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Structure and function of chloroplast proteins
SRCHIVES
OF
BIOCHEMISTRY
AND
Structure
BIOPHYSICS
and
Function
II. Effect of p-Chloromercuribenzoate Carboxylase
Activity
T. SUGIYAMA, Research
Institute
98-106
126,
of Chloroplast Treatment
of Spinach
N. NAKAYAMA,
for Biochemical
(1968)
Proteins’
on the Ribulose Leaf Fraction
M. OGAWA,
Regulation, Nagoya University, h’agoya, Japan
1, 5Diphosphate
I Protein*
T. AKAZAWA3 School
of Agriculture,
Chikusa,
AND
T. ODA Cancer
Institute, Received
Okayama October
University 16,1967;
School accepted
of Medicine, November
Okayama,
Japan
25,1967
Fraction I protein was purified from spinach leaves by Sephadex gel filtration and DEAE-cellulose column chromatography. To study the role of SH-groups in the enzyme molecule, p-chloromercuribenzoate (PCMB) titration of SH-groups was carried out with a parallel determination of enzyme activity. It was found that the rate and extent of reaction with PCMB was identical in the presence or absence of urea (4.5 M), sodium dodecyl sulfate (2 X 1OW M), and ribulose 1,5-diphosphate (RuDP) (1.1 X lo+ M). The total number of SW-groups per mole protein was 96, in agreement with chemical data. Approximately 10 SH-groups were blocked before an appreciable loss of the RuDP-carboxylase activity occurred, and complete inhibition of enzyme activity was associated with the blocking of about 30 SH-groups. The possible role of SH-groups in the structural rigidity of the protein molecule was suggested by the finding that proteolytic digestibility (chymotrypsin and Nagarse) of the protein was greatly enhanced by PCMB pretreatment as measured by decrements of RuDP-carboxylase activity. Full restoration of the enzyme activity by the additionof cysteine to the PCMB-inactivated enzyme protein was accompanied by restoration of resistance to proteolytic attack. That the molecule is restored to a conformation possibly identical with that of the native protein in tertiary structure was supported by electron microscopic observations of the reconstituted protein.
Ribulose 1, 5diphosphate4 carboxylase, which may be identical with fraction I protein, occupies a pivotal role in the photosynthetic carbon reduction cycle in chloroplasts of green leaf tissues (4). The uniquely
large molecular size (5) and conformation of the protein from subunits (6, 7) have prompted us to investigate its structure and function more precisely. Previous studies with SH-blocking agents such as PCMB and IAA have shown the important role of SHgroups in RuDP-carboxylase (7-11). A particularly notable study by Trown and Rabin (11) demonstrated that only two SH-groups are available for the substrate (RuDP) binding site per enzyme molecule. Our own study on wheat fraction I protein has shown that there is a close correlation between
‘This research was supported in part by USPHS research grant AM-10792-01. 2Parts of the present investigation have appeared in preliminary forms (l-3). 3To whom requests for reprints should be sent. ‘Abbreviations used : IAA, iodoacetamide ; RuDP, ribulose 1,5-diphosphate; SDS, sodium dodecyl sulfate; PCMB, p-chloromercuribenzoate. 98
STRUCTURE
AND
FUNCTION
decay of enzyme activity and the number of PCMB titratable SH-groups (7). Although RuDP protects the enzyme from PCMB inactivation, the number of total SH-group(s) titratable by PCMB was essentially the same regardless of the pretreatment of the protein withRuDP. Itisthuspossibletopostulatethat only a minor portion of the SH-groups is catalytically essential to the enzyme reaction. However, it is often difficult to demonstrate the catalytic role of SH-groups in an enzyme reaction, particularly when conformational changes of the molecule result from treatment with the SH-blocking agents (12). To test the possible conformation change of fraction I protein induced by the PCMBtreatment, we investigated the selective proteolytic digestion of spinach fraction I protein in a parallel determination of the RuDPcarboxylase activity. Also the reversible structural reconstitution of protein molecule by cysteine addition has been examined by high-resolution electron microscopy. MATERIALS
AND
METHODS
PuriJication of protein. The methods of extraction and fractionation of spinach leaf fraction I protein were based on the previous report for purifying the protein from rice and wheat leaf, with some modifications (7, 13). Crude leaf protein extract precipitated with (NH,)aS04 at 50yo saturation was first applied to a Sephadex G-25 column, and then to Sephadex G-200 gel filtration. The eluting solution was 0.025 M Tris buffer (pH 7.5) containing 0.1 mM EDTA. Protein fractions were collected and reprecipitated at 50°jc, saturation of (NH&SO*. After being desalted by passage through a column of Sephadex G-25, the protein was next applied to a column of DEAEcellulose and eluted with 0.01 M Tris buffer (pH 7.5) containing 0.1 M EDTA and NaCl in a linear gradient (O-l M). A typical elution pattern is shown in Fig. 1, which represents an improvement over previous reports of the protein resolution (5, 7). A colorless enzymically active sample (tubes 60-71) showed a typical ultraviolet spectra of protein, with an absorbance ratio (280/260 rnp) of 1.6-1.7 and a shoulder at 292 rnp. It was homogeneous by analytical ultracentrifugation, with a single boundary of ~20,~ = 17S, and showed a single band in polyacrylamide-gel electrophoresis. Titration of SH-groups. The method used was that of Boyer (14). Unless otherwise indicated,
OF
CHLOROPLAST
PROTEINS
99
!a
FRACTION
NUMBER
1. DEAE-Cellulose column chromatography of spinach leaf fraction I protein. Experimental details are described in the text. In this experiment 18 ml (&c = about 17) of protein sample was added to a column (3.6 X 33 cm). The contents of tubes 60-71 were collected and concentrated in a cellophane tubing containing Carbowax 6000 powder up to a concentration of approximately 5 mg/ml. FIG.
0.02 ml of PCMB solution of different concentrations dissolved in 0.1 M glycylglycine buffer (pH 7.8) was added to 0.18 ml of the protein sample. The mixture was incubated at 25”, and the time dependent increase of OD at 255 rnp was measured. To calculate the titratable SH-groups per mole enzyme protein, the molecular weight of spinach fraction I protein (5.15 X 105) as determined by Trown (5) was used. Assay method of RuDP-carboxylase. The standard reaction mixture contained the following compositions (in micromoles): Tris buffer (pH 7.8), 50; MgC12, 5; RuDP, 0.35; NaHW03, 25 (2 &); enzyme preparation, 0.1 ml; in a total volume of 0.5 ml. After a lo-minute incubation at 25” (gas phase, air) 0.05 ml of glacial acetic acid was added to stop the reaction. The mixture was immersed in a boiling water bath, and a 0.2.ml aliquot of the centrifuged supernatant fluid was plated on a planchet. Radioactivity was measured in a Nuclear-Chicago windowless gas-flow counter. All enzyme assays were duplicated, and the averaged values are presented. Proteolytic digestion of enzyme protein. Unless otherwise indicated, 1.8 ml of fraction I protein
100
SUGIYAMA
ET
was treated with 0.2 ml of PCMB solution of varying concentrations dissolved in 0.1 M glycylglycine buffer (pH 7.8) and incubated at 25”. At appropriate time intervals, an aliquot of the reactant was withdrawn for the assay of RuDPcarboxylase activities employing the reaction mixture as described above. Either Nagarse or 01. chymotrypsin dissolved in 0.05 M Tris buffer (pH 7.8) was added to the PCMB-treated fraction I protein samples, and incubation was continued for different periods at 25”. Afterward, aliquots were taken for the assay of RuDP-carboxylase activity. To examine reconstitution of the protein molecule by cysteine, 0.2 ml of freshly prepared 0.6 M cysteine solution was added to the PCMB-treated protein sample, and incubation was continued for another 30 minutes at 25”. Proteolytic digestion of the cysteine-restored protein molecule, as measured by RuDP-carboxylase activity, was examined by the method described in the section, Assay method of RuDP-carboxylase. Polyacrylamide-gel electrophoresis. Horizontal polyacrylamide-gel electrophoresis was carried out in a pH 9.3 system at 400 V for 4 hours at 2” (7). Protein samples applied were treated either with SDS or with PCMB alone as well as with both. Electron microscopy. Protein samples were brought to Okayama University Medical School in a cold chamber, and negatively stained [l% phosphotungstic acid (pH 7)] specimens were prepared for electron microscopic examination
AL.
(15). An Hitachi model 11D electron microscope was used for the ultrastructural study. Reagents. Ribulose 1,5-diphosphate was prepared by the method of Horecker et al. (16) and converted to the sodium salt just before the enzyme assay. Purity was estimated to be 57%. Examination of preparations by high-voltage paper electrophoresis indicated that the major compound was RuDP. Also detectable were negligible amounts of ribose-5-P and Pi. All other reagents used were commercial products: (Ychymotrypsin, BShringer GmbH Mannheim, Germany; PCMB, Sigma Chemical Co., St. Louis, Missouri; and NaHldCO 3, The Radiochemical Centre, Amersham, England. RESULTS
We first examined the SH-titration reaction with different PCMB-protein concentration ratios (moles/mole). Figure 2 shows that regardless of the concentration of PCMB, the saturation level of the mercaptide formation was nearly complete within 30 minutes. In the presence of excess PCMB, the equilibrium position for the mercaptide formation was equivalent to 96 SH-groups in accord with chemical data (17). However, the rate and extent of PCMB titration was found to be essentially independent of the presence of urea (4.5 M) and SDS (2 X 1OV’ M) in the protein sample (Fig. 3). Our pre-
120
0
30
90 INCLLTION
120 TIME
150 (MINUTES)
MO
FIG. 2. Sulfhydryl titration of fraction I protein at varying PCMB-protein concentration ratios. PCMB solutions (0.3 ml) of different concentrations, which were disolved in 0.1 M glycylglycine buffer (pH 7.8), were added at 2.7 ml of fraction I protein preparation containing different amounts of the protein. The reacion mixture was then incubated at 25”. After appropriate reaction periods, a 0.2-ml aliquot was withdrawn and made up to 4 ml with 0.05 M Tris buffer (pH 7.8). The absorbance increase at 255 rnp wfts measured against a blank solution of PCMB and protein. Calculation of SH-groups per mole protein was based on the method described in text. The protein content of the reaction mixture was as follows: 18.05 mg, X; 9.03 mg, q ; 8.75 mg, A; 3.62 mg, 0.
STRUCTURE
AND
FUNCTION
FIG. 3. Effect of urea, SDS, and RuDP on SH-titration of fraction I protein. Conditions of the pretreatment system were as follows. (A) Urea: 1.5 ml of protein solution (6.37 mg) plus 1.3 ml of 10 M urea dissolved in 0.05 M Tris buffer (pH 7.8). (B) SDS: 2 ml of protein solution (8.5 mg) plus 0.8 ml of 7.0 X 1OV M SDS solution dissolved in 0.05 M Tris buffer (pH 7.8). (C) RuDP: 2 ml of protein solution (8.5 mg) plus 0.8 ml of RuDP (3.12 pmoles) dissolved in 0.05 M Tris buffer (pH 7.8). As a control, 0.8 ml of 0.05 M Tris buffer (pH 7.8) was incubated with 2 ml of protein sample, which was used in systems (B) and (C). In each case, after the incubation for 30 minutes at 2.5O, 0.2 ml of 1OW M PCPvlB solution dissolved in 0.1 M glycylglycine buffer (pH 7.8) was added to the incubated reaction mixture. The time-dependent absorbance increase at 255 rnr was read by withdrawing a 0.2-ml aliquot of the reactant and making it up to 4 ml by adding 3.8 ml of 0.05 M Tris buffer (pH 7.8). 0, Native protein; q , 4.5 M urea; A, 2 X 1OP M SDS; 0, 1.1 X 1OW M RuDP.
vious experiment’s have shown that RuDP pretreatment protects the enzyme of wheat leaf from the PCRIB inact’ivation (7). But addition of excess RuDP (1 .l X 10e4 M) did not affect t’he level of SH-groups titration, which is consistent with the results obtained by wheat leaf fraction I protein (7). Figure 4 shows t,he relationship between t,he extent of blocking of SH-groups by PCMB and residual RuDP-carboxylase activity. An antisigmoidal type curve was observed for the decline of enzyme activity in relation to the gradual masking of SHgroups. Before an appreciable loss of the enzyme activity occurred, about 10 SH-groups were titrated, and complete loss of enzyme activity was associated with about 30 SHgroups out of a total of 96 SH-groups. It should be noted that even in the presence
OF
CHLOROPLAST
101
PROTEINS
of a nonsaturating level of PCMB, as reflected in incomplete blocking of the SHgroups, a drastic decline in enzvme act’ivity occurs. Consistent with our pre;ious findings \vit,h wheat leaf fraction I protein (i), the results of polyacrylamide-gel electrophoresis showed diffuse banding patterns, moving slightly faster t,han the native protein, by the addition of PCMB to protein sample. Furt’her addition of SDS (2 X lOA M) caused the protein molecule to split with the formation of three major bands. However, the banding patterns of these subunits are identical with those produced by the direct treatment with SDS. Alterations in the order of addition of reagents to the protein sample did not cause any change in t)he electrophoretogram. All these findings indicate the possible conformational change of the prot’ein decline in molecule in a subsequent, enzyme activit,y. To test’ “unfolding” or in the st,ructural organiznt,ion “loosening”
E“tz 0
30
60
90 PCMB [x10?
M)
,;,i.1s
FIG. 4. Relationship between HH-blocking of fraction I protein and the residual RuDP-carboxylase activity. To 0.18 ml of fraction I protein (0.51 mg protein) was added 0.02 ml of either 0.1 M glycylglycine buffer (pH 7.8) or PC1\IB solution of varying concentrations. After incnbation at 25” for 60 minutes, the reaction mixture was made up to 4 ml with 0.05 Y Tris buffer (pH 7.8), and the absorbance increase at 255 rnp was measured against a blank to estimate the number of SH-groups blocked per mole protein. Separately, 0.45 ml protein solution (1.25 mg) was incubated with 0.05 ml each of either 0.1 M glycylglycine buffer (pH 7.8) or PCXB solution of varying concentrations for 60 minutes at 25”. For the determination of RuDP-carboxylase activity, a 0.2-ml aliquot was withdrawn and added to a standard react,ion mixtllre as described in text.
102
SUGIYAMA
of the teolytic ing the 5 shows
ET
AL.
protein, the method of selective prodigestion was employed by measurRuDP-carboxylase activities. Figure the effect of chymotryptic digestion
1
0
30
60 INCUBATION
90 TIME
120 (MINUTES)
150
180
+CHYMOTRYFSIN
0
I
I 180
30 INC”&ON
‘:ME
(‘;N”TE:jS
FIG. 5. Chymotryptic digestion of fraction I protein. To 1.8ml of fraction I protein (2.8mg/ml) w&sadded0.2ml eachof 0.1Mglycylglycine buffer (pH 7.8) or PCMB solution dissolved in 0.1 M glycylglycine buffer (pH 7.8) or PCMB solution dissolved in 0.1 M glycylglycine (pH 1W or 1.7 X 1W3 M) and incubated 60 minutes. At the end of incubation,
7.8) at
(1.5 X 25” for a 0.2-ml
aliquot was taken out for the enzyme assay at zero time. Two-tenths ml each of 0.05 M Tris buffer (pH 7.8) (dotted lines) or or-chymotrypsin solution dissolved separately in 0.05 M Tris buffer (straight lines) was added to 1.8 ml of the preincubated enzyme preparation, and the incubation was continued. After appropriate reaction intervals, a O.l-ml aliquot was withdrawn to the assay of RuDP-carboxylase activity by using a standard reaction mixture as described in text. Top figure: 0.4 pg chymotrypsin/5 mg protein. Control, 0, 0; 1.5 X lo+M PCMB, A, A; 1.7 X 10-M PCMB, q , W. Bottom $gure: 5 rg chymotrypsin/5 mg protein. Control, 0; 1.5 X l(r’ M PCMB, A; 1.7 X 10-4M PCMB, Kl.
FIG. 6. Nagarse digestion of fraction I protein. Experimental conditions were essentially the same as those shown in Fig. 5, except that Nagarse solution dissolved in 0.05 M Tris buffer (pH 7.8) was added to 1.8 ml of the preincubated enzyme preparation to determine the proteolytic digestion. Top Jigure: 5 rg Nagarse/5 mg protein. Control, 0; 1.5 X W4 M PCMB, A; 1.7 X 10m4 M PCMB, EI. Bottom figure: 20 rg Nagarase/S mg protein. Control, 0 ; 1.5 X lo+ M PCMB, A; 1.7 X lo-"M PCMB, l3.
at two different concentration levels. PCMB (1.5 X lo4 M, 1.7 X 10e4M) alone did not cause great decline in RuDP-carboxylase activity. The subsequent addition of chymotrypsin (chymotrypsin’protein, l/12,500) caused a marked decline of the carboxylase activities. In 180 minutes of incubation, some 80 % loss of the enzyme activity occurred. By increasing the amount of chymotrypsin about tenfold, the effect was made even more pronounced. Native protein itself was susceptible to proteolytic attack but became more so after PCMB-treatment. Since Nagarse has been known to exhibit a broad reaction pattern compared with chymotrypsin, an analogous experiment was
STRUCTURE
AND
FUNCTION
undertaken at two concentration levels. As can be seen in Fig. 6, the carboxylase activity of the native fraction I protein was not affected by Nagarse treatment (1: 1000) up t,o 60 minut,es. After 180 minutes, however, less than 10 % of the original enzyme activity remained. The dat’a suggest that, after several bonds have been split by t’he proteolyt,ic enzyme, without much loss of act,ivity, others then became more susceptible. On the other hand, a sharp decline of enzyme activity occurred with PCMB treatment at the two different concentrations. Nearly complete loss of the enzyme activity occurred with the proteinase by 60 minutes of treatment. By increa,sing the concent,ration of Nagarse 5 fold (l/200), the decline of RuDP-carboxylase activity was still more pronounced.
FIG. 7. Effect of cysteine addition to the PCMB-treated fraction I protein. Experimental condition was basically the same as that shown in Fig. 6. To 1.8 ml of fraction I protein (2.8 mg/ml) was added 0.2 ml each of 0.1 M glycylglycine (pH 7.8) or PCMB solution dissolved in 0.1 M glycylglycine of different concentrations (1.5 X 10-a or 1.7 X 1W3 M), and incubation was carried out for 60 minutes at 25”. Then 0.2 ml of either 0.05 M Tris buffer (pH 7.8) (straight lines) or 0.6 M cysteine (dotted lines) was added to the reaction mixture, and incubation was continued for another 30 minut,es. An aliquot (0.1 ml) was withdrawn for the enzyme assay at zero time. To 1.8 ml of the residual reactant was added 0.05 ml of 0.0170 Nagarse solution dissolved in 0.05 M Tris buffer (pH 7.8). After appropriate incubation periods, a O.l-ml aliquot was withdrawn to the assay of RuDP-carboxylase activity by using a standard reaction mixture as described in text. Five pg Nagarase/4.6 mg protein. Control, 0; 1.3 X 10-d M PCMB, A; 1.7 X 1O-4 M PCMB, q.
OF
CHLOROPLAST
PROTEISS
103
FIG. 8. Protective effect of RuDP on Nagarse digestion of fraction I protein in relation to residual RuDP-carboxylase activity. The experimental condition was basically the same as that shown in Fig. 6, except that 2.8 pmoles of RuDP was added to the preincubated reaction mixture (25” for 60 minutes) with or without addition of PCMB. The effect of Nagarse digestion was tested by adding 0.1 ml each of 0.05 M Tris buffer (straight lines) or 0.001670 enzyme solution (dotted lines) to 0.8 ml of the preincubated enzyme mixture; a 0.1.ml aliquot was withdrawn for the RuDP-carboxylase assay. Five pg Nagarse/5 mg protein. Control, 0;lX10-4~PCMB,A;1.5X10-4~PCMB,m; 6 X lo-.’ M PCMB, 0.
It is evident that splitting of the polypeptide chain of the fraction I protein molecule caused the loss of the carboxylase activity, which was not’ restored by addition of cysteine. However, when the PCMB-inactivated enzyme was restored by the addition of cysteine, it restored the resistance against the Nagarse hydrolysis in the 30minute incubation and lost its RuDPcarboxylase activity in the go-minute treatment. The overall reaction patterns were remarkably similar to the one observed by the native protein (Fig. 7). It was previously demonstrated that the preincubation of wheat fraction I proteinwith RuDP effectively protected the enzyme from the PCRIB-inactivat’ion (7). We next examined the effect of RuDP on the proteolytic digestibility of the PCMB-titrated fraction I protein. The results shown in Fig. 8 clearly demonstrate a prominent protective effect of RuDP. It will be noted that even under the treatment withPCRIB of the highest concentration (6 X 1OWh*) RuDP-preincubated
FIG. 9. Electron micrographs of fraction I protein. (A) Native protein. (B treated 1 PI .otein. (C) Cysteine-reversed protein. Magnification, X225,000. Native I prote iin, about 4.2 mg. (A) was treated with 600 mfimoles of PCMB at 25” for After r em oval of the aggregated protein by centrifugation, the supernatant fluid for elec :trc m microscopy (B). Cysteine (60 mmoles) was added to the PCMB-treate and inc :ub ated at 25’ for 30 minutes. After centrifugation, the supernatant fluid for elec :trc m microscopy (C). 104
) PC>MB: fra ction 12 h IOUrs. was used !d sa ,mple was used
STRUCTURE
AND
FUNCTION
prot,ein still retained an appreciable carboxylase activity, which was hardly affected by the Nagarse digestion. Electron microscopic studies have partly supported the reversible structural constitution of fraction I protein molecule by PCMB-cyst#eine reaction [Fig. 9 (A-C)]. After a prolonged treatment of protein with PCMB, t’he ordered subunit structure (A), typical for the original native protein, is completely lost, and disordered aggregates (B) are predominant. However, an ultrastructural picture (C) of protein particles reconstituted by the addition of cysteine resembled t’hat of the native protein and support,s in essence the data of the enzyme experiments (cf. Fig. 7).
OF
CHLOROPLAST
PROTEINS
105
that are opposed to this concept, because even if SH-groups had no relation to the conformation, the changes in protein conformation required to allow reaction with PCMB could lead to the same results (12). Restoration of RuDP-carboxylase activity of the PCMB-inactivated protein molecule by the addition of cysteine indicates that a reversible association mechanism may operate in our system. Yet we have been unable to characterize subunits in a transition state of the protein molecule by physicochemical techniques. The only detectable change induced in t’he protein structure was the production of diffuse protein bands on a polyacrylamide-gel electrophoretogram. It will also be noted t,hat the subsequent, cleavage of the PCRIB-treated protein with SDS did DISCUSSION not cause any change in the mobility patstern The essential point of the present experiof the “degradomers,” compared with those ment is that PCMB caused an increased produced by the direct treatment of protein susceptibility of the structure of fraction I with SDS. There is thus a possibility that protein t,o the proteolytic attack, as reflected another mechanism might control the conin t’he RuDP-carboxylase activity. Although formational change of the mercurial-treated this could be clearly shown at a chymotrypprot’ein. No decisive answer has been drawn as to sin concemration of 1: 12,500, it’ was more pronounced at Nagarse (1: 1000)) where whether only minor SH-groups of the prolarge decrements of the activity occurred in tein molecule are catalytically essential in the PCMB-treated protein during 40 and SO the RuDP-carboxylase activity. In this conminutes of incubation. Results thus strongly text, our experimental results appear to be indicate t,hat PCMB binding at the level of similar to the ones obtained for rabbit muscle 30-34 moles/mole protein which caused a aldolase (26)) in which the role of SH-groups 10-50s loss of enzyme activity produced in the molecule appears to be primarily a “loosening” of the structure in the viciniby structural. It has been shown that 10 SHof the enzymic site which made it more susgroups per mole of aldolase were reacted with ceptible t,o proteolytic attack. Analogous PC!UB without’ a decrease of the catalytic experimental techniques have been employed activity. Further, it has been established by other workers for different enzymes; hexot,hat, t,he additional reaction with PCMB kinase (1X) , 3-phosphoglyceraldehyde dehycaused reversible loss of enzyme activity drogenase (19), aldolase (20, al), and alkawhich is associated with a structural modifiline phosphat’ase (22). All these enzymes cation of the enzyme protein. It is thus have been shown t,o consist of subunits, and irueresting that. the direct participation of PC?tIB treatment was proved to produce the SH-groups in the RuDP-carboxylase activity unstable enzyme form, with a concomitant has been refuted recently by Akoyunoglou enhancement of the proteolytic hydrolysis. and his associates (27, 2s). Their conclusion Because of recent studies pertaining to the was based on the fact that not only RuDP subunit organization of regulatory enzymes but also other sugar phosphates and car(23-25), it, would be of value t,o speculate bamyl phosphate were able to protect the about, the role of SH-groups in the subunit) enzyme protein against IAA inhibition. Yet organization. Our present study may suggest, t’hese compounds did not compete with that SH-groups play a role in maintaining RuDP in t’he enzyme reaction. Our recent the conformation of fraction I protein. How experiment, which n-ill be reported in a sucever, arguments will nat’urally be advanced ceeding paper (29), has shown that RuDP
SUGIYAMA
specifically protects the alkylation of fraction I protein with IAA, and the numbers of SH-groups titrated have been calculated to be about 10 per mole protein. We have to reserve our final conclusion as to the exact numbers of tiatalytically essential SH-group in the RuDP-carboxylase molecule until we can clearly disclose the subunit structure of the protein molecule. Nonetheless, it appears that SH-groups have structural and catalytic roles in the fraction I protein.
ET AL.
19.
B. R., AND TROWN, P. W., Proc. Natl. Sci. U.S. 61, 497 (1964). TROWN, P. W., AND R.~BIN, B. R., Proc. iVatZ. Acad. Sci. U.S. 62, 88 (1964). BOYER, P. D., in “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrbiick, eds.), 2nd edition, Vol. I, pp. 511-588. Academic Press, New York (1959). MENDIOLA, L., AND AKAZAWA, T., Biochemistry 3, 174 (1964). BOYER, P. D., J. Am. Chem. Sot. 76, 4331 (1954). ODA, T., AND SEKI, S., Proc. Intern. Congr. Electron Microscopy, 6th, Kyoto. pp. 369, 387 (1966). HORECKER, B. L., HURWITZ, J., AND WSISSBACH, A., J. Biol. Chem. 218, 785 (1956). RIDLEY, S. M., THORNBER, J. D., .~ND BAILEY, J. L., Biochim. Biophys. Acta 140, 62 (1967). KAJI, A., Arch. Biochem. Biophys. 112, 54 (1965). SZABOLCSI, G., BISZKN, E., AND SZORENYI, E.,
20.
SZABOLCSI,
10. RABIN, Acad.
11. 12.
13. 14. 15.
ACKNOWLEDGMENT The authors sincerely thank Professors T. Murachi and Y. Morita for their counsel and for their many stimulating discussions in connection with this work. The skilled assistance of Mr. T. Itaya for taking electron micrographs is also gratefully acknowledged. REFERENCES 1. SUGIYAMA, T., AND AKAZA~A,
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AND
TANAKA,
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Y.,
Arch.
Biochem.
Biophys.
OF
BIOCHEMISTRY
AND
Structure
BIOPHYSICS
and
Function
II. Effect of p-Chloromercuribenzoate Carboxylase
Activity
T. SUGIYAMA, Research
Institute
98-106
126,
of Chloroplast Treatment
of Spinach
N. NAKAYAMA,
for Biochemical
(1968)
Proteins’
on the Ribulose Leaf Fraction
M. OGAWA,
Regulation, Nagoya University, h’agoya, Japan
1, 5Diphosphate
I Protein*
T. AKAZAWA3 School
of Agriculture,
Chikusa,
AND
T. ODA Cancer
Institute, Received
Okayama October
University 16,1967;
School accepted
of Medicine, November
Okayama,
Japan
25,1967
Fraction I protein was purified from spinach leaves by Sephadex gel filtration and DEAE-cellulose column chromatography. To study the role of SH-groups in the enzyme molecule, p-chloromercuribenzoate (PCMB) titration of SH-groups was carried out with a parallel determination of enzyme activity. It was found that the rate and extent of reaction with PCMB was identical in the presence or absence of urea (4.5 M), sodium dodecyl sulfate (2 X 1OW M), and ribulose 1,5-diphosphate (RuDP) (1.1 X lo+ M). The total number of SW-groups per mole protein was 96, in agreement with chemical data. Approximately 10 SH-groups were blocked before an appreciable loss of the RuDP-carboxylase activity occurred, and complete inhibition of enzyme activity was associated with the blocking of about 30 SH-groups. The possible role of SH-groups in the structural rigidity of the protein molecule was suggested by the finding that proteolytic digestibility (chymotrypsin and Nagarse) of the protein was greatly enhanced by PCMB pretreatment as measured by decrements of RuDP-carboxylase activity. Full restoration of the enzyme activity by the additionof cysteine to the PCMB-inactivated enzyme protein was accompanied by restoration of resistance to proteolytic attack. That the molecule is restored to a conformation possibly identical with that of the native protein in tertiary structure was supported by electron microscopic observations of the reconstituted protein.
Ribulose 1, 5diphosphate4 carboxylase, which may be identical with fraction I protein, occupies a pivotal role in the photosynthetic carbon reduction cycle in chloroplasts of green leaf tissues (4). The uniquely
large molecular size (5) and conformation of the protein from subunits (6, 7) have prompted us to investigate its structure and function more precisely. Previous studies with SH-blocking agents such as PCMB and IAA have shown the important role of SHgroups in RuDP-carboxylase (7-11). A particularly notable study by Trown and Rabin (11) demonstrated that only two SH-groups are available for the substrate (RuDP) binding site per enzyme molecule. Our own study on wheat fraction I protein has shown that there is a close correlation between
‘This research was supported in part by USPHS research grant AM-10792-01. 2Parts of the present investigation have appeared in preliminary forms (l-3). 3To whom requests for reprints should be sent. ‘Abbreviations used : IAA, iodoacetamide ; RuDP, ribulose 1,5-diphosphate; SDS, sodium dodecyl sulfate; PCMB, p-chloromercuribenzoate. 98
STRUCTURE
AND
FUNCTION
decay of enzyme activity and the number of PCMB titratable SH-groups (7). Although RuDP protects the enzyme from PCMB inactivation, the number of total SH-group(s) titratable by PCMB was essentially the same regardless of the pretreatment of the protein withRuDP. Itisthuspossibletopostulatethat only a minor portion of the SH-groups is catalytically essential to the enzyme reaction. However, it is often difficult to demonstrate the catalytic role of SH-groups in an enzyme reaction, particularly when conformational changes of the molecule result from treatment with the SH-blocking agents (12). To test the possible conformation change of fraction I protein induced by the PCMBtreatment, we investigated the selective proteolytic digestion of spinach fraction I protein in a parallel determination of the RuDPcarboxylase activity. Also the reversible structural reconstitution of protein molecule by cysteine addition has been examined by high-resolution electron microscopy. MATERIALS
AND
METHODS
PuriJication of protein. The methods of extraction and fractionation of spinach leaf fraction I protein were based on the previous report for purifying the protein from rice and wheat leaf, with some modifications (7, 13). Crude leaf protein extract precipitated with (NH,)aS04 at 50yo saturation was first applied to a Sephadex G-25 column, and then to Sephadex G-200 gel filtration. The eluting solution was 0.025 M Tris buffer (pH 7.5) containing 0.1 mM EDTA. Protein fractions were collected and reprecipitated at 50°jc, saturation of (NH&SO*. After being desalted by passage through a column of Sephadex G-25, the protein was next applied to a column of DEAEcellulose and eluted with 0.01 M Tris buffer (pH 7.5) containing 0.1 M EDTA and NaCl in a linear gradient (O-l M). A typical elution pattern is shown in Fig. 1, which represents an improvement over previous reports of the protein resolution (5, 7). A colorless enzymically active sample (tubes 60-71) showed a typical ultraviolet spectra of protein, with an absorbance ratio (280/260 rnp) of 1.6-1.7 and a shoulder at 292 rnp. It was homogeneous by analytical ultracentrifugation, with a single boundary of ~20,~ = 17S, and showed a single band in polyacrylamide-gel electrophoresis. Titration of SH-groups. The method used was that of Boyer (14). Unless otherwise indicated,
OF
CHLOROPLAST
PROTEINS
99
!a
FRACTION
NUMBER
1. DEAE-Cellulose column chromatography of spinach leaf fraction I protein. Experimental details are described in the text. In this experiment 18 ml (&c = about 17) of protein sample was added to a column (3.6 X 33 cm). The contents of tubes 60-71 were collected and concentrated in a cellophane tubing containing Carbowax 6000 powder up to a concentration of approximately 5 mg/ml. FIG.
0.02 ml of PCMB solution of different concentrations dissolved in 0.1 M glycylglycine buffer (pH 7.8) was added to 0.18 ml of the protein sample. The mixture was incubated at 25”, and the time dependent increase of OD at 255 rnp was measured. To calculate the titratable SH-groups per mole enzyme protein, the molecular weight of spinach fraction I protein (5.15 X 105) as determined by Trown (5) was used. Assay method of RuDP-carboxylase. The standard reaction mixture contained the following compositions (in micromoles): Tris buffer (pH 7.8), 50; MgC12, 5; RuDP, 0.35; NaHW03, 25 (2 &); enzyme preparation, 0.1 ml; in a total volume of 0.5 ml. After a lo-minute incubation at 25” (gas phase, air) 0.05 ml of glacial acetic acid was added to stop the reaction. The mixture was immersed in a boiling water bath, and a 0.2.ml aliquot of the centrifuged supernatant fluid was plated on a planchet. Radioactivity was measured in a Nuclear-Chicago windowless gas-flow counter. All enzyme assays were duplicated, and the averaged values are presented. Proteolytic digestion of enzyme protein. Unless otherwise indicated, 1.8 ml of fraction I protein
100
SUGIYAMA
ET
was treated with 0.2 ml of PCMB solution of varying concentrations dissolved in 0.1 M glycylglycine buffer (pH 7.8) and incubated at 25”. At appropriate time intervals, an aliquot of the reactant was withdrawn for the assay of RuDPcarboxylase activities employing the reaction mixture as described above. Either Nagarse or 01. chymotrypsin dissolved in 0.05 M Tris buffer (pH 7.8) was added to the PCMB-treated fraction I protein samples, and incubation was continued for different periods at 25”. Afterward, aliquots were taken for the assay of RuDP-carboxylase activity. To examine reconstitution of the protein molecule by cysteine, 0.2 ml of freshly prepared 0.6 M cysteine solution was added to the PCMB-treated protein sample, and incubation was continued for another 30 minutes at 25”. Proteolytic digestion of the cysteine-restored protein molecule, as measured by RuDP-carboxylase activity, was examined by the method described in the section, Assay method of RuDP-carboxylase. Polyacrylamide-gel electrophoresis. Horizontal polyacrylamide-gel electrophoresis was carried out in a pH 9.3 system at 400 V for 4 hours at 2” (7). Protein samples applied were treated either with SDS or with PCMB alone as well as with both. Electron microscopy. Protein samples were brought to Okayama University Medical School in a cold chamber, and negatively stained [l% phosphotungstic acid (pH 7)] specimens were prepared for electron microscopic examination
AL.
(15). An Hitachi model 11D electron microscope was used for the ultrastructural study. Reagents. Ribulose 1,5-diphosphate was prepared by the method of Horecker et al. (16) and converted to the sodium salt just before the enzyme assay. Purity was estimated to be 57%. Examination of preparations by high-voltage paper electrophoresis indicated that the major compound was RuDP. Also detectable were negligible amounts of ribose-5-P and Pi. All other reagents used were commercial products: (Ychymotrypsin, BShringer GmbH Mannheim, Germany; PCMB, Sigma Chemical Co., St. Louis, Missouri; and NaHldCO 3, The Radiochemical Centre, Amersham, England. RESULTS
We first examined the SH-titration reaction with different PCMB-protein concentration ratios (moles/mole). Figure 2 shows that regardless of the concentration of PCMB, the saturation level of the mercaptide formation was nearly complete within 30 minutes. In the presence of excess PCMB, the equilibrium position for the mercaptide formation was equivalent to 96 SH-groups in accord with chemical data (17). However, the rate and extent of PCMB titration was found to be essentially independent of the presence of urea (4.5 M) and SDS (2 X 1OV’ M) in the protein sample (Fig. 3). Our pre-
120
0
30
90 INCLLTION
120 TIME
150 (MINUTES)
MO
FIG. 2. Sulfhydryl titration of fraction I protein at varying PCMB-protein concentration ratios. PCMB solutions (0.3 ml) of different concentrations, which were disolved in 0.1 M glycylglycine buffer (pH 7.8), were added at 2.7 ml of fraction I protein preparation containing different amounts of the protein. The reacion mixture was then incubated at 25”. After appropriate reaction periods, a 0.2-ml aliquot was withdrawn and made up to 4 ml with 0.05 M Tris buffer (pH 7.8). The absorbance increase at 255 rnp wfts measured against a blank solution of PCMB and protein. Calculation of SH-groups per mole protein was based on the method described in text. The protein content of the reaction mixture was as follows: 18.05 mg, X; 9.03 mg, q ; 8.75 mg, A; 3.62 mg, 0.
STRUCTURE
AND
FUNCTION
FIG. 3. Effect of urea, SDS, and RuDP on SH-titration of fraction I protein. Conditions of the pretreatment system were as follows. (A) Urea: 1.5 ml of protein solution (6.37 mg) plus 1.3 ml of 10 M urea dissolved in 0.05 M Tris buffer (pH 7.8). (B) SDS: 2 ml of protein solution (8.5 mg) plus 0.8 ml of 7.0 X 1OV M SDS solution dissolved in 0.05 M Tris buffer (pH 7.8). (C) RuDP: 2 ml of protein solution (8.5 mg) plus 0.8 ml of RuDP (3.12 pmoles) dissolved in 0.05 M Tris buffer (pH 7.8). As a control, 0.8 ml of 0.05 M Tris buffer (pH 7.8) was incubated with 2 ml of protein sample, which was used in systems (B) and (C). In each case, after the incubation for 30 minutes at 2.5O, 0.2 ml of 1OW M PCPvlB solution dissolved in 0.1 M glycylglycine buffer (pH 7.8) was added to the incubated reaction mixture. The time-dependent absorbance increase at 255 rnr was read by withdrawing a 0.2-ml aliquot of the reactant and making it up to 4 ml by adding 3.8 ml of 0.05 M Tris buffer (pH 7.8). 0, Native protein; q , 4.5 M urea; A, 2 X 1OP M SDS; 0, 1.1 X 1OW M RuDP.
vious experiment’s have shown that RuDP pretreatment protects the enzyme of wheat leaf from the PCRIB inact’ivation (7). But addition of excess RuDP (1 .l X 10e4 M) did not affect t’he level of SH-groups titration, which is consistent with the results obtained by wheat leaf fraction I protein (7). Figure 4 shows t,he relationship between t,he extent of blocking of SH-groups by PCMB and residual RuDP-carboxylase activity. An antisigmoidal type curve was observed for the decline of enzyme activity in relation to the gradual masking of SHgroups. Before an appreciable loss of the enzyme activity occurred, about 10 SH-groups were titrated, and complete loss of enzyme activity was associated with about 30 SHgroups out of a total of 96 SH-groups. It should be noted that even in the presence
OF
CHLOROPLAST
101
PROTEINS
of a nonsaturating level of PCMB, as reflected in incomplete blocking of the SHgroups, a drastic decline in enzvme act’ivity occurs. Consistent with our pre;ious findings \vit,h wheat leaf fraction I protein (i), the results of polyacrylamide-gel electrophoresis showed diffuse banding patterns, moving slightly faster t,han the native protein, by the addition of PCMB to protein sample. Furt’her addition of SDS (2 X lOA M) caused the protein molecule to split with the formation of three major bands. However, the banding patterns of these subunits are identical with those produced by the direct treatment with SDS. Alterations in the order of addition of reagents to the protein sample did not cause any change in t)he electrophoretogram. All these findings indicate the possible conformational change of the prot’ein decline in molecule in a subsequent, enzyme activit,y. To test’ “unfolding” or in the st,ructural organiznt,ion “loosening”
E“tz 0
30
60
90 PCMB [x10?
M)
,;,i.1s
FIG. 4. Relationship between HH-blocking of fraction I protein and the residual RuDP-carboxylase activity. To 0.18 ml of fraction I protein (0.51 mg protein) was added 0.02 ml of either 0.1 M glycylglycine buffer (pH 7.8) or PC1\IB solution of varying concentrations. After incnbation at 25” for 60 minutes, the reaction mixture was made up to 4 ml with 0.05 Y Tris buffer (pH 7.8), and the absorbance increase at 255 rnp was measured against a blank to estimate the number of SH-groups blocked per mole protein. Separately, 0.45 ml protein solution (1.25 mg) was incubated with 0.05 ml each of either 0.1 M glycylglycine buffer (pH 7.8) or PCXB solution of varying concentrations for 60 minutes at 25”. For the determination of RuDP-carboxylase activity, a 0.2-ml aliquot was withdrawn and added to a standard react,ion mixtllre as described in text.
102
SUGIYAMA
of the teolytic ing the 5 shows
ET
AL.
protein, the method of selective prodigestion was employed by measurRuDP-carboxylase activities. Figure the effect of chymotryptic digestion
1
0
30
60 INCUBATION
90 TIME
120 (MINUTES)
150
180
+CHYMOTRYFSIN
0
I
I 180
30 INC”&ON
‘:ME
(‘;N”TE:jS
FIG. 5. Chymotryptic digestion of fraction I protein. To 1.8ml of fraction I protein (2.8mg/ml) w&sadded0.2ml eachof 0.1Mglycylglycine buffer (pH 7.8) or PCMB solution dissolved in 0.1 M glycylglycine buffer (pH 7.8) or PCMB solution dissolved in 0.1 M glycylglycine (pH 1W or 1.7 X 1W3 M) and incubated 60 minutes. At the end of incubation,
7.8) at
(1.5 X 25” for a 0.2-ml
aliquot was taken out for the enzyme assay at zero time. Two-tenths ml each of 0.05 M Tris buffer (pH 7.8) (dotted lines) or or-chymotrypsin solution dissolved separately in 0.05 M Tris buffer (straight lines) was added to 1.8 ml of the preincubated enzyme preparation, and the incubation was continued. After appropriate reaction intervals, a O.l-ml aliquot was withdrawn to the assay of RuDP-carboxylase activity by using a standard reaction mixture as described in text. Top figure: 0.4 pg chymotrypsin/5 mg protein. Control, 0, 0; 1.5 X lo+M PCMB, A, A; 1.7 X 10-M PCMB, q , W. Bottom $gure: 5 rg chymotrypsin/5 mg protein. Control, 0; 1.5 X l(r’ M PCMB, A; 1.7 X 10-4M PCMB, Kl.
FIG. 6. Nagarse digestion of fraction I protein. Experimental conditions were essentially the same as those shown in Fig. 5, except that Nagarse solution dissolved in 0.05 M Tris buffer (pH 7.8) was added to 1.8 ml of the preincubated enzyme preparation to determine the proteolytic digestion. Top Jigure: 5 rg Nagarse/5 mg protein. Control, 0; 1.5 X W4 M PCMB, A; 1.7 X 10m4 M PCMB, EI. Bottom figure: 20 rg Nagarase/S mg protein. Control, 0 ; 1.5 X lo+ M PCMB, A; 1.7 X lo-"M PCMB, l3.
at two different concentration levels. PCMB (1.5 X lo4 M, 1.7 X 10e4M) alone did not cause great decline in RuDP-carboxylase activity. The subsequent addition of chymotrypsin (chymotrypsin’protein, l/12,500) caused a marked decline of the carboxylase activities. In 180 minutes of incubation, some 80 % loss of the enzyme activity occurred. By increasing the amount of chymotrypsin about tenfold, the effect was made even more pronounced. Native protein itself was susceptible to proteolytic attack but became more so after PCMB-treatment. Since Nagarse has been known to exhibit a broad reaction pattern compared with chymotrypsin, an analogous experiment was
STRUCTURE
AND
FUNCTION
undertaken at two concentration levels. As can be seen in Fig. 6, the carboxylase activity of the native fraction I protein was not affected by Nagarse treatment (1: 1000) up t,o 60 minut,es. After 180 minutes, however, less than 10 % of the original enzyme activity remained. The dat’a suggest that, after several bonds have been split by t’he proteolyt,ic enzyme, without much loss of act,ivity, others then became more susceptible. On the other hand, a sharp decline of enzyme activity occurred with PCMB treatment at the two different concentrations. Nearly complete loss of the enzyme activity occurred with the proteinase by 60 minutes of treatment. By increa,sing the concent,ration of Nagarse 5 fold (l/200), the decline of RuDP-carboxylase activity was still more pronounced.
FIG. 7. Effect of cysteine addition to the PCMB-treated fraction I protein. Experimental condition was basically the same as that shown in Fig. 6. To 1.8 ml of fraction I protein (2.8 mg/ml) was added 0.2 ml each of 0.1 M glycylglycine (pH 7.8) or PCMB solution dissolved in 0.1 M glycylglycine of different concentrations (1.5 X 10-a or 1.7 X 1W3 M), and incubation was carried out for 60 minutes at 25”. Then 0.2 ml of either 0.05 M Tris buffer (pH 7.8) (straight lines) or 0.6 M cysteine (dotted lines) was added to the reaction mixture, and incubation was continued for another 30 minut,es. An aliquot (0.1 ml) was withdrawn for the enzyme assay at zero time. To 1.8 ml of the residual reactant was added 0.05 ml of 0.0170 Nagarse solution dissolved in 0.05 M Tris buffer (pH 7.8). After appropriate incubation periods, a O.l-ml aliquot was withdrawn to the assay of RuDP-carboxylase activity by using a standard reaction mixture as described in text. Five pg Nagarase/4.6 mg protein. Control, 0; 1.3 X 10-d M PCMB, A; 1.7 X 1O-4 M PCMB, q.
OF
CHLOROPLAST
PROTEISS
103
FIG. 8. Protective effect of RuDP on Nagarse digestion of fraction I protein in relation to residual RuDP-carboxylase activity. The experimental condition was basically the same as that shown in Fig. 6, except that 2.8 pmoles of RuDP was added to the preincubated reaction mixture (25” for 60 minutes) with or without addition of PCMB. The effect of Nagarse digestion was tested by adding 0.1 ml each of 0.05 M Tris buffer (straight lines) or 0.001670 enzyme solution (dotted lines) to 0.8 ml of the preincubated enzyme mixture; a 0.1.ml aliquot was withdrawn for the RuDP-carboxylase assay. Five pg Nagarse/5 mg protein. Control, 0;lX10-4~PCMB,A;1.5X10-4~PCMB,m; 6 X lo-.’ M PCMB, 0.
It is evident that splitting of the polypeptide chain of the fraction I protein molecule caused the loss of the carboxylase activity, which was not’ restored by addition of cysteine. However, when the PCMB-inactivated enzyme was restored by the addition of cysteine, it restored the resistance against the Nagarse hydrolysis in the 30minute incubation and lost its RuDPcarboxylase activity in the go-minute treatment. The overall reaction patterns were remarkably similar to the one observed by the native protein (Fig. 7). It was previously demonstrated that the preincubation of wheat fraction I proteinwith RuDP effectively protected the enzyme from the PCRIB-inactivat’ion (7). We next examined the effect of RuDP on the proteolytic digestibility of the PCMB-titrated fraction I protein. The results shown in Fig. 8 clearly demonstrate a prominent protective effect of RuDP. It will be noted that even under the treatment withPCRIB of the highest concentration (6 X 1OWh*) RuDP-preincubated
FIG. 9. Electron micrographs of fraction I protein. (A) Native protein. (B treated 1 PI .otein. (C) Cysteine-reversed protein. Magnification, X225,000. Native I prote iin, about 4.2 mg. (A) was treated with 600 mfimoles of PCMB at 25” for After r em oval of the aggregated protein by centrifugation, the supernatant fluid for elec :trc m microscopy (B). Cysteine (60 mmoles) was added to the PCMB-treate and inc :ub ated at 25’ for 30 minutes. After centrifugation, the supernatant fluid for elec :trc m microscopy (C). 104
) PC>MB: fra ction 12 h IOUrs. was used !d sa ,mple was used
STRUCTURE
AND
FUNCTION
prot,ein still retained an appreciable carboxylase activity, which was hardly affected by the Nagarse digestion. Electron microscopic studies have partly supported the reversible structural constitution of fraction I protein molecule by PCMB-cyst#eine reaction [Fig. 9 (A-C)]. After a prolonged treatment of protein with PCMB, t’he ordered subunit structure (A), typical for the original native protein, is completely lost, and disordered aggregates (B) are predominant. However, an ultrastructural picture (C) of protein particles reconstituted by the addition of cysteine resembled t’hat of the native protein and support,s in essence the data of the enzyme experiments (cf. Fig. 7).
OF
CHLOROPLAST
PROTEINS
105
that are opposed to this concept, because even if SH-groups had no relation to the conformation, the changes in protein conformation required to allow reaction with PCMB could lead to the same results (12). Restoration of RuDP-carboxylase activity of the PCMB-inactivated protein molecule by the addition of cysteine indicates that a reversible association mechanism may operate in our system. Yet we have been unable to characterize subunits in a transition state of the protein molecule by physicochemical techniques. The only detectable change induced in t’he protein structure was the production of diffuse protein bands on a polyacrylamide-gel electrophoretogram. It will also be noted t,hat the subsequent, cleavage of the PCRIB-treated protein with SDS did DISCUSSION not cause any change in the mobility patstern The essential point of the present experiof the “degradomers,” compared with those ment is that PCMB caused an increased produced by the direct treatment of protein susceptibility of the structure of fraction I with SDS. There is thus a possibility that protein t,o the proteolytic attack, as reflected another mechanism might control the conin t’he RuDP-carboxylase activity. Although formational change of the mercurial-treated this could be clearly shown at a chymotrypprot’ein. No decisive answer has been drawn as to sin concemration of 1: 12,500, it’ was more pronounced at Nagarse (1: 1000)) where whether only minor SH-groups of the prolarge decrements of the activity occurred in tein molecule are catalytically essential in the PCMB-treated protein during 40 and SO the RuDP-carboxylase activity. In this conminutes of incubation. Results thus strongly text, our experimental results appear to be indicate t,hat PCMB binding at the level of similar to the ones obtained for rabbit muscle 30-34 moles/mole protein which caused a aldolase (26)) in which the role of SH-groups 10-50s loss of enzyme activity produced in the molecule appears to be primarily a “loosening” of the structure in the viciniby structural. It has been shown that 10 SHof the enzymic site which made it more susgroups per mole of aldolase were reacted with ceptible t,o proteolytic attack. Analogous PC!UB without’ a decrease of the catalytic experimental techniques have been employed activity. Further, it has been established by other workers for different enzymes; hexot,hat, t,he additional reaction with PCMB kinase (1X) , 3-phosphoglyceraldehyde dehycaused reversible loss of enzyme activity drogenase (19), aldolase (20, al), and alkawhich is associated with a structural modifiline phosphat’ase (22). All these enzymes cation of the enzyme protein. It is thus have been shown t,o consist of subunits, and irueresting that. the direct participation of PC?tIB treatment was proved to produce the SH-groups in the RuDP-carboxylase activity unstable enzyme form, with a concomitant has been refuted recently by Akoyunoglou enhancement of the proteolytic hydrolysis. and his associates (27, 2s). Their conclusion Because of recent studies pertaining to the was based on the fact that not only RuDP subunit organization of regulatory enzymes but also other sugar phosphates and car(23-25), it, would be of value t,o speculate bamyl phosphate were able to protect the about, the role of SH-groups in the subunit) enzyme protein against IAA inhibition. Yet organization. Our present study may suggest, t’hese compounds did not compete with that SH-groups play a role in maintaining RuDP in t’he enzyme reaction. Our recent the conformation of fraction I protein. How experiment, which n-ill be reported in a sucever, arguments will nat’urally be advanced ceeding paper (29), has shown that RuDP
SUGIYAMA
specifically protects the alkylation of fraction I protein with IAA, and the numbers of SH-groups titrated have been calculated to be about 10 per mole protein. We have to reserve our final conclusion as to the exact numbers of tiatalytically essential SH-group in the RuDP-carboxylase molecule until we can clearly disclose the subunit structure of the protein molecule. Nonetheless, it appears that SH-groups have structural and catalytic roles in the fraction I protein.
ET AL.
19.
B. R., AND TROWN, P. W., Proc. Natl. Sci. U.S. 61, 497 (1964). TROWN, P. W., AND R.~BIN, B. R., Proc. iVatZ. Acad. Sci. U.S. 62, 88 (1964). BOYER, P. D., in “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrbiick, eds.), 2nd edition, Vol. I, pp. 511-588. Academic Press, New York (1959). MENDIOLA, L., AND AKAZAWA, T., Biochemistry 3, 174 (1964). BOYER, P. D., J. Am. Chem. Sot. 76, 4331 (1954). ODA, T., AND SEKI, S., Proc. Intern. Congr. Electron Microscopy, 6th, Kyoto. pp. 369, 387 (1966). HORECKER, B. L., HURWITZ, J., AND WSISSBACH, A., J. Biol. Chem. 218, 785 (1956). RIDLEY, S. M., THORNBER, J. D., .~ND BAILEY, J. L., Biochim. Biophys. Acta 140, 62 (1967). KAJI, A., Arch. Biochem. Biophys. 112, 54 (1965). SZABOLCSI, G., BISZKN, E., AND SZORENYI, E.,
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SZABOLCSI,
10. RABIN, Acad.
11. 12.
13. 14. 15.
ACKNOWLEDGMENT The authors sincerely thank Professors T. Murachi and Y. Morita for their counsel and for their many stimulating discussions in connection with this work. The skilled assistance of Mr. T. Itaya for taking electron micrographs is also gratefully acknowledged. REFERENCES 1. SUGIYAMA, T., AND AKAZA~A,
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