Free subunits of ribulose-1,5-bisphosphate carboxylase in pea leaves

Pea leaves, supplied with [³⁵S]methionine, were homogenized and a crude hypotonic soluble fraction was centrifuged on sucrose gradients to separate fully assembled ribulose-1,5-biphosphate (RuBP) carboxylase from any free or partially assembled carboxylase subunits. Slowly sedimenting subunits of the enzyme were identified in upper fractions of the sucrose gradient, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), isoelectric focussing, and immune precipitation. The presence of these subunits in low molecular weight form was shown not to be due to artefactual dissociation of the enzyme. It is suggested that these subunits are related to the assembly of RuBP carboxylase.     

Isolation and characterization of wheat ribulose-1,5-diphosphate carboxylase

Wheat ribulose-1,5-diphosphate carboxylase purified to homogeneity had a MW of 540 000, sedimentation coefficient (S_{20, W}) of 18.5 S, apparent diffusion constant (D_app) of 3.07 × 10^?7 cm²/sec, Stoke's radius 5.44 nm, and fractional ratio of 1.17. Electron microscopy revealed particles of 10–12 nm diameter. The enzyme was dissociated by sodium dodecyl sulphate into two subunits of MW 53 000 (S_{20, W} = 3.0 S) and 13 500 (S_{20, W} = 1.7 S). The total amino acid residues in the large and small subunits were 481 and 117, respectively. Tryptic peptide maps of the two subunits confirmed the estimated numbers of Arg and Lys residues. Although the amino acid pattern of the large subunit closely resembled that from barley, rather than that for spinach, beet or tobacco, the pattern of the small subunit was markedly different from those of all the other species.     

Development of ribulose-1,5-diphosphate carboxylase in castor bean cotyledons

Light was not essential for the development of ribulose-1,5-diphosphate carboxylase protein or catalytic activity in the photosynthetic cotyledons of germinating castor beans (Ricinus communis). Cotyledons developing in the dark showed higher activity than those in the light. Returning cotyledons developing in the light to darkness resulted in a significant increase in ribulose-1,5-diphosphate carboxylase activity compared to cotyledons in continuous light.     

Homotropic effect of CO 2 in ribulose-1,5-diphosphate carboxylase reaction

The concentration effect of aqueous CO₂ on the reaction velocity of spinach leaf ribulose-1,5-diphosphate carboxylase has been reevaluated. The homotropic effect of CO₂ in the enzyme reaction supports the previously reported allosteric nature of the enzyme in the CO₂-fixation process in chloroplasts. The concentration of CO₂ giving the half maximal reaction velocity, S_0.5, has been calculated to be 1.47 × 10⁻⁵M.     

Specific inhibition of oxygenase activity of ribulose-1,5-diphosphate carboxylase by hydroxylamine

Ribulose-1,5-diphosphate oxygenase activity of ribulose-1,5-diphosphate carboxylase was completely inhibited by preincubation of the enzyme with 5mM hydroxylamine in presence of the substrate ribulose-1,5-diphosphate. Inhibition by hydroxylamine was uncompetitive with respect to ribulose-1,5-diphosphate and noncompetitive with respect to magnesium. Carboxylase activity was not affected by hydroxylamine. These results suggest that the two activities of the enzyme can be regulated differentially and that inhibiting the oxygenase activity does not stimulate the carboxylase activity of the enzyme. The data further suggest that the inhibition by hydroxylamine may be through its interaction with carbonyl groups of the enzyme exposed on the binding of ribulose-1,5-diphosphate to the protein.     

Catalytic role of subunit A in ribulose-1,5-diphosphate carboxylase from Chromatium strain D

The oligomeric form of the larger subunit designated as A_m produced by alkali treatment of ribulose-1,5-diphosphate carboxylase from the purple sulfur bacterium, Chromatium strain D, retained partial enzymic activity in the absence of the small subunit (B). Supporting evidence was obtained by polyacrylamide gel electrophoresis at pH 8.9 and Sephadex G-200 gel filtration equilibrated with alkaline buffer at pH 9.2. The specific enzyme activity of A_m (45 nmoles CO₂ fixed/mg protein/min) was approximately 15% of the native intact enzyme molecule. By sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the A_m preparation was proved to be free from contamination of subunit B. With reservation of the sensitivity limit of this particular technique we concur that the larger subunit is the catalytic entity of the carboxylase reaction. The optimum pH of the ribulose-1,5-diphosphate carboxylase reaction catalyzed by isolated A_m lies on the alkaline side at about pH 8.3 with or without Mg²⁺. The undissociated native enzyme possesses an optimum pH on the alkaline side in the absence of Mg²⁺, which shifts to the acidic side in the presence of Mg²⁺. From this behavior it is inferred that the association of the smaller subunit with the larger subunit causes conformational stabilization of the enzyme molecule with an accompanying change in the pH optimum due to Mg²⁺.     

Properties of ribulose-1,5-bisphosphate carboxylase from dark- and light-exposed soybean leaves

The low activity of ribulose bisphosphate carboxylase from darkened soybean (Glycine max [L.] Merr. cv. Bragg) leaves was not raised to the level of that from leaves in the light by CO₂ and Mg²⁺, even after a 4-h incubation. The extract of darkened leaves, unlike the extract from illuminated leaves, was not fully CO₂/Mg²⁺-activatable after Sephadex gel filtration in the absence of Mg²⁺. (NH₄)₂SO₄ fractionation eliminated the inhibition effect found in the dark extracts resulting in similar rates for the extracts obtained from leaves in the dark and light. Although the V_max values of the gel-filtered extracts from dark and light leaves differed by 3-fold, the K_m(CO₂)-values were the same (12.7 μM), as were the K_m(RuBP)-values (250 μM). These data support the hypothesis that for soybean leaves in the dark a tightly-binding inhibitor renders much of the ribulose bisphosphate carboxylase enzyme catalytically non-functional.     

Solubilization of ribulose-1,5-bisphosphate carboxylase from the membrane fraction of pea leaves

The solubilization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the membrane fraction was studied in whole leaf extracts and chloroplasts from pea. The amount of membrane-bound Rubisco was dependent on the pH of the chloroplastic lysate buffer. Maximum binding was found at pH 8.0, with about 8% of total leaf Rubisco being bound. The binding of Rubisco to the membranes was strong, and it was not released by repeated washing with hypotonic buffer or by changing ionic strength. Detergents such as Triton X-100, Tween 20, deoxycholate and dodecylsulfate were effective in solubilizing the membrane-bound Rubisco. Triton X-100 was most effective in the range of 0.04% to 0.2% and it solubilized Rubisco from the membrane without any decrease in enzyme activity.Abbreviations BSA bovine serum albumin - CABP carboxyarabinitol-1,5-bisphosphate - DTT dithiothreitol - LDS lithium dodecylsulfate - LHC light-harvesting chlorophyll protein complex - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase - SDS sodium dodecylsulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

A one-step method for the isolation and determination of leaf ribulose-1,5-diphosphate carboxylase

A convenient method is described for the isolation in one step of Rumex leaf ribulose-1,5-diphosphate carboxylase (RuDPCase) by sucrose density gradient centrifugation. The amount of RuDPCase in a sample of leaf tissue is determined by quantification of the enzyme peak. RuDPCase prepared by this method is shown to be highly pure by criteria including light absorption at 260 and 280 nm, RuDPCase enzyme activity, and polyacrylamide gel electrophoresis at pH 9.2 and at pH 7.1 in the presence of sodium dodecylsulfate. The procedure has been successfully applied to various additional species including bean, maize, and spinach with little or no modification. This method should prove useful for determination of in vivo levels of RuDPCase protein in physiological studies, for measurements of synthesis and breakdown of RuDPCase using radioisotope labeling, and for the preparation of milligram amounts of RuDPCase for further purification and in vitro studies of enzyme structure and function.     

In vitro reassembly of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase from fully denatured subunits

It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from fully denatured subunits in vitro in higher plant,because large subunit of fullydenatured Rubisco is liable to precipitate when the denaturant is removed by common methods of directdilution and one-step dialysis.In our experiment,the problem of precipitation was resolved by an improvedgradual dialysis method,which gradually decreased the concentration of denaturant.However,fully denaturedRubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60was added.The restored activity of reassembled Rubisco was approximately 8% of natural enzyme.Thequantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassemblyprocess.ATP and Mg~(2 ) were unnecessary for in vitro reassembly of Rubisco,and Mg~(2 ) inhibited the reassemblyprocess.The reassembly was weakened when ATP,Mg~(2 ) and K~ existed together in the reassembly process.