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SUBUNITS FROM REDUCED .AND S-CARBOXYMETHYLATED RIBULOSE DIPHOSPHATE CARBOXYLASE (FRACTION I PROTEIN) By K. E. MOON* and E. O. P. THOMPSONt [Manuscript received August 21, 1968] Summary Fraction I protein isolated from spinach beet chloroplasts was purified by ammonium sulphate fractionation and Sephadex G·200 gel filtration. The isolated protein was reduced and S-carboxymethylated, and the dissociated protein resolved into two distinct subunits by gel filtration on Sephadex G-200 in an 8M urea buffer at pH 10·0. A comparison of the amino acid composition of the two subunits showed distinct differences between them. The molecular weights of the two protein subunits ""ere estimated to be 54,000 and 16,000 by comparison of their elution volume on gel filtration with elution volumes of reduced and carboxymethylated proteins of known molecular weight. I. INTRODUCTION The enzymic properties of fraction I protein have been studied extensively since its isolation from green leaves by Wildman and Bonner (1947). However, only recently has the subunit structure of fraction I protein been investigated in any detail Haselkorn et al. (1966) suggested, from the amino acid composition and number of tryptic peptides of cabbage leaf fraction I, that it consists of24 identical subunits each of molecular weight 22,500. From electron micrographs they were able to propose a model of a cube with three subunits along each edge, and one in the centre of each of four faces. Furthermore, Ridley, Thornber, and Bailey (1967) derived a minimum molecular weight of 24,427 from the amino acid analysis of fraction I protein isolated from spinach beet. However, there is increasing evidence to suggest the non-identical subunit organization of the protein. Rutner and Lane (1967) dissociated the protein with sodium dodecyl sulphate and isolated, by gel filtration, two subunits having different electrophoretic mobilities, sedimentation velocities, and amino acid compositions. Sugiyama and Akazawa (1967) also observed multiple bands when samples offraction I protein (isolated from wheat leaves) were cleaved with sodium dodecyl sulphate and examined in acrylamide gels. '" School of Botany, University of New South Wales, Kensington, N.S.W. 2033. School of Biochemistry, University of New South Wales, Kensington, N.S.W. 2033. t Aust. J. biol. Sci., 1969,22, 463-70 464 K. E. MOON AND E. O. P. THOMPSON This paper reports the isolation of two non-identical subunits from fraction I protein of spinach beet chloroplasts using an 8M urea buffer solution as a disaggregating agent. II. MATERIALS AND METHODS (a) Plant Material Young loaves from spinaoh beet (Bcta 'Vulgaris) grown out-of-doors were used. (b) Chemicals Acrylamide monomer, N,N-methylenebisacrylamide, and ammonium persulphate were purchased from Cyanimid Australia Pty Ltd., and N,N,N',N'-tetramethylethylenediamine from Eastman Organic Chemicals; all were used without further purification. D-Ribulose-l,5-diphosphate tetrasodium salt was purchased from Sigma Chemical Co. All urea solutions were deionized by passage through a mixed bed of ion-exchange resin before adding the buffer salts. The 18/32 Visking cellulose tubing was boiled in changes of distilled water before use (Hughes and Klotz 1956). The proteins used for calibrating the Sephadex G-200 column were commercial crystalline samples and consisted of bovine plasma albumin (Sigma Chemical Co.), lysozyme, ovalbumin, and ,B-lactoglobulin (Pentex Incorp.). (c) Preparation of Chloroplasts and Isolation of Fraction I Protein The chloroplasts were isolated from the leaves of spinach beet (160 g) using the method of Ridley, Thornber, and Bailey (1967). The isolated chloroplasts were ruptured in o· 01M Tris-HCIo·IM KCI-O' OOlM EDTA-O' 01M mercaptoethanol (pH 8·3) and left for 30 min. The resultant slurry was centrifuged at 38,000 g for 20 min and the supernatant made 35% saturated with ammonium sulphate. "Saturated" was defined as being 70 g of ammonium sulphate per 100 ml of original solution (Trown 1965). After 1 hr the precipitate was removed by centrifuging at 38,000 g for 10 min. The supernatant was made 45% saturated with ammonium sulphate and left for 1 hr. The precipitate was collected by centrifuging at 12,000 g for 10 min dissolved in the minimum amount of homogenizing buffer (3-5 ml), and submitted to gel filtration through a Sephadex G-200 column (44 by 2·5 cm). The temperature was maintained at 0-4°C throughout the preparation. Elution was effected using the homogenizing buffer, and fractions monitored for protein at 280 m", and ribulose-l,5-diphosphate carboxylase activity. The peak corresponding to carboxylase activity was collected and aliquots taken for electrophoresis and protein estimation. The remainder of the fraction I solution was freeze-dried in the presence of an amount of urea to facilitate solution of the material and to give an 8M urea solution with the added water. Samples for electrophoresis were dialysed against electrophoresis buffer, whilst samples for protein estimation were dialysed against water. Protein was determined by the method of Lowry et al. (1951) using bovine plasma albumin as a standard. (d) Preparation of Reduced and Carboxymethylated Fraction I Protein The fraction I protein freeze-dried in the presence of urea was reduced and carboxymethylated by a method similar to that of Crestfield, Moore, and Stein (1963). To samples of freeze-dried fraction I protein (30-70 mg) the correct amount of water was added to make the final solution 8M with respect to urea. The flask was flushed out with nitrogen and redistilled mercaptoethanol was added to give a final concentration of 0 ·14M. '1'he pH was taken to 10·5 with 5M potassium hydroxide and left under nitrogen for 3 hr at room temperature. After 3 hr iodoacetic acid (2'68 g per 1· 0 ml of mercaptoethanol) was dissolved in 3M Tris-HCl (pH 8·5) (8·3 ml 3M Tris-HCl per 2·68 g iodoacetic acid), added to the flask, and the pH adjusted to 8·5 SUBUNITS FROM FRACTION I PROTEIN 465 with 5M potassium hydroxide if necessary. The alkylation was allowed to proceed 5-10 min at pH 8·5. The reagents were removed by dialysis for 3-4 hr against 8M urea. The pH was raised to 10 by the addition of concentrated ammonia and the reaction mixture submitted to gel filtration through Sephadex G-200 containing 8M urea buffer at 4°C. For gel filtration a column (2, 5 by 44 cm) of Sephadex G-200 (that retained by a 300-mesh sieve) was used. The urea buffer used for equilibrating and developing was 8M urea-O· 05M Tris-HCl-O· OObr EDTA-O ·lM KCI-0·2M ammonia (pH 10·0). At pH 10 there should be no carbamylation of IX- and <-amino groups (Thompson and O'Donnell 1966). The fractions were monitored for protein at 280 mfl-. The required fractions were pooled and dialysed against water, 0 ·lM KCl-O· 001M borate, and finally water before freeze-drying. The S-carboxymethyl (SCM) derivates of bovine plasma albumin, ,a-lactoglobulin, lysozyme, and ovalbumin were prepared in essentially the same manner as for SCM-fraction 1. (e) Gel Electrophore8i8 Disk electrophoresis in 5· 0 % polyacrylamide gels was performed using the following buffer systems: (1) The glycine-Tris discontinuous buffer system of Davis (1964); (2) 0·05M Tris-HCI (pH 8·3); (3) 0'05M 2-methyl-2-aminopropanol-HCl (pH 10'3). All buffers contained O' 001M sodium thioglycollate to prevent oxidation of -SH groups. As an added precaution gels run in the homogeneous buffer systems 2 and 3 were equilibrated before use by application of a potential difference of 120 V for 25 min. Urea gels were prepared to give a final urea concentration of 8M. Electrophoresis in urea was performed using buffer l. Electrophoresis was carried out for 60 min at 120 V and 4-5 rnA per tube. After electrophoresis the gels were stained for 15 min in a solution of 1 % amido black in 7% acetic acid and destained by washing for 24 hr with numerous changes of 7% acetic acid. (1) Ribulo8e-l,5-dipho8phate Carboxyla8e A88ay This determination involved the measurement of acid-stable radioactivity produced in the reaction between ribulose-l,5-diphosphate and NaH14C03 (Rabin and Trown 1964). The components of the reaction mixture and the procedure followed were the same as that used by Ridley, Thornber, and Bailey (1967) except that 1 fl-Ci of NaH14C0 3 was used. Aliquots of the acid-stable radioactivity were counted in a Nuclear Chicago gas-flow counter. (g) Amino Acid AnalY8i8 Freeze-dried samples were hydrolysed with redistilled 6N Hel in vacuo by the method of Crestfield, Moore, and Stein (1963) for 24 hr and analysed using a Beckman amino acid analyser, model 120C. III. RESULTS (a) Preparation and Purity of Fraction I Protein A typical elution curve of the 35-45% ammonium sulphate fraction from the ruptured chloroplasts, together with ribulose-l,5-diphosphate carboxylase activities across the protein peak, are shown in Figure 1. The elution pattern after the main protein peak has been observed to be variable; this is probably due to the variation in the concentration of soluble proteins in the chloroplasts over the different seasons. 466 K. E. MOON AND E. o. P. THOMPSON The ribulose-1,5-diphosphate carboxylase activity is associated with the main protein peak, but has a slight tendency to be closer to the trailing boundary, a result similar to that found by Ridley, Thornber, and Bailey (1967) and Thornber, Ridley, and Bailey (1966). Electrophoretic examination of the main peak from the gel ffitration revealed that the bulk of the protein migrated as a single band [Figs. 3(a), 3(b), and 3(c)]. ! Fig. 1 0-6 ::l.. 0-5 E ~ 04 0; .~ ~ ] 8- '0·3 02 0-1 24 20 Fig. 2 .c :~ 0-7 ~ & =: ,.!g-€. '"Ii J III :f~ / 1 12 ~ ;1 -0_ 20 40 60 0 80 ~ !J ::l.. E o<:f) 0-4 of 03 -15 0-2 6" /\ 0-5 '"0; -c -;;; j\ 0-6 0 I \ l ° 0-1 oj ~ o la..", \ / 10 Tube number 20 \ B A \ \--0-0_0_0 I t . 30 40 50 I 0 . 0 60 70 80 Tube number Fig. I.-Gel filtration on Sephadex G-200 of 22 mg of a 35-45% ammonium sulphate fraction of the chloroplast extract. 0 Absorbance at 280 mI'. D. Ribulose-1,5-diphosphate carboxylase activity. See text for assay procedure. Fraction size 6 ml; flow rate 24 ml/hr; eluent O·OlM Tris-HCI-0·1M KCI-O·OOIM EDTA-0·01M mercaptoethanol, pH 8·3. Column 2·5 by 44 cm. Fig. 2.-Gel filtration on Sephadex G-200 of 30 mg of SCM-fraction I in 8M urea-O· 05M Tris-HCI-0·001M EDTA-O ·lM KCI-O· 2M ammonia, pH 10· o. Fraction size 4 ml; column dimensions 2 5 by 44 cm. 0 (b) SCM-fraction I Examination of the reduced and carboxymethylated protein in 8M ureaacrylamide gels revealed that the protein migrated as one band at high loadings of protein [Fig. 3(d)], but at lower protein concentrations there was evidence of splitting (Fig. 4). A band which is not stained intensely by amido black is seen to move slightly faster than the major band. Gel filtration of SCM-fraction I on Sephadex G-200 in the presence of 8M urea resolved the protein into two major subunits of different molecular weights (Fig. 2). Both these peaks, when electrophoretically analysed, revealed their correspondence to the two bands seen in the light-loaded 8M urea gels [Figs. 3(e) and 3(f)J. An estimate ofthe molecular weight of reduced and carboxymethylated proteins in 8M urea solutions may be obtained by comparison of its elution volume on gel ffitration with the elution volume of SCM-proteins whose molecular weights are known. In the presence of 8M urea the proteins are likely to behave as randomly coiled molecules (Thompson and O'Donnell 1965). SUBUNITS FROM FRACTION I PROTEIN 467 The relationship between the elution volume and the logarithm of molecular weight for a series of reduced and carboxymethylated proteins of known molecular Fig. 3 Fig. 4 Fig. 3.-Polyacrylamide gel electrophoresis patterns of fraction I protein at various pH values (a, b, and c) and after S-carboxymethylation (d, e, andf). (a) o· 05M Tris-HCl, pH 8·3; (b) O· 05M 2-methyl-2-aminopropanol-HCl, pH 10·3; (e) glycine-Tris discontinuous system, which gives a running pH of about 9·5. Approximately 60 (Lg of protein loaded in each case. (d) 8M urea gel of c. 100 (Lg of SCM-fraction 1. (e) and (f) 8M urea gels of c. 50 (Lg of peaks A and B respectively as indicated in Figure 2 from the gel filtration of SCM-fraction I on Sephadex G-200. Fig. 4.-8M urea-acrylamide gel pattern of a light-loaded (c. 50 (Lg) SCM-fraction I, showing distinct splitting of the band into two major components. Origin is not shown; arrow indicates the direction of protein migration. weight is shown in Figure 5. The molecular weights taken for these proteins were: plasma albumin 67,000 (Putman 1965), ovalbumin 45,000 (Warner 1954), lysozyme 14,300 (Canfield 1963), and ,B-lactoglobulin 18,300 (Piez et al. 1961). Allowances were made for the introduction of the carboxymethyl groups. The elution volumes of the 200 "'--] 150 E Lysozyme .~ SCM-fraction I (peak B) ~-LactOglo~~ ..g,. ~·~_SCM-fraction Ovalbumin c v ..2 ::::: I ~. (peak A) Bovine plasma albumin ~ 100 W 50! 4·0 ! H 4·2 4·3 4A 4·5 4·6 4·7 log 10 (Molecular weight) 4·8 4·9 5·0 Fig. 5.-Graph of elution volume against logarithm of the molecular weight for a series of S-carboxymethyl proteins chromatographed on Sephadex G-200 in 8M urea buffer. Approximately 5 ml containing 30-60 mg of proteins was loaded in each case. K. E. MOON AND E. O. P. THOMPSON 468 two peaks (A and B) are consistent with proteins having molecular weights of approximately 54,000 and 16,000 respectively. The tubes indicated by the bars in Figure 2 were pooled, and after dialysis and freeze-drying were subjected to amino acid analyses. The results are given in Table 1 and clearly indicate that the two peaks constitute different protein chains, and not aggregates of some basic unit. TABLE 1 AMINO ACID COMPOSITIOX OF THE SUBUNIT FRACTIOXS OF REDUCED AXD CARBOXYMETHYLATED FRACTION I PROTEIN Time of hydrolysis 24 hr at 110 0 e. Values are the number of residues per mole taking the molecular weights of peaks A and B as 54,000 and 16,000 respectively. Average values from one hydrolysatc from three different preparations are given. Values were calculated from the number of /-Lmoles of amino acids recovered from the column, assuming an average residue weight of 1l0. Values in parentheses are calculated from the data of Rutner and Lane (1967) for subunits obtained from fraction I protein treated with sodium dodecyl sulphate assuming identical phenylalanine contents Amino Acid Peak A Peak B Amino Acid Lysine Histidine Arginine S-Carboxymethylcysteine Aspartic acid Threonine* Serine* Glutamic acid 25·9 (26·6) 14·0 (15·1) 28·3 (32·4) 11· 7 (9·8) 1·7 (3·7) 4·7 (8·0) 7·9 43·9 31·6 19·9 51·8 2·8 1l·7 (17·3) 6·2 (9·6) 7·4 (6·2) 17·6 (18·3) Proline Glycine Alanine Valino Methionine Isoleucine Leucine Tyrosine* Phenylalanine (49 ·1) (39·4) (18·2) (49·5) Peak A 23·9 50·1 47·9 35·3 7·5 19·7 43·0 17·1 22·5 (25·4) (47·2) (48·6) (9·5) (19·8) (47·2) (20·7) (22·5) Peak B 1l·6 (12·6) 12·8 (9·3) 8·8 (7·0) 1l·4 2·3 (3·7) 5·7 (4·5) 12·5 (13·2) 8·0 (12·6) 8·1 (8·1) * Uncorrected for decompositions. IV. DISCUSSION Fraction I protein is a major protein of chloroplasts, comprising at least 25% by weight of the dry chloroplasts (Kupke 1962). The function of this large amount of protein is that of an essential enzyme in the fixation of carbon dioxide by ribulose-l,5-diphosphate to produce phosphoglyceric acid. As in the case of other enzyme systcms, e.g. aspartate transcarbamylase (Gerhart and Schachman 1965), or zymogens, e.g. procarboxypeptidase (Brown et al. 1963), there is some evidence suggesting that fraction I protein is comprised of different subunits (Rutner and Lane 1967; Sugiyama and Akazawa 1967). In this paper we have reported values for the molecular weight and amino acid composition of two subunit species of fraction I prepared under disaggregating conditions after blocking the sulphydryl groups. Previous work on the separation of subunits by Rutner and Lane (1967) gave two fractions for which only preliminary amino acid analyses and sedimentation coefficients were reported. The values for SUBUNITS FROM FRACTION I PROTEIN 469 amino acid analyses reported here (Table 1) are in general agreement with those reported by the above workers. So far as molecular weight is concerned, our data support the observed difference in S values reported by Rutner and Lane (1967) for sedimentation in sodium dodecyl sulphate solutions, and give particular values based on the assumption that in 8M urea the proteins behave as random coils and give a valid molecular weight by comparison of their elution volumes on gel ffitration with those of other well-characterized proteins. The electrophoretic separation reported here is not as good as that of Rutner and Lane (1967), but in this case the separation is a true indication of charge difference compared with the molecular weight basis of the separation in sodium dodecyl sulphate. The large molecular weight of fraction I protein (values in the range 5-6 X 105 have been reported) has hindered concise biochemical studies. The preparation of the subunits of fraction I protein by the use of disaggregating agents such as urea should allow definitive chemical studies to be performed. However, a study of the enzymic or possible regulatory role of the subunits will only be possible when the protein can be dissociated by agents which do not irreversibly denature the enzyme, and investigations along these lines are in progress. V. ACKNOWLEDGMENTS The authors wish to thank Mr. R. Whittaker for performing some of the amino acid analyses and Dr. R. S. Vickery for assistance with acrylamide gel electrophoresis. The work was supported in part by the Australian Research Grants Committee. VI. REFERENCES BROWN, J. R., GREENSHIELDS, R. N., YAMASAKI, M., and NEURATH, H. (1963}.-Biochemistry 2, 867. CANFIELD, R. E. (l963}.-J. biol. Chem. 238, 2691. CRESTFIELD, A. M., MOORE, S., and STEIN, W. H. (1963}.-J. biol. Chem. 238, 622. DAVIS, B. J. (1964}.-Ann. N.Y. Acad. Sci. 121, 404. GERHART, J. C., and SCHACHMAN, H. K. (1965}.-Biochemistry 4, 1054. HASELKORN, R., FERNANDEZ.MoRAN, H., KIERAS, F. J., and BRUGGEN, E. F. J. VAN (1966).Science, N. Y. 150, 1598. HUGHES, T. R., and KLOTZ, 1. M. (1956}.-"Methods of Biochemical Analysis." Vol. 3, p. 265. (Interscience Publishers, Inc.: New York.) KUPKE, D. W. (1962}.-J. biol. Chem. 237, 3287. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951}.-J. biol. Chem. 193, 265. PIEZ, K. A., DAVIE, E. W., FOLK, J. E., and GLADNER, J. A. (1961}.-J. biol. Chem. 236, 2912. PUTMAN, F. W. (1965}.-In "The Proteins". (Ed. H. Neurath.) Vol. 3, p. 153. (Academic Press, Inc.: New York.) RABIN, B. R., and TROWN, P. W. (1964}.-Proc. natn. Acad. Sci. U.S.A. 51, 497. RIDLEY, S. M., THORNBER, J. P., and BAILEY, J. L. (1967).-Biochim. biophys. Acta 140, 62. RUTNER, A. C., and LANE, M. D. (1967}.-Biochem. biophys. Res. Comm. 28, 531. SUGIYAMA, T., and AKAZAWA, T. (1967}.-J. Biochem., Tokyo 62, 474. THOMPSON, E. O. P., and O'DONNELL, 1. J. (1965}.-Aust. J. biol. Sci. 18, 1207. 470 K. E. MOON AND E. O. P. THOMPSON THOMPSON, E. O. P., and O'DONNELL, I. J. (1966).-Aust. J. biol. Sci. 19, 1139. THORNBER, J. P., RIDLEY, S. M., and BAILEY, J. L. (1966).-N.A.T.O. Advanced Study Institute, Biochemistry of Chloroplasts, Aberystwyth. Vol. I, p. 275. TROWN, P. W. (1965).-Biochemistry 4, 908. WARNER, R. C. (1954).-In "The Proteins". (Eds. H. Neurath and K. Bailey.) Vol. 2, pt. A, p. 435. (Academic Press, Inc.: New York.) WILDMAN, S. G., and BONNER, J. (1947).-Archs Biochem. 14, 381.