RuBP carboxylase

H,COPO Ribulose-1,5-bis-phosphate (RuBP) carboxylase HCOH 1, H,C0P05 Two 3-Phospho- kinase 1,3-Blsphospho-glvcerate (BPG) dehydrogenase Glyceraldehydi 3-phosphate (G3P)... 

The classic work by Park and Epstein (1960) and others (see O Leary, 1981, and Fogel and Cifuentes, 1993, for review) established that the enzyme ribulose 1,5-bisphosphate carboxylase (RuBP carboxylase) controls fractionation of carbon... 

Unlike C3 or C4 land plants, diffusion of CO2 limits photosynthesis in aquatic plants. Consequently, algae have adapted a mechanism that allows them to actively transport or pump either CO2 or HCO3 across their membrane, thereby accumulating DIC in the cell (Lucas and Berry, 1985). Although carbon fixation is through RuBP carboxylase,... 

Most plants reduce CO2 to carbohydrate according to the well-known Calvin-Benson or C3 pathway, where the initial product of photosynthesis is the 3C compound phosphoglycerate. Fixation of CO 2 to phosphoglycerate occurs with the assistance of the enzyme ribulose bisphosphate (RuBP) carboxylase, which discriminates heavily against C02 (11). Consequently, plants with C3 photosynthesis have 6 values that average -27.0 (12). Plants with the Hatch-Slack or Ci,... 

The dark reaction, known as the Calvin cycle, uses the reducing power of NADPH as well as the free energy stored in the ATP to assimilate carbon dioxide in the form of carbohydrates. The way by which Nature achieves carbon fixation is via the reaction of CO2 with ribulosebiphosphate (RuBP) to give two molecules of 3-phosphoglycerate, a process which is catalyzed by the enzyme RuBP-carboxylase. The phosphogylcerate is converted further to fructose 6-phosphate, the final product of the Calvin cycle. The overall reaction, despite its complex mechanism, corresponds to the simple Eq. (16) above. 

Ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase) catalyzes the carboxylation of ribulose bisphosphate to give 3-phosphoglyceric acid [Eq. (14)] (7/. 72). 

RuBP carboxylase is the central C02-fixing enzyme in all plants thus, it is the most abundant enzyme in the biosphere (71, 72). The enzyme requires a divalent metal ion but has no other cofactors. CO2 is the substrate, rather than HCOs". 

RuBP carboxylase has a number of unusual features. First, the enzyme must be activated by reaction with CO2 and metal prior to catalysis. Activation involves reaction of CO2 with an e-amino group of a lysine residue (Lys-191 in the enzyme from Rhodospirillum rubrum, Lys-201 in that from spinach) to form a carbamate that is then coordinated to the metal (75). This bound metal then forms part of the substrate-binding site. 

Hall, N.P., P. Koivuiuemi, and N.E. Tolbert The ratio of RuBP carboxylase to RuBP oxygenase in crude and purified preparations from tobacco leaves Plant Physiol. (1978) Suppl, 544. 

Crude Extract Assay In Rg. 3, the response of crude extract fractions to RuBP is shown. One milli-unit of RuBisCO is that amount which catalyzes the RuBP-dependent fixation of 1.0 nmole of C02toinute at 30° C. Specific activity is expressed as milli-units/mg protein. As evident, both mutant enzymes were similarly saturated with RuBP in the 39-52 mM range. In contrast, wild-type RuBP carboxylase saturated in a lower concentration range of 13-26 mM. Quantitative comparisons were not possible because the Km for RuBP markedly increases with increasing ionic strength. The major effect of Ae substitution of leu and lys for arg at position 292 was, however, upon the specific activity of the enzymes. 

After irradiation, an indicated amount of NaBH4, prepared in 0.1 N NaOH, was added to the photomodified sample. The samples were incubated for one hour at 25 C. Fructose was then added to a final concentration of 23 mM to quench the remaining NaBH4. The samples were allowed to react for an additional hour at 25° C. The samples were then activated and assayed for RuBP carboxylase activity (3). 

CHARACTERIZATION OF RuBP CARBOXYLASE/OXYGENASE OF N. TABACUM BY INHIBITION OF THE OXYGENASE FUNCTION BY MEANS OF SPECIFIC ANTIBODIES... 

In the present publication we compare the inhibitory effect of an antiserum to RuBP carboxylase/oxygenase of Spinacia oleracea on the oxygenase activity of the wild type enzyme of tabacum var. JWB with the inhibitory effect of the homologous antiserum and that of the antiserum to the above described tabacum mutant Su/su. Furthermore, we compare the amount of antibodies bound out of the respective antisera. As shown earlier, RuBP carboxylase/oxygenase from tobacco species shows only partial immunochemical identity, when compared to the enzyme of Spinacia oleracea (1,3). 

Comparative immunological studies of RuBP carboxylase/oxygenase in the tandem crossed immuno electrophoresis have shown that immunochemical identity exists between the enzymes of the wild type tabacum var. 

Fig. 1 Comparison of RuBP carboxylase/oxygenase of tabacum var. JVJB with that of the tobacco mutant Su/su, Su/su var. Aurea, Spinacia oleracea by tandem crossed immuno electrophoresis.

Antigen Chloroplast preparation of J, tabacum var. JWB Su, N. tabacum Su/su Sa, tabacum Su/su var. Aurea Sp, Spinacia oleracea. Antiserum AS, mixed antiserum to RuBP carboxylase/oxygenase of tabacum var. JWB and of tabacum Su/su, 1.4% serum in the gel ASo, mixed antiserum to RuBP carboxylase/oxygenase of tabacum var. JWB and of Spinacia oleracea A, antiserum to RuBP carboxylase/oxygenase of N. tabacum var. JWB... 

Fig. 2 Binding of antibodies onto RuBP carboxylase/oxygenase of N.. var. JWB, of the mutant from N.. Su/su and of Spinacia oleracea out of homologous and non-homologous antisera in the region of equivalence. The values give the number of antibody molecules bound onto one enzyme molecule.

Fig 3 Dependence of oxygenase activity of RuBP carboxylase/oxygenase of tabacum var, JWB on the binding of antibodies out of the homologous and of the non-homologous antiserum,... 

A, treatment of RuBP carboxylase/oxygenase from tabacum var. JWB with the homologous antiserum and B, with the antiserum to the enzyme of N tabacum Su/su and C, with the antiserum to the enzyme of Spinacia oleracea. 

IMMUNOLOGICAL DEMONSTRATION OF STRUCTURAL DIFFERENCES OF RuBP CARBOXYLASE/OXYGENASE IN MUTANTS OF NICOTIANA TABACUM... 

Okabe et al. (11) and Bhagwat et al. (12) have shown that hydroxylamine specifically inhibits the oxygenase activity of RuBP carboxylase/oxygenase. Moreover, Okabe et al. (11) have shown that a temperature treatment to 50°C increases both activities of the enzyme by about 60%. In the following we investigate, whether chemical modifications of the enzyme, induced by hydroxylamine or heat treatment, are immunochemically characterizable. 

Fig. 2 Comparison of RuBP carboxylase/oxygenase of N.t. var. JWB with that of the mutants of N.. var. Consolation by means of the tandem crossed immuno electrophoresis.

REACTIVATION OF DARK-INACTIVATED RUBP CARBOXYLASE FROM PHASEOLUS VULGARIS IN VITRO... 

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