A New Purification Procedure for Fumarase Based of Affinity Chromatography. Isolation and Characterization of Pig-Liver Fumarase

European Journal of Biochemistry

1989

Self Cite

Evidence is growing that the citric acid cycle, like many other metabolic pathways, might exist in vivo as a more or less tightly organized multi-enzyme cluster. The term 'metabolon' [Robinson, J. B. & Srere, P. A. (1985) J. Biol. Chem. 260, 10800-108051 was recently introduced to describe such a complex of sequential metabolic enzymes.We adopted the technique of affinity electrophoresis for the study of interactions between the cycle enzymes fumarase and malate dehydrogenase. This approach offers several advantages over our previously described affinity chromatographic technique [Beeckmans, S. & Kanarek, L. (1981) Eur. J . Biochem. 117, 527-5351, one of which is the fact that the interaction can be directly visualized. The observed association is specific since both metabolically unrelated proteins and the cytoplasmic isoenzyme of malate dehydrogenase do not interact with fumarase.Several metabolites (citrate, isocitrate, 2-oxoglutarate, succinate, fumarate, malate, oxaloacetate, Pi, AMP, ADP, NAD', NADH) were found not to affect the association between fumarase and mitochondrial malate dehydrogenase. Both ATP, Mg2+-ATP and GTP disrupt the association when they are present at 1 mM concentrations. Lower non-physiological ATP concentrations do not, however, disturb the interaction. The presence of 1 mM ADP was found to abolish the disrupting effect of 1 mM ATP. The latter findings are suggestive of an interruption of the citric acid cycle at the level of fumarase under conditions of high energy load (i.e. high ATP/ ADP ratios).Whereas it was generally assumed in the past that the enzymes of a metabolic pathway are randomly distributed in their particular subcellular compartment or organelle, evidence is accumulating that, on the contrary, a cell should rather be considered to be a fully organized entity where all the constituent pathways are segregated as multi-enzyme complexes or multi-enzyme clusters, which very often seem to be associated with cellular structural elements (see [l -171 for recent reviews). Such an organization of pathways as 'metabolons' [I81 offers several advantages for a cell: (a) intermediates are passed directly to the next enzyme of the pathway, thereby circumventing diffusion problems arising from the organization of cellular water in layers along the surface of ubiquitous membranous and skeletal structures [19], and from the apparent crowdedness, in terms of protein content inside cells and organelles [20,21]; (b) intermediates can easily and efficiently be channeled by switching on and off of welldefined enzyme-enzyme interactions in response to changing local metabolite concentrations, allowing the cell to regulate meticulously its overall metabolism according to its momentary needs and, moreover, avoiding competition between opposite pathways; (c) energetically difficult steps in a pathway will readily be overcome; (d) a special environment is created around a pathway so that a high flux can be maintained with a moderate number of intermediate molecules; (e) labile intermediates are ...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Enzymes Proteins and Metabolitesmentioning

confidence: 99%

Section: Immobilisation Of Proteinsmentioning

confidence: 99%

“…This approach offers several advantages over our previously described affinity chromatographic technique [Beeckmans, S. & Kanarek, L. (1981) Eur. J .…”

mentioning

confidence: 99%

See 3 more Smart Citations

The visualization by affinity electrophoresis of a specific association between the consecutive citric acid cycle enzymes fumarase and malate dehydrogenase

European Journal of Biochemistry

1989

Self Cite

Clustering of sequential enzymes in the glycolytic pathway and the citric acid cycle

J of Cellular Biochemistry

1990

Self Cite

In recent years, evidence has been accumulating that metabolic pathways are organized in vivo as multienzyme clusters. Affinity electrophoresis proves to be an attractive in vitro method to further evidence specific associations between purified consecutive enzymes from the glycolytic pathway on the one hand, and from the citric acid cycle on the other hand. Our results support the hypothesis of cluster formation between the glycolytic enzymes aldolase, glyceraldehydephosphate dehydrogenase, and triosephosphate isomerase, and between the cycle enzymes fumarase, malate dehydrogenase, and citrate synthase. A model is presented to explain the possibility of regulation of the citric acid cycle by varying enzyme-enzyme associations between the latter three enzymes, in response to changing local intramitochondrial ATP/ADP ratios.

Immobilized enzymes as tools for the demonstration of metabolon formation. A short overview

J of Molecular Recognition

1993

Self Cite

In recent years it has become clear that a cell cannot be visualized as a 'bag' filled with enzymes dissolved in bulk water. The aqueous-phase properties in the interior of a cell are, indeed, essentially different from those of an ordinary aqueous solution. Large amounts of water are believed to be organized in layers at the surface of intracellular structural proteins and membranes. Such considerations prompt us to reconsider the operation and regulation of metabolic pathways. Enzymes of metabolic pathways are nowadays thought to be clustered and operate as 'metabolons'. Very often interactions between enzymes of a pathway can exclusively be evidenced in vitro in media which are known to reduce the water concentration in the vicinity of the proteins. Immobilized enzyme preparations have been shown to be excellent tools for this type of research. We describe here some recent studies where immobilized enzymes have been used in various applications to investigate associations among enzymes of a number of different metabolic pathways (glycolysis/gluconeogenesis, citric acid cycle and its connection to the electron transport chain, aspartate-malate shuttle, glyoxylate cycle). Advantages and disadvantages of the different techniques are also discussed.