Enzyme Co-Localization with Rubisco in Pea Leaf Chloroplasts

Regular paper

Enzyme co-localization with Rubisco in pea leaf chloroplasts

Louise E. Anderson* & Andrew A. CarolDepartment of Biological Sciences, m/c 066, University of Illinois-Chicago, 845 West Taylor, Chicago, IL60607-7060, USA; *Author for correspondence (e-mail: [email protected]; fax: +1-312-413-2435)

Received 9 January 2004; accepted in revised firm 26 March 2004

Key words: carbonic anhydrase, co-localization, functional enzyme complexes, glyceraldehyde-3-P dehydr-ogenase, nearest neighbor analysis, P-glycerate kinase, phosphoribulokinase, photosynthetic CO2-fixation,Rubisco, Rubisco activase

Abstract

Immunocytolocalization experiments indicate that carbonic anhydrase, phosphoribulokinase, and P-gly-cerate kinase are near neighbors of Rubisco in the pea leaf chloroplast stroma. Direct transfer of ribulosebisphosphate and gaseous CO2 from phosphoribulokinase and carbonic anhydrase to Rubisco, and directtransfer of P-glycerate from Rubisco to P-glycerate kinase in the chloroplast stroma, is then a possibility.Rubisco activase, responsible for the removal of inhibitory sugar phosphates that bind to the active site ofRubisco in the dark, also appears to be co-localized with Rubisco.

Introduction

Ribulosebisphosphate carboxylase (EC 4.1.1.39,Rubisco) catalyzes the first step in the reductivepentose phosphate (Calvin) cycle, the carboxyla-tion of ribulose bisphosphate with the formation oftwo molecules of P-glycerate. Rubisco makes up asmuch as 70% of the soluble protein in the chloro-plast stroma. It forms stable complexes with severalother stromal enzymes, including the enzymes thatprovide its substrates, namely carbonic anhydrase(EC 4.2.1.1) (Jebanathirajah and Coleman 1998)and phosphoribulokinase (EC 2.7.1.19) (Gonteroet al. 1988; Sainis and Harris 1986; Suss et al. 1993;Jebanathirajah and Coleman 1998; Babadzhanovaet al. 2002). Direct interaction could enable chan-neling of the one carbon substrate CO2 from car-bonic anhydrase, and the five carbon sugarbisphosphate substrate ribulose-1,5-P2 from phos-phoribulokinase, respectively, to the Rubisco ac-tive site. While it makes sense that these enzymesshould interact in the chloroplast stroma, not allproteins that form complexes in vitro are locatedtogether and are able to form functional complexes

in vivo. Rubisco is activated by Rubisco activase.The activase must, therefore, interact directly withRubisco during activation. We have developed asimple graphical method for analysis of the dis-tances separating nearest neighbor immunogoldparticles on electron micrographs (Anderson et al.2003). Here we present evidence that Rubiscoactivase, carbonic anhydrase, phosphoribuloki-nase, and the enzyme 3-P-glycerate kinase (EC2.7.2.3), which catalyzes the next reaction in theCalvin cycle, are co-localized with Rubisco in situ.Glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13),the enzyme that utilizes the P-glycerate kinaseproduct, 1,3-bisphosphoglycerate, is also co-local-ized with Rubisco. Triose-P isomerase (EC 5.3.1.1),the next enzyme in the sequence, is not.

Materials and methods

Plant material

Pea (Pisum sativum L., var Little Marvel) plantswere grown from seed in the University of Illinois

Photosynthesis Research 82: 49–58, 2004.� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

49

at Chicago greenhouse as described previ-ously (Anderson et al. 1995a). Seeds were pur-chased from Old’s Seed Company, Madison,Wisconsin.

Antibodies

Mouse anti-spinach Rubisco large subunit andrabbit anti-spinach Rubisco activase were kindlyprovided by Archie Portis, University of Illinois-Urbana, Illinois; rabbit anti-tobacco phosphori-bulokinase was provided by Michael Salvucci,USDA-ARS Western Cotton Research Labora-tory, Phoenix, Arizona; rabbit anti-pea chloroplastcarbonic anhydrase was provided by John Cole-man, University of Toronto, Canada; and rabbitanti-rye chloroplastic triose-P isomerase was pro-vided by Jurgen Feierabend, Johann WolfgangGoethe-Universitat, Frankfurt am Main,Germany.

Isozyme-directed anti-chloroplast P-glyceratekinase and anti-glyceraldehyde-3-P dehydrogenasesubunit A and B antibodies (Anderson et al. 2003)were produced by Bethyl Laboratories, Mont-gomery, Texas. The antisera were raised againstpeptides corresponding to unique external stret-ches of sequence on the respective enzymes.

Immunoblots

SDS-treated stromal extracts were prepared asdescribed in Anderson et al. (2003). Membranesfrom the lysed chloroplasts (mainly thylakoids)were collected by centrifugation. Chlorophyll wasremoved by extraction into 80% acetone. Theresidual pale orange material was suspended inSDS sample buffer and incubated overnight at37 �C. Starch was removed by centrifugation (1min) in a microfuge. The proteins were separatedelectrophoretically on 8 to 25% Phast gels(Amersham-Pharmacia Phast gel apparatus). AnImmobilon-P membrane (Millipore, Bedford,Massachusetts) was laid over the gel, and theproteins were transferred to the membrane byraising the bed temperature to 70 �C for 40 min.Immunodetection was as described in (Andersonet al. 2004) except that the initial exposure to theblocking solution was for an hour at room tem-perature and the blot was exposed to antibody(diluted 1–2500) overnight at room temperature.

When the blot was probed with the anti-rye chlo-roplast triose-P isomerase antibody there was asingle band at 26.5 kDa in the stromal lane (datanot shown). The known molecular masses of thechloroplast enzymes from rye and spinach are 27(Schmidt et al. 1995) and 27.2 kDa (Henze et al.1994), respectively. The molecule mass of peachloroplast triose-P isomerase has not been re-ported. We conclude that the anti-rye chloroplasttriose-P isomerase antibody is recognizing the peastromal triose-P isomerase on the protein blot andthat the antibody is monospecific with respect torecognition of pea chloroplast proteins.

Immunoblots of pea chloroplast stromal pro-teins probed with the anti-Rubisco, anti-Rubiscoactivase, anti-phosphoribulokinase and pea car-bonic anhydrase antibodies (Anderson et al.1996a) and with the isozyme-directed chloroplastP-glycerate kinase and glyceraldehyde-3-P dehy-drogenase antibodies (Anderson et al. 2003) havebeen published.

Fixation and immunolabeling

Thin sections were prepared from pea leaf tissuefixed in 1% acrolein, 0.1% glutaraldehyde,embedded in LR White resin, and immunolabeledas described in Anderson et al. (1995b, 2003). Thegrids were floated on solution containing the pri-mary antibodies overnight. Exposure to thesecondary antibodies was for 4 h the followingmorning. Details of the labeling experiments aregiven in Table 1. The secondary antibodies wereimmunogold labeled IgGs obtained from TedPella, Inc., Redding California, Electron Micros-copy Sciences, Fort Washington, Pensylvania andNanoprobes, Stony Brook, New York. Nor-mal serum from the species used to elicit the sec-ondary antibody was used in all of the blockingsolutions.

For micrographs of chloroplast sections treatedwith preimmune serum for the anti-Rubisco, anti-Rubisco activase, anti-phosphoribulokinase andanti-pea carbonic anhydrase antibodies see(Anderson et al. 1996a). It is impossible to quan-titatively assess non-specific labeling for the iso-zyme-directed antibodies, as the componentsresponsible for such labeling would be removedduring the affinity purification. In order to assesspotential non-specific labeling we treated tissue

50

sections with IgGs isolated from the animal speciesused to produce those antibodies. We found neg-ligible labeling (not shown). Note that any

non-specific labeling should be random and willnot then interfere with the nearest neighbor anal-ysis as discussed below (next section).

Table 1. Details of labeling

Primary

antibody pair

Figure Primary antibody,

dilution or

concentrationa

Secondary

antibodybGold

particle size(nm)

Maximum distance

between gold particles

marking interacting

enzymesc(nm)

M > Rubisco large subunit 1,2A 1fi1000 G > M 10 84

R > Rubisco Activase 1fi500 G > R 20

2B 1fi5000 G > M 20 84

1fi1000 G > R 10

M > Rubisco large subunit 3A 1fi2000 G > M 10 80

R > Carbonic Anhydrase 1fi500 G > R 20

3B 1fi5000 G > M 20 80

1fi50 G > R 10


R > Phosphoribulokinase 1fi500 G > R 20

4B 1fi500 G > M 20 84

1fi500 G > R 10


R > Chloroplastic P-glycerate

Kinase

2.52 lg/ml G > R 10

5B 1fi2000 G > M 10 80

2.52 lg/ml G > R 20

M > Rubisco large subunit 6A 1fi2000 R > M 10 90

S > Glyceraldehyde-3-P 200 lg/ml R > S 20

Dehydrogenase A

6B 1fi2000 D > M 25 93

1 mg/ml D > S 10


Ck > Glyceraldehyde-3-P 24 lg/ml G > Ck 25

Dehydrogenase B

7B 1fi2000 G > M 20 90

60 lg/ml G > Ck 10

M > Rubisco large subunit 8 1fi2000 G > M 10 80

R > Triose-P Isomerase 1fi200 G > R 20

1fi2000 D > M 25 83

1fi10 D > R 10

aPrior to passage through a 20-l filter. Some antibody protein may have been removed during filtration.bCk – chicken; D – donkey, G – goat; M – mouse; R – rabbit; S – sheep.cBased on the diameters of 4 IgG molecules (4 · 12 nm = 48 nm), 1 Rubisco molecule (10.5 nm), the second protein molecule, and the

radii of the gold particles. These maximum estimates for the distances between gold particles in the putative complexes are based on the

assumption that the complexes are flat and linear. This is very unlikely, and, therefore, the actual distance between the gold particles

marking interacting proteins will almost certainly be considerably less than these estimates.

51

Nearest neighbor analysis

We used the method of Anderson et al. (2003) foranalysis of nearest neighbor distances on themicrographs from the double labeling experi-ments. For a population of two differentnon-interacting species the expression n/N ¼ 1)exp()pr2q) gives the fraction n/N corresponding toposition in an ordered list of samples withincreasing nearest-neighbor distance r, where n isthe number of the measurement in rank order, N isthe total number of measurements, r is the distancebetween nearest neighbors, and q is the speciesdensity (Anderson et al. 2003). A plot of )ln(1)n/N) versus r2 produces a straight line, if the twospecies are distributed randomly. Where there ispositive interaction the initial data points will bedisplaced towards the )ln(1)n/N) axis and thecurve will balloon out toward that axis. We mea-sured the distance from the center of each largegold particle to the center of the nearest small goldparticle using Scion Image (Scion Corporation,Frederick, Maryland) and plotted )ln(1)n/N)against r2. It is not necessary to correct for non-specific labeling, as it will simply add data pointsto the portion of the curve representing non-interacting species.

Results and discussion

Rubisco and Rubisco activase

Figure 1 is a micrograph of a chloroplast in a leafsection doubly labeled with the anti-Rubisco andanti-Rubisco activase antibodies. There are severalinstances of small and large gold particles occur-ring together, suggesting that the enzymes mightinteract directly in vivo. When we measured thenearest neighbor distances between gold particlesmarking the anti-Rubisco activase antibody andthe gold particles marking the anti-Rubisco anti-body and plotted )ln(1)n/N) against r2, the datadid not fit a straight line (Figure 2a). The datapoints balloon out from the trend line at low val-ues of r. These results indicate that Rubisco andRubisco activase are co-localized in the pea leafchloroplast. When the particle sizes were reversedthere was almost no deviation from the trendline(Figure 2b), indicating that while most of theRubisco activase is co-localized with Rubisco, a

large part of the Rubisco is not co-localized withthe activase. Consistent with the first experiment,when the nearest neighbor distances were mea-sured from activase to Rubisco a biphasic datacurve was obtained (not shown).

Although a complex composed of Rubisco andRubisco activase has not been isolated, a change inthe diameter of Rubisco protein particles, ob-served by electron microscopic imaging, occurswhen Rubisco activase and an ATP analog areadded to Rubisco (Buchen-Osmond et al. 1992). Itis suggested that the activase encircles the Rubiscomolecule (Portis 2003). While co-localization doesnot necessarily indicate direct contact, our

Figure 1. Chloroplast in a pea leaf section doubly labeled with

antibodies raised against Rubisco activase (20-nm particles) and

with antibodies raised against Rubisco (10-nm particles). The

large white area in the center of the chloroplast is a starch grain.

Part of a second chloroplast is visible in the upper left corner of

the figure. The maximum possible distance between the centers

of two gold particles marking Rubisco and Rubisco activase

molecules that are in direct contact with one another would be

about 84 nm (see Table 1). Bar = 200-nm. S – stroma;

T – thylakoid; CW – cell wall; Cy – cytosol.

52

experiments are completely consistent with thepossibility that these enzymes are located near oneanother and do interact in the chloroplast.

Rubisco and carbonic anhydrase

Carbonic anhydrase converts HCO�3 in the stroma

to the Rubisco substrate, gaseous CO2 (CO2(g)).When we measured the nearest neighbor distancesfrom gold particles marking carbonic anhydrase tothe gold particles marking Rubisco and plotted)ln(1 ) n/N) against r2, the data points rose abovethe trend line towards the )ln(1 ) n/N) axis at low

values of r (Figure 3a). When the particle sizeswere reversed and distances were measured fromparticles marking Rubisco to particles markingcarbonic anhydrase, deviation from the trendlinewas less pronounced (Figure 3b). These resultsindicate that Rubisco and carbonic anhydrase arelocated near one another in the chloroplast stro-ma. They strongly suggest that the complex con-taining these two enzymes isolated byJebanathirajah and Coleman (1998) occurs in situ.Rubisco could, then, be positioned to acceptCO2(g) directly from carbonic anhydrase, as sug-gested by those workers and by Kachru andAnderson (1974). Direct interaction between thetwo enzymes, or release of CO2(g) from carbonicanhydrase near Rubisco, should increase thesteady state concentration of CO2(g) available atthe Rubisco CO2 binding site. This should result inan increase in the rate of CO2 fixation when CO2 islimiting. It is also possible that the actual substratefor Rubisco in vivo is the carbonic anhydrase–CO2

complex. There are a number of potential advan-tages to utilization of enzyme-bound product inmetabolic systems, including an overall increase invelocity as a result of elimination of a slow productrelease step and elimination of the need to build uphigh levels of intermediates. In photosynthetic andchemoautotrophic prokaryotes Rubisco and car-bonic anhydrase are located together in carboxy-somes (reviewed in Badger and Price 2003). Theabsence of carboxysomes from green plants mayreflect changes in the surface of the enzymes dur-ing evolution that would allow an increase ininteraction and complex formation in higherplants, and/or simply the need for Rubisco tointeract with other stromal enzymes.

Carbonic anhydrase might also protect Rubi-sco from the alternate substrate oxygen. If CO2 ispassed directly from carbonic anhydrase to Rubi-sco in the chloroplast stroma, O2 will be a lesseffective competitive substrate for Rubisco in vivothan in vitro. Co-localization of carbonic anhydr-ase and Rubisco may help to protect C-3plants from the deleterious effects of photorespi-ration.

Rubisco and phosphoribulokinase

Phosphoribulokinase produces the 5-carbon sugarbisphosphate substrate, ribulose-1,5-P2, utilized byRubisco. Phosphoribulokinase was a component

Figure 2. (a) Plot of the negative log of 1–fraction in the ordered

list of measurements against the square of the distance between

20-nm gold particles marking Rubisco activase and 10-nm gold

particles marking Rubisco, from the experiment in Figure 1.

There were 395 measurements. The first 391 data points are

shown. (b). When the particle sizes were reversed the displace-

ment above the trendline was much less pronounced. There were

89 measurements. The first 81 data points are shown.

53

of the carbonic anhydrase-Rubisco complex iso-lated by Jebanathirajah and Coleman (1998). Co-localization of Rubisco and phosphoribulokinasemight be expected to enhance photosynthetic CO2

fixation. The higher local concentration of ribu-lose-1,5-P2 near Rubisco, or direct passage ofribulose bisphosphate from phoshoribulokinase toRubisco, if the two enzymes are in contact withone another, should increase the production of P-glycerate. When the nearest neighbor distanceswere measured from gold particles marking phos-phoribulokinase to the gold particles markingRubisco and plotted as )ln(1 ) n/N) against r2, thedata curve bulged out from the trend line towardsthe )ln(1 ) n/N) axis at low values of r (Figure 4a).

Similar results were obtained when the particlesizes and the relative antibody concentrations werereversed (Figure 4b). These results indicate thatthe enzymes are located near one another in thechloroplast stroma. Therefore, productive inter-action between the enzymes is a possibility.

Rubisco and P-glycerate kinase

P-glycerate kinase catalyzes the step that followsRubisco in the Calvin cycle, namely the transfer ofphosphate from ATP to P-glycerate with the for-mation of 1,3-P2-glycerate. We are not aware ofany experimental evidence for direct interactionbetween these two enzymes. Neither Suss et al.(1993) or Jebanathirajah and Coleman (1998) re-ported the presence of P-glycerate kinase in thecomplexes they isolated. Gontero et al. (1988) andBabadzhanova et al. (2002), however, found bothP-glycerate kinase and Rubisco in multi-enzymecomplexes isolated from spinach and cotton,respectively. When we measured the nearestneighbor distances from the gold particles markingRubisco to the gold particles marking P-glyceratekinase and plotted )ln(1 ) n/N) against r2, thedata did not fit a straight line (Figure 5a). Devia-tion from the trendline was also observed when theparticle sizes and the relative antibody concentra-tions were reversed (Figure 5b). These resultsindicate that Rubisco and P-glycerate kinase arelocated in the same vicinity in the pea leaf chlo-roplast. This does not necessarily imply that thetwo enzymes are located adjacent to one another.Localization in the same complex, even withoutdirect contact between the two enzymes, might stillallow the kinase first access to P-glycerate gener-ated by nearby Rubisco molecules.

Rubisco and glyceraldehyde-3-P dehydrogenase.

Higher plant chloroplast glyceraldehyde-3-Pdehydrogenase is a tetramer composed of two verysimilar A and B subunits. The enzyme utilizes theNADPH generated by the photochemical appa-ratus to reduce the 1,3-P2-glycerate produced byP-glycerate kinase. We have shown that peachloroplast P-glycerate kinase and glyceraldehyde-3-P dehydrogenase interact in vitro (Macioszek etal. 1990; Wang et al. 1996) and that they are co-localized in situ (Anderson et al. 2003). Whennearest neighbor measurements were plotted as

Figure 3. (a) Plot of the negative log of 1–fraction in the or-

dered list of measurements against the square of the distance

between 20-nm gold particles marking carbonic anhydrase and

10-nm gold particles marking Rubisco. There were 152 mea-

surements. The first 146 data points are shown. (b). When the

particle sizes were reversed the displacement above the trendline

was less pronounced. There were 113 measurements. The first

103 data points are shown.

54

)ln(1 ) n/N) against r2 for the distances separatinggold particles marking subunit A and Rubisco, inthe data points rose above the trend line towardsthe )ln(1 ) n/N) axis at low values of r (Figure 6a).Deviation from the trendline was also observedwhen the particle sizes and relative antibody con-centrations were reversed (Figure 6b). Theseresults indicate that glyceraldehyde-3-P dehydro-genase molecules containing subunit A, like P-glycerate kinase, are located near Rubisco, in thechloroplast stroma. In contrast, there is no obvi-ous deviation from linearity when distances sepa-rating the gold particles are measured from theparticles marking subunit B to the particles

marking Rubisco and plotted (Figure 7a). Whenparticle sizes and antibody concentrations are re-versed, deviation is evident (Figure 7b). It seemspossible that there are differences in the distribu-tion of the glyceraldehyde-3-P dehydrogenase tet-ramers containing mostly A subunits and thedehydrogenase tetramers containing mostly Bsubunits in the chloroplast stroma.

Rubisco and triose-P isomerase

Triose-P isomerase follows glyceraldehyde-3-Pdehydrogenase in the reductive pentose phosphate



between 20-nm gold particles marking phosphoribulokinase

and 10-nm gold particles marking Rubisco. There were 715

measurements. (b) When the particle sizes were reversed the

displacement above the trendline was less pronounced. There

were 660 measurements. The first 636 data points are shown.

Figure 5. (a) Plot of the negative log of 1–fraction in the ordered


20-nm gold particles marking P-glycerate kinase and 10-nm gold

particles marking Rubisco. There were 216 measurements. (b).

Plot of the negative log of 1–fraction in the ordered list of

measurements against the square of the distance between nearest

neighbor gold particles when the particle sizes were reversed.

There were 162 measurements. The first 152 are included.

55

pathway. When we measured the nearest neighbordistances from gold particles marking triose-Pisomerase to gold particles marking Rubisco andplotted )ln(1 ) n/N) against r2, the data pointsfollowed a straight line (Figure 8). Similar resultswere obtained when the particle sizes were reversedand distances were measured from particlesmarking Rubisco to particles marking triose-Pisomerase (not shown). Triose-P isomerase, then,does not appear to be a near neighbor of Rubisco.Apparently the two enzymes are distributed ran-

domly, with respect to one another, in the chlo-roplast stroma.

Conclusions

Clearly the complexes involving Rubisco, carbonicanhydrase (Jebanathirajah and Coleman 1998)and phosphoribulokinase (Gontero et al. 1988;Sainis and Harris 1986; Suss et al. 1993;

Figure 6. (a). Plot of the negative log of 1–fraction in the or-


between 20-nm gold particles marking the A subunit of glyc-

eraldehyde-3-P dehydrogenase and 10-nm gold particles

marking Rubisco. There were 352 measurements. The first 316

are shown. (b). Plot of the negative log of 1–fraction in the

ordered list of measurements against the square of the distance

between nearest neighbor gold particles when the particle sizes

and relative antibody concentrations were reversed. There were

65 measurements. The first 49 data points are shown.



between 25-nm gold particles marking subunit B of glyceral-

dehyde-3-P dehydrogenase and 10-nm gold particles marking

Rubisco. There were 555 measurements. The first 547 are in-

cluded. (b). Plot of the negative log of 1–fraction in the ordered


nearest neighbor gold particles when the particle sizes and rel-

ative antibody concentrations were reversed. There were 563

measurements. The first 539 data points are shown.

56

Jebanathirajah and Coleman 1998; Babadzhanovaet al. 2002) do not represent simple artifactsresulting from fortuitous protein associationin vitro, but rather represent functioning com-plexes that facilitate photosynthetic CO2 fixationin the chloroplast. P-glycerate kinase and glycer-aldehyde-3-P dehydrogenase may also be part ofthat complex. We have previously shown thatP-glycerate kinase and glyceraldehyde-3-P dehy-drogenase, and aldolase and glyceraldehyde-3-Pdehydrogenase, are co-localized in the chloroplaststroma (Anderson et al. 2003) and that the isolatedenzymes form complexes with one another (Wanget al. 1996; Anderson et al. 1995a, 1996b). Thesecomplexes are almost certainly responsible, inpart, for the very short initial lag period in pho-tosynthetic CO2 fixation and allow the Calvin cycleto operate with low levels of intermediates (seeMarques et al. 1987). Net CO2 fixation has yet to beaccomplished in a reconstituted system, possiblybecause not all of the enzymes are able to re-asso-ciate into productive complexes in such systems.

Acknowledgements

This work was supported by National ScienceFoundation Grant MCB-0079913. We thankLawrence Sykora and Jim Scios for cultivating the

pea plants, and Archie Portis, Michael Salvucci,John Coleman and Jurgen Feierabend for gener-ously providing antibodies.

References

Anderson LE, Goldhaber-Gordon IM, Li D, Tang X-Y, Xiang

M and Prakash N (1995a) Enzyme-enzyme interaction in the

chloroplast: glyceraldehyde-3-phosphate dehydrogenase, tri-

ose phosphate isomerase and aldolase. Planta 196: 245–255

Anderson LE, Wang X and Gibbons JT (1995b) Three enzymes

of carbon metabolism, or their antigenic analogs, in pea

nuclei. Plant Physiol 108: 659–667

Anderson LE, Gibbons JT and Wang X (1996a) Distribution of

ten enzymes of carbon metabolism in pea (Pisum sativum)

chloroplasts. Int J Plant Sci 157: 525–538

Anderson LE, Tang X-Y, Johansson G, Wang X, Marques IA

and Macioszek J (1996b) Interaction between chloroplast

phosphoglycerate kinase and glyceraldehyde-3-phosphate

dehydrogenase. Adv Mol Cell Biol 15A 273–279

Anderson JB, Carol AA, Brown VK and Anderson LE (2003)

A quantitative method for assessing co-localization in

immuno-labeled thin section electron micrographs. J Struct

Biol 143: 95–106

Anderson LE, Ringenberg MR and Carol AA (2004) Cytosolic

glyceraldehyde-3-P dehydrogenase and the B subunit of the

chloroplast enzyme are present in the pea leaf nucleus.

Protoplasma 223: 33–43

Babadzhanova MP, Babadzhanova MA and Aliev KA (2002)

Free and membrane-bound multienzyme complexes with

Calvin cycle activities in cotton leaves. Rus J Plant Physiol

49: 592–597

Badger MR and GD Price (2003) CO2 concentrating mecha-

nisms in cyanobacteria: molecular components, their diver-

sity and evolution. J Exp Bot 54: 609–622

Buchen-Osmond C, Portis Jr AR and Andrews TJ (1992)

Rubisco activase modifies the appearance of Rubisco in the

electron microscope. In: Murata N (ed) Research in Photo-

synthesis, IX International Congress on Photosynthesis, Vol

3, pp 653–656. Kluwer Academic Publishers, Dordrecht, The

Netherlands

Gontero B, Cardenas ML and Ricard J (1988) A functional

five-enzyme complex of chloroplasts involved in the Calvin

cycle. Eur J Biochem 173: 437–443

Henze K, Schnarrenberger C, Kellermann J and Martin W

(1994) Chloroplast and cytosolic triosephosphate isomerases

from spinach: purification, microsequencing and cDNA

cloning of the chloroplast enzyme. Plant Mol Biol 26:

1961–1973

Jebanathirajah JA and Coleman JR (1998) Association of

carbonic anhydrase with a Calvin cycle enzyme complex in

Nicotiana tabacum. Planta 204: 177–182

Kachru RB and Anderson LE (1974) Chloroplast and cyto-

plasmic enzymes V Pea leaf carbonic anhydrases. Planta 118:

235–240

Macioszek J, Anderson JB and Anderson LE (1990) Isolation

of chloroplastic phosphoglycerate kinase Kinetics of the two

enzyme phosphoglycerate kinase/glyceraldehyde-3-phosphate

dehydrogenase couple. Plant Physiol 94: 291–296

Figure 8. Plot of the negative log of 1–fraction in the ordered


20-nm gold particles marking triose-P isomerase and 10-nm

gold particles marking Rubisco. There were 112 measurements.

The first 101 are included. The data points also described a

straight line when the particle sizes and relative antibody con-

centrations were reversed (not shown).

57

Marques IA, Ford DM, Muschinek G and Anderson LE (1987)

Photosynthetic carbon metabolism in isolated pea chloro-

plasts: metabolite levels and enzyme activities. Arch Biochem

Biophys 252: 458–466

Portis Jr AR (2003) Rubisco activase – Rubisco’s catalytic

chaperone. Photosynth Res 75: 11–27

Sainis JK and GC Harris (1986) The association of ribulose-

1,5-bisphosphate carboxylase with phosphoriboisomerase

and phosphoribulokinase. Biochem Biophys Res Commun

139: 947–954

Schmidt M, Svendsen I and Feierabend J (1995) Analysis of the

primary structure of the chloroplast isozyme of triosephos-

phate isomerase from rye leaves by protein and cDNA

sequencing indicates a eukaryotic origin of its gene. Biochim

Biophys Acta 1261: 257–264

Suss KH, Arkona C, Manteuffel R and Adler K (1993) Calvin

cycle multienzyme complexes are bound to chloroplast

thylakoid membranes of higher plants in situ. Proc Natl

Acad Sci 90: 5514–5518

Wang X, Tang X-Y and Anderson LE (1996) Enzyme-

enzyme interaction in the chloroplast: physical evidence for

association between phosphoglycerate kinase and glyceral-

dehyde-3-phosphate dehydrogenase in vitro. Plant Sci 117:

45–53

58

Documents

Enzyme Co-Localization with Rubisco in Pea Leaf Chloroplasts