Rubisco activation

Substrate RuBP binds much more tightly to the inactive E form of rubisco (An = 20 nM) than to the active ECM form (A, for RuBP = 20 ixM). Thus, RuBP is also a potent inhibitor of rubisco activity. Release of RuBP from the active site of rubisco is mediated by rubisco activase. Rubisco activase is a regulatory protein it binds to A-form rubisco and, in an ATP-dependent reaction, promotes the release of RuBP. Rubisco then becomes activated by carbamylation and Mg binding. Rubisco activase itself is activated in an indirect manner by light. Thus, light is the ultimate activator of rubisco. 

In C4 Plants, C02 Fixation and Rubisco Activity Are Spatially Separated... 

Average RubisCO activity in plant materials Average SOD activity in plant materials Average Chi content in plant materials... 

In the case of culture under plain air, as shown in Fig. 3A, both the Chi content, Cchl, and RubisCO activity, AR, were kept at appreciable levels as long as sucrose was included in the medium (1st to 3rd stages). While being kept in sucrose-free medium (4th stage), however, the hairy roots turned brown and the values of Cchl and AR were ultimately extinguished. 

Fig. 3A,B. Changes in RubisCO activity and Chi content during acclimation cultures of photomixotrophic pak-bung hairy roots to photoautotrophy. Each culture was carried out using the conical flasks illuminated at I= 11 W nr2 and shaken at 100 rpm with A plain air (0.03 % C02) supply B 3.0% C02-enriched air supply...

Flores et al. [34] reported that RubisCO activity of A. oppositifolia hairy roots increased with decreasing sucrose concentration during acclimating cultures for photo autotrophy, and RubisCO activity of the hairy roots cultivated with sugar-free medium was 1.7 times that cultivated with medium containing 30 kg nr3 sucrose. 

Table 3 compares the values of Cchb apparent C02 fixation rate, RubisCO activity, and some other enzyme activities among the hairy roots of different types and different parts of the parent plants of pak-bung. The Cchl and AR values in the hairy roots cultivated photomixotrophically with 30 kg m 3 sucrose for 14 days were somewhat lower than those found in parent plant stems. Those values in the photoautotrophs were in excess of the values in the stems although even lower than those in parent plant leaves. Likewise, the C02 fixation rate of photoautotrophic hairy roots reached more than 3.0 times that of photo-mixotrophic ones. 

Table 4 summarizes properties of various plant cells with photosynthetic ability, indicating the Chi contents, RubisCO activities, and apparent C02 fixation rates of photoautotrophic cells derived from various plants [34,38,40]. The callus of G. max possessed the Chi content of 1.4-1.9 x 103 mg (kg-FW)-1, the value of which was about 28-fold over that in the callus of N. tabacum (58 mg (kg-FW)1). The photoautotrophic hairy roots of pak-bung exhibited relatively high Chi content of 6.1 x 102 mg (kg-FW)-1 though the value was lower than that of G. max callus. On the other hand, as shown in Table 4, the levels of RubisCO activity and Chl-based C02 fixation rate of the pak-bung hairy roots were comparable to those of the photoautotrophic plant cells reported in the literatures. These values, therefore, can be used as indicators for the potentials of photoautotrophic growth of plant cells. 

Table 4. Chi contents, RubisCO activities and apparent C02 fixation rates of photo autotrophic cells derived from various plants... 

Cells C02 and light conditions Chi content [mg (kg-FW) ] RubisCO activity [pmol-CO, (h mg-Chl)-1] Apparent C02 fixation rate [pmol-CO, (h mg-Chl) ] Ref. 

To correlate the Chi content and the photosynthetic potential of the hairy roots in detail, RubisCO activity was investigated because it is a key enzyme of C02 fixation in photosynthetic reactions. Figure 22 shows the relationship between the average RubisCO activity, AR, and Cchl of pak-bung hairy roots cultivated under various conditions. The AR value was directly proportional to the CChi value in pak-bung hairy roots and correlated by the following equation ... 

Fig. 22. Relationship between average Chi content and average RubisCO activity in hairy roots cultivated under various conditions...

Figure 6.8. The effects of four organic osmolytes, glycerol, sarcosine, glycine betaine, and TMAO on salt-inhibited ribulose-1,5-bisphosphate-carboxylase-oxygenase (RuBisCO) activity of the cyanobacterium Aphanothece halophytica. The enzyme was placed in a solution containing KC1 at a concentration sufficient to inhibit activity by 50%. Then, increasing concentrations of organic osmolytes were added to the assay medium, leading to restoration of activity. (Modified after Incharoensakdi et al., 1986.)...

Gerard, V. A., and DriscoU, T. (1996). A spectrophotometric assay for Rubisco activity Application to the icelp Laminaria saccharina and implications for radiometric assays. J. Phycol. 32, 880—884. 

There are many other phenomena specifically observed in H-COj conditions. For example, H-COj cells of C. littorale exhibit higher Rubisco activity than L-COj cells, due to the increase in the amount of this enzyme detected by antibody [11]. Rubisco activase is also higher, but phosphoenolpyruvate carboxylase activity is lower in H-... 

Table 1. RubisCO activity of R. palustris No. 7, Forml and Formll deficient mutant...

To analyze RuBisCO activities, 200 pi of the soluble protein sample (Img/ml) was added to 0.5ml of a buffer containing lOOmM HEPES-KOH (pH 8.0), 20mM MgCl2, O.lmM... 

Soluble proteins from the transformed and Sp25 plants, as well as from wild type N. tabacum plants, were separated by SDS-PAGE. As illustrated m Figure 2, RuBisCO L and S amounts are nearly the same in the wild type and transformed plants. This contrasts with then-virtual absence in Sp25. RuBisCO activity assays revealed that PGA is detectable after about 6 min in the products of the transformed plants, but that it is not present in Sp25 (data not shown). Taken together, these data indicate that RuBisCO content and activity are restored to wild type levels in the transformed plants. 

The results in this report strongly support the hypothesis that the conversion of Gly-322 to Ser-322 leads to the loss of RuBisCO activity in Sp25. These results also pave the way for the Introduction of mutations into the tobacco rbcL gene. This will allow us to investigate the... 

Rubisco activity was quantified in extracts of cells carrying various combinations of the two sets of plasmids. The amounts of soluble active Rubisco... 

Figure 2 Chaperonin- and Mg-ATP-dependent in vitro reconstitution of active dimeric Rubisco from denatured Rubisco. a) Rubisco activity, expressed as a percentage of activity observed with an equal quantity of native Rubisco. b) Western blot after non-denaturing PAGE of the reaction mixtures used in a and probed with antibody raised against Rubisco. c) as in b, but probed with antibody raised against cpn60. d) Summary of the reaction conditions. Reprinted by permission from NATURE vol. 342 pp. 884-889. Copyright (C) 1989 Macmillan Magazines Ltd.

PHOTOAFFINITY LABELING OF THE RUBISCO ACTIVE-SITE AND SMALL SUBUNIT WITH 8-AZIDOADENOSINE 5 -TRIPHOSPHATE... 

Photoaffinity labeling of the Rubisco active-site Two lines of indirect evidence showed that binding of 8-N3ATP to the LSu of Rubisco takes place in the active-site. First, covalent modification of Rubisco by incorporation of the photoprobe reduced enzyme activity by 14.4%, an amount equivalent to the amount of [32p]8-N ATP incorporated into the LSu (data not shown). Second, phosphorylated effectors afforded a level of protection to the site which correlated with the apparent affinity of each for the active-site of Rubisco (data not shown). Photoaffinity labeling of the SSu was unaffected by active-site-directed effectors (data not shown). 

The X-ray structure of spinach Rubisco identifies 10 loop regions in the active-site (8). The major photolabeled peptide isolated by ion-exclusion and ion-exchange chromatography, and reverse phase HPLC corresponded to the Rubisco LSu tryptic peptide Val-42 to Arg-79 (Table 1), an active-site peptide (9). Two portions of the tryptic peptide Asp-95 to Lys-128, the peptide which comprises loop B in the crystal structure (8) were also recovered. Also recovered were two peptides (Table 1) which include residues in two other loop regions of the Rubisco active-site (8). The peptide Asp-202 to Arg-213 (loop 2 in ref. 8) is adjacent to the activator lysine (10). The peptide Gln-259 to His-267 is the N-terminal fragment of Gln-259 to Arg-285 the tryptic peptide which encompasses loop 4. Thus, all four of the photoaffinity labeled LSu peptides have been previously shown to be active-site peptides based on X-ray crystallography or chemical modification or by both techniques. 

The dissociation and reconstitution of RubisCO subunits from prokar yotes(l,2) and eucaryotes(3) have been investigated recently. It has been demonstrated that the small subunit is a necessary component for RubisCO catalysis, and it is inferred that the role of the small subunit in maintenance of RubisCO activity is probably related to its spatial arrangment in the molecule. Up to now, insights into the quaternary structure of the LgSg RubisCO are almost solely based on X-ray crystallographic and electron microscopic studies(4,5). 

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