, 1966; RIDLEY et al., 1967; P o ~ , 1967). This inf o r m a t i o n on t h e Stokes r a d i u s a n d p a r t i a l specific volume, t o g e t h e r

Fraction I Protein from Avena

259

Table. Amino acid analysis of Avena Fraction I protein. Residues marked with an asterisk were estimated by extrapolation to zero time assuming /irst order brealcdown kinetics. Maximum values were taken in all other cases (except Trp). Column 3 gives the molar ratios in a hypothetical protomer of molecular weight one twenty/ourth that o/the Fraction I protein. Alongside (in brae]cets) are the corresponding values/ound by RIDLEY et al. (1967) in the minimum molecular weight o/ spinach beet Fraction I protein Amino acid residue Asp Thr* Ser* Glu Pro Gly Ala Cys* Val Met Ile Leu Tyr* Phe Lys His Arg Trp

Residue per 100 g protein (gms) 8.58 4.84 2.78 12.26

6.51 4.60 5.42 1.95 5.45 1.94 4.73 7.38 4.81 5.78 5.58 3.19 8.05 1.85

Moles per 23,750 g protein 17.7 (17.8) 11.4 (14.2) 7.8 (10.9) 21.0 (21.0) 13.5 (11.8) 19.2 (19.7) 18.4 (18.2) 4.5 (4.0) 13.1 (18.2) 3.5 (4.0) 10.0 (10.1) 15.5 (19.1) 7.0 (9.0) 9.3 (10.1) 10.4 (12.0) 5.5 (5.9) 12.3 (10.2) 2.6 (3.0)

with the S~ value of 18.2 (obtained by correcting the experimentally determined S20w value of 17.7 for a 0.75 mg/ml solution) imply a molecular weight of 5.7 • l0 B. If the amide nitrogen content is the same as in spinach beet Fraction I protein (RIDLEu et al., 1967), the percentage nitrogen calculated from the amino acid analysis is i6.36 %. RIDL~u et al. (1967) found that the molar ratios of amino acid residues in the spinach beet protein approximate to integral numbers when corrected to 3.0 moles of tryptophane. They point out that the minimum molecular weight they observed is very nearly one twenty fourth of the protein molecular weight. While the analysis of the A r e n a protein is not so precise, it is nevertheless in close agreement with this work. Column 3 of the table compares the molar quantities of residues in one twenty fourth of the calculated molecular weight of A r e n a protein with the quantities found by I~IDL~,Y et al. (1967), setting tryptophane at 3.0. There is a possibly significant difference with regard to the pereentage of amino acids with strongly hydrophobie groups (proline, valine, leueine, isoleueine and phenylalanine, VAN HOLDS, i966). A r e n a Fraction I protein has 30.3%, and spinach beet 27.I %.

260

M.W. STEER, B. E. S. GV~INO, T. A. GRAHAM,and D. J. CA~R:

Figs. 3--9. General: Fraction I protein molecules negatively stained with sodium phosphotungstate. All • Figs. 3--7 pure preparations from Avencb; Figs. 8 and 9 from crude preparations of Phaseolus leaves

Fraction I Protein from Arena

261

b) Negative Staining o/Avena Fraction I Protein The information in this description concerns both pure protein and presumed Fraction I protein in crude extracts. In-focus micrographs provide comparatively little information other t h a n on the outline shape of the particles (Fig. 3). Depending on the depth to which t h e y are embedded in phosphotungstate t h e y m a y also show t h a t the molecules c o m m o n l y have one or occasionally two (Figs. 5--7) dark regions t h a t are presumably due to penetration of phosphotungstate into pits or depressions in the protein surface. This feature is very much more obvious in the case of Phaseolus Fraction I protein (Figs. 8 and 9). The outline m a y be almost square, rectangular, approximately hexagonal, or irregular (Figs. 3 and 4). Portions of the edges (often straight edges) are sometimes seen to be scalloped, with usually not more t h a n two indentations visible along an edge suggesting t h a t at least p a r t of the molecule consists of a row of three subunits (Fig. 3). A few of the molecules in pure preparations present circular, or somewhat distorted ring-shaped profiles (Figs. 6 and 7). Dimers are occasionally seen. High magnification under-focus images (Figs. 10 and 11) are of somew h a t debatable value in t h a t the background granularity is of the same order of magnitude as in the patterns within the particles. Nevertheless a tentative interpretation is possible. Figs. 12--19 compare these patterns with portions of the surface of a model consisting of t w e n t y four spheres arranged in 4 : 3 : 2 s y m m e t r y on the surface of an octahedral shell. Several reasonably "good fits" are shown. I t is suggested t h a t in underfocus images the dark regions of the molecules are less conspicuous simply because the surrounding subunits are more obvious (CHv,sco]~ and AGA~, 1966). I n an in-focus image, particularly if the phosphotungstate layer is thick enough to embed most of a particle, the dark region(s) are more obvious, and scalloped profiles are the only good indication of the presence of subunits. Disposition of subunits is most clearly discerned in particles t h a t must be in an extremely thin layer of phosphotungstate,

Fig. 3. Small arrowheads indicate molecules showing a scalloped edge suggestive of the presence of a row of three subunits. Large arrowheads indicate molecules possessing a dark (negatively-stained) area. Fig. 4. Shows one molecule with an approximately hexagonal profile (small arrowhead), one with a scalloped circular outline (large arrowhead), and others with dark centres Fig. 5. One molecule possesses two dark regions (arrowhead) Figs. 6 and 7. Ring-shaped molecules are present (large arrowheads), as are molecules each with two dark regions (small arrowheads) Figs. 8 and 9. Nearly all PhaseolusFraction I protein molecules have a dark region 19

P l a n t a (BerL), Bd. 79

262

M . W . STEER, B. E. S. G u y i n G , T. A. G~A~A~, and D. J. C A ~ :

Figs. 10--19. General: Avena Fraction I protein molecules, all • 1,500,000. Figs. 17 and 19 from pure preparations, the remainder from crude preparations

Fraction I Protein from Avena

263

as judged by the very low contrast and by comparison with other images (Figs. 12 and 13, cf. Figs. l0 and 11).

Discussion Judging by physical and biochemical data, Fraction I protein from Avena is not markedly different from that of the other species that have been investigated. I t possesses ribnlose diphosphate carboxylase activity, and it can be separated from ribosephosphate isomerase and phosphoribulokinase by the gentle process of molecular sieve chromatography on Sephadex gels. The argument against the formation of a multi-enzyme complex of these three enzymes (CgIDDLE, 1966) is thereby supported. The estimate of its molecular weight, 5.7 • 105, is remarkably close to values given by more accurate procedures for other Fraction I proteins (PAULSEN and LANE, 1966; Port, 1967). Its Stokes radius (74A) is surprisingly large considering the electron microscope observations. Thus, if the model discussed below is correct, then the diameter of the particle as seen in negatively-stained preparations is only in the region of 105 A. The large Stokes radius also implies a high frictional ratio. As measured by the method of SIEGEL and MOnTu (1966), the ratio is in fact 1.35. Again, this high value is unusual in that the electron micrographs show the molecule to be more or less isodiametric. The amino acid analysis of Avena Fraction I protein is similar to that of the spinach beet enzyme (table). Our results do not justify calculation of a minimum molecular weight and hence the number of subunits (protomers - - 1VIONOD,WYNA~ and CHANGEUX, 1965) in the molecule. The work of t{ees [cited by HASELKOan, FEanAnD~z-MoRin, KIE~AS and VAn BI~UGGEN (1965)] and the minimum molecular weight determined by gIDLEY et al. (1967) remain the only biochemical evidence that the molecule consists of twenty four protomers. Other workers have, however, succeeded in dissociating Fraction I protein into components with low sedimentation coefficients (Tnow~, 1965 ; RIDLEY et al., 1967 ; Po~, 1967).

Figs. I0 and l I. Two fields from underfocus micrographs. The background granularity is of the same order of magnitude as the subunits in the molecules Figs. 12--16. Selected molecules together with out-of-focus photographs of a 24-subunit model with 4:3:2 symmetry oriented so as to attempt to match the patterns in the corresponding negatively-stained images Figs. 17--19. These three photographs of the model show views along the fourfold, three-fold and two-fold axes of symmetry. The corresponding electron micrographs are of molecules with somewhat similar symmetry relationships. That shown in Fig. 18 is under focus, while those in Figs. 17 and 19 are closer to focus 19"

264

M.W. STEER, B. E. S. GUNNING,T. A. G R A ~ , and D. J. CARa:

HASELKORN c t a l . (1965) interpreted their electron mierographs of negatively stained chinese cabbage Fraction I protein as indicating t h a t the molecules were cubical, with one subunit in the centre of each of four faces, and three along each edge. They take the propensity of the protein to form linear aggregates as evidence for a unique two-fold axis of symmetry, and suggest t h a t this axis is provided by the existence of an axial hole piercing the molecule from the centre of one to the centre of the opposite face. This hole might also account for the dark regions seen b y other observers (PA~I(, 1966; MILL~, KA~LSSON, and BOArDMAN, 1966; t~IDL~u eta]., 1967; GunNInG et al., in press). Although the square, rectangular, and hexagonal profiles shown b y Arena Fraction I protein are compatible with this model, other features are not. Hollow rings cannot be explained, except by assuming t h a t they represent impurities present in low concentration. More significant, perhaps, is the frequency with which dark regions appear, for on the above cubical model, they should only be seen when the axis of the hole is not too far off the electron optical axis of the microscope. HAS]~LKO~N et al. (1965) acknowledged t h a t their proposed model was not in accord with the principle of quasi-equivalence (CAsPA~ and KLUr 1962). On purely theoretical grounds the suggestion t h a t twenty four protomers are disposed on the surface of an octahedral shell with 4 : 3 : 2 s y m m e t r y is attractive (C~ICK and WATSON, 1956; HOaNE and WILDY, 1961 ; CASPAR and KLUG, 1962). They could be grouped as eight trimers or six tetramers by combinations of isologous and heterologous associations (JAcoB et al., 1965; H ~ s o ~ , 1966). Such a model accounts for several of the features observed in negatively stained preparations of Avena Fraction I protein. Dark regions could be the points where the three four-fold axes intersect the surface of the shell (Figs. 17 and 19). Since there are six such points, the frequency with which the dark regions are observed is no longer remarkable. A view along a two-fold axis could show two dark regions within one particle (e.g. Fig. 19). Hollow rings could be given if phosphotungstate penetrated and filled the interior of the shell. I t m a y be t h a t the molecules have to be damaged for this to occur, but whatever the mechanism, the process has been observed to occur in other small shell structures (T~oMANS and Hogtie, 1961). We have a t t e m p t e d to obtain photographically reinforced images of these ring views, using the rotational method of MA~KUAM, F ~ Y and HILLS (1963), and in different instances, have obtained either no reinforcement, or rotation values ranging between seven and ten subunits, with nine as the most frequent result. Finally there is the question of the overall outline of the molecules. An octahedron is basically a body with cubic symmetry, and in views, e.g. along the four-fold axes (Fig. 17), very high resolution would be

Fraction I Protein from Mvena

265

needed to show that its outline is not quite square. The phage 0 X 1 7 4 studied by TROMANS and HORNE (1961) may again be taken to illustrate this point. Although this ieosahedron is some two and a half times the diameter of the Fraction I protein molecule, it too can appear to have a nearly square outline, especially in low magnification views. With the smaller particle, correspondingly higher resolution would be required to detect the true shape. Hexagonal outlines are possible, and not necessarily on the threefold axes only. I t may be surmised that rectangular outlines are given by particles that are just touching a very thin layer of negative stain, i.e. are only partially embedded. At almost any orientation there are edges which could be seen as three subunits separated by two indentations (eft Figs. 12--19). While the above model accounts for many of the features of negatively stained Avena Fraction I protein, it should not be taken as proven. The resolution in the micrographs is too low to justify any such claim, and the likelihood of distortion when the grids are being air-dried remains as an unknown quantity. Neither can the model be applied in detail to the published mierographs of protein from spinach (PARK, 1966), spinach beet (~:~IDLEY et al., 1967), Phaseolus (HITCHBORlV and HILLS, 1965; GUSNI~O et al., in press), and tobacco (MILLEl~et al., 1966). There is no compelling evidence to show that Fraction I protein from different plants has the same composition and construction. The rather striking difference between Avena and Phaseolus protein is significant in this connection (Figs. 3, 8 and 9). Finally, it should be pointed out that the proposed models all assume the presence of twenty four identical protomers. The evidence for this is slender, and if the assumption turns out to be incorrect, then interpretations of the shell structure of the protein will have to be reconsidered, and will be much more difficult. The financial support of the ScienceResearch Council is gratefully acknowledged. We wish also to thank the staff of the Queen's University Electron Microscopy Laboratory and Drs. J. LEGGETTBAILEY,P. THORNBERand S. I:~IDLEY,Of Twyford Research Laboratories, for advice and for the use of their analytical ultracentrifuge.

References

AXELROD,B., and R. JANG: Purification and properties of phosphoriboisomerase from alfalfa. J. biol. Chem. 209, 847--856 (1954). BEAVEI%G. H., and E. R. HOLIDAY:Advanc. Protein Chem. 7, 319--328 (1952). CASPAR,D. L. ]~., and A. KLUG: Physical principles in the construction of regular viruses. Cold Spr. Harb. Syrup. on quant. Biol. 27, 1--24 (]962). CHESCOE,D., et A.W. AGAR: Interpretation des mierographies en tr~s haute r6solution. J. Mieroscopie 5, 91--94 (1966). CR~CK,F. It., and J. D. WATSON:Structure of small viruses. Nature (Lond.) 177, 473--475 (1956).

266

M.W. STEE~, B. E. S. GUNNING, T. A. GRAHAm, and D. J. C~m~:

CRIDDLE, R. S. : Protein and lipoprotein organisation in the chloroplast. In: Biochemistry of chloroplasts, vol. I, p. 203--232 (T. W. GOODWIN, ed.). New York and London: Academic Press 1966. GELOTTE, B. J. : Studies on gel filtration: sorption properties of the bed material " S e p h a d e x " . J. Chromatogr. 3, 330--342 (1960). GUYING, B. E. S. : The fine structure of chloroplast stroma following aldehyde osmium-tetroxide fixation. J. Cell Biol. 24, 79--93 (1965). --IVL W. STEER, and M.P. COCI-YRANE: Occurrence, molecular structure and induced formation of the "stromacentre" in plastids. J. Cell Sci. (in press). HANSON, K. R. : Symmetry of protein oligomers formed by isologous association. J. molec. Biol. 22, 405--409 (1966). HASELKORN,1~., H. FERNJ~NDEZ-MoRJ~N,F. J. KIElCAS,and E. F. J. VAN BRVGGEN: Electron microscopic and biochemical characterisation of Fraction I protein. Science 150, 1598--1601 (1965). HITO~BO~N, J. H., and G. J. HILLS: The use of negative staining in the electron microscopic examination of plant viruses in crude extracts. Virology 27, 528-540 (1965). K. E. VAN HOLDE: The molecular architecture of multichain proteins. In: Molecular architecture in cell physiology, p. 81--96 (T. HAYAS~I and A. G. SZENT-GYoRGI, eds). New Jersey: Prentice-Hall Inc. 1966. I-IoRNE, R. W., and P. WILDY: Symmetry in virus architecture. Virology 15, 348-373 (1961). ]-IURWITZ,J., A. WEISSBACH, B.L. HORECKER, and P. Z. SMYRNIOTIS: Spinach phosphoribulokinase. J. biol. Chem. 218, 769--783 (1956). LAURENT, T., and J. KILLANDER: A theory of gel filtration and its experimental verification. J. Chromatogr. 14, 317--330 (1964). M A R ~ : ~ , 1~., S. FaEY, and G. J. HILLS: Methods for the enhancement of image detail and accentuation of structure in electron microscopy. Virology 20, 88--102 (1963). MENDIOLA, L., and T. AKAZAWA:Partial purification and the enzymatic nature of Fraction I protein of rice leaves. Biochemistry 3, 174--179 (1964). MILLER, A., V. KAtCLSSO~I,and N. K. BOA~DMAN:Electron microscopy of ribosomes isolated from Tobacco leaves. J. molee. Biol. 17, 487--489 (1966). MONOD, J., J. WYMAN, and J. P. C~ANG~.VX: On the nature of allosteric transitions: A plausible model. J. molec. Biol. 12, 88--118 (1965). PARK, R. : Chloroplast structure. The chlorophylls, (L. P. VERNON and G. R. SEELY, p. 283---312. New York and London: Academic Press 1966. PAULSEN, J . M . , and M.D. LANE: Spinach ribulose diphosphate carboxylase. I. Purification and properties of the enzyme. Biochemistry 5, 2350--2357 (1966). Po~, N. G. : Some physical properties of purified fraction I protein from spinach chloroplasts. Arch. Biochem. 119, 179--193 (1967). I~ABIN, B. R., and P. W. TROWS: Mechanism of action of carboxydismutase. Nature (Lond.) 292, 1290--1293 (1964). •IDLEY, S. M., J. P. THORNBER, and J. L. BAILEY: A study of the water-soluble proteins of spinach beet chloroplasts with particular reference to fraction I protein. Biochim. biophys. Acta (Amst.) 140, 62--79 (1967). SIEGEL, L. M., and K. J. MONTY: Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Biochim. biophys. Acta (Amst.) 112, 346--362 (1966). SPENCER, D., and P. R. WHITFIELD : The nature of the ribonucleie acid of isolated chloroplasts. Arch. Biochem. 117, 337--346 (1966).

Fraction I Protein from Avena

267

STEER, ~/i. W. : The loealisation and biochemical properties of Fraction I protein in Arena sativa seedlings. Thesis Queen's University Belfast 1966. -

B. E. S. Gv~I~rG, and D. J. CA~R: Fraction I protein from oat leaves. In: Biochemistry of chloroplasts, vol I, p. 285--291 (T. W. Gool)wI~, ed.). New York and London: Academic Press 1966. TROMA~S, W. J., and R . W . HORNE: The structure of bacteriophage OX 174. Virology 15, 1--7 (1961). TRows, P . W . : An improved method for the isolation of Carboxydismutase. Probable identity with Fraction I protein and the protein moiety of protochlorophyll holoehrome. Biochemistry 4, 908--918 (1965). WEISSBACII, A., B. L. HORECKER, and J. HURWITZ: The enzymatic formation of phosphoglyeeric acid from ribulose diphosphate and carbon dioxide. J. biol. Chem. 218, 795--8]0 (1956). -

Dr. B. E. S. GUNNING Department of Botany The Queen's University David Keir Building, Stranmillis Road Belfast 9, Northern Ireland