Regulation of synthesis of pyruvate carboxylase in the photosynthetic bacterium Rhodobacter capsulatus

Abstract

The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C 4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C 4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis.

A Luminol/Iodophenol Chemiluminescent Detection System for Western Immunoblots

“…Stock solutions used were: luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, ICN Biochemicals), 10 mM solution in 50% dimethyl sulfoxide (Me 2 SO); 4-iodophenol (Aldrich Chemical Co., U.S.A.), 50 mM solution in 50% Me 2 SO; H 2 O 2 (Fisher Scientific), 30%. Antisera raised against purified proteins from Rhodobacter capsulatus were as described (7,8,9).…”

Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum

“…In most varieties of pyruvate carboxylases examined so far, the enzyme appears to be constitutive, with regulation accomplished either through effector modulation of holoenzyme activity (pyruvate carboxylase from animal sources, yeast, or several species of bacteria) or through control of the biotinylation of the apoenzyme by biotin ligase (Bacillus stearothermophilus) (4,5,38). Finally, two other organisms where the carbon source controls the pyruvate carboxylase expression are Azotobacter vinelandii (37) and Rhodobacter capsulatus (25,44). Most interesting are the pyruvate carboxylase activity differences obtained on pyc overexpression in two different host strains and in two different carbon sources.…”

Structure, function and regulation of pyruvate carboxylase

“…In lower organisms, such as S. cere isiae [46], Methanobacterium thermoautotrophicum [47], Bacillus stearothermophilus [48] and Rhizobium etli [5,49], the availability of biotin in the medium has been shown to greatly enhance PC activity, and this effect is thought to be mediated through biotinylation of apoenzyme to holoenzyme, rather than by gene induction. In contrast, changes in PC specific activity of Rhodobacter capsulatus under different growth conditions are mediated at the level of enzyme synthesis [50]. -Aspartate is known to inhibit PC activity and biotinylation in yeast through an allosteric effect [46].…”