D-Ribulose-1,5-bisphosphate (RuBP) carboxylase has been purified from glutamate-C02-S203 2-_grown Thiobacillus intermedius by pelleting the enzyme from the high-speed supernatant and by intermediary crystallization followed by sedimentation into a discontinuous 0.2 to 0.8 M sucrose gradient. The enzyme was homogeneous by the criteria of electrophoresis on polyacrylamide gels of several acrylamide concentrations, sedimentation velocity and equilibrium measurements, and electron microscopic observations of negatively stained preparations. The molecular weights of the enzyme determined by sedimentation equilibrium and light-scattering measurements averaged 462,500 + 13,000. The enzyme consisted of closely similar or identical polypeptide chains of a molecular weight of 54,500 5,450 determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The s°2,, of the enzyme was 18.07S + 0.22. Electron microscopic examination suggested that the octomeric enzyme (inferred from the molecular measurements mentioned) had a cubical structure. The specific activity of the enzyme was 2.76 ,tmol of RuBP-dependent CO2 fixed/min per mg of protein (at pH 8 and 30 C), and the turnover number in terms of moles of CO2 fixed per mole of catalytic site per second was 2.6. The enzyme was stable for 3 months at -20 C and at least 4 weeks at 0 C. The apparent Km for CO2 was 0.75 mM, and Km values for RuBP and Mg2+ were 0.076 and 3.6 mM, respectively. Dialyzed enzyme could be fully reactivated by the addition of 20 mM Mg2+ and partially reactivated by 20 mM Co2+, but Cd2+, Mn2+, Ca2+, and Zn2+ had no effect. The compound 6-phosphogluconate was a linear competitive inhibitor with respect to RuBP when it had been preincubated with enzyme, Mg2+, and HCO3r. D-Ribulose-1,5-bisphosphate carboxylase (RuBPCase; 3-phospho-D-glycerate carboxylyase Ldimerizing], EC 4.1.1.39), the cardinal enzyme of the reductive pentose phosphate pathway, catalyzes the condensation of CO2 and ribulose-1,5-bisphosphate (RuBP) and the cleavage of the intermediate 2-carboxy-3-ketoribitol-1,5-bisphosphate (31) into 3-phosphoglycerate. RuBPCase is present in all photolithotrophic life forms including purple sulfur and nonsulfur bacteria and the green sulfur bacterium Chlorobium limicola f. thiosulfatophilum (37). The distribution, purification, and properties of RuBPCases have been recently reviewed (22,25). RuBPCase is known to be present in several chemolithotrophic bacteria (1,16,19,21,22,23,29,30,32,42), and pure enzymes have been isolated from fructose-grown Hydrogenomonas eutropha, Hydrogenonormas facilis (13,14), and autotrophically grown Thiobacillus novellus (19,20). RuBPCases isolated from plants, algae, and bacteria have been classified into three size categories (22, 25): large (molecular weight 550,000), intermediate (molecular weight 360,000), and small (molecular weight 120,000). With one exception, large RuBPCases isolated from plants, algae, or bacteria consist of two types of subunits, large (molecular weight 51,000 to 58,000) an...

No abstract

The growth-related parameters of Thiobacillus intermedius, cultured in glutamate-CO.2-S20O2medium, have been determined. After centrifugation at 48,000 x g for 1 h, 24% of the n-ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity ofthe disrupted-cell suspensions obtained from C02-S2032-and glutamate-CO.,-S.,203-grown cells could be sedimented, and the specific activities of this enzyme in the supernatant fractions were almost equivalent. The enzyme was stable in T. intermedius starved of thiosulfate in the presence and absence of glutamate, but a progressive decrease was evident in several growth cycles, each cycle supported by resupplementation of cells with thiosulfate. Polyhedral inclusion bodies were present in CO.._S.,O:2-and glutamate-C02-S 0:1-grown cells. The number of polyhedral bodies per cell increased during mixotrophic growth approximately in proportion to the observed increase in the specific activity of RuBPCase. RuBPCase could not be detected in T. intermedius grown heterotrophically on yeast extract, nor could polyhedral bodies be found.

No abstract

Electrophoretically homogeneous ribulose-1,5-bisphosphate (RuBP) carboxylase was obtained from autotrophically grown Hydrogenomonas eutropha by sedimentation of the 105,000 x g supernatant in a discontinuous sucrose gradient and by ammonium sulfate fractionation followed by another sucrose gradient centrifugation. The molecular weight of the enzyme determined by light scattering was 490,000 + 15,000. The enzyme could be dissociated by sodium dodecyl sulfate into three types of subunits, and the molecular weights (±10%) could be measured. There were two species of large subunits, L and L' (molecular weight 56,000 and 52,000, respectively) and one species of small subunits (molecular weight, 15,000). The mole ratio of L to L' was 5:3, and the overall mole ratio of the small to large subunits was 1.08. The simplest quaternary structure of the enzyme is L5L'3S8. The enzyme contained RuBP oxygenase activity as evidenced by the 02-dependent production of phosphoglycolate and 3phosphoglyceric acid in equimolar quantities from RuBP.