Download SUBUNITS FROM REDUCED .AND S

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)].
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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
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150
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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.
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