Further characterization of the Krebs tricarboxylic acid cycle metabolon.

A preparation of gently disrupted rat liver mitochondria which shows exposed and easily sedimented Krebs tricarboxylic acid cycle enzyme activities has been characterized further. The exposed malate dehydrogenase is inhibited by high molecular weight blue dextran which indicates the availability of the enzyme to the bulk solvent. Further, mitoplasts are not permeable to citrate synthase antibodies ruling out the possibility of vesicularization of high molecular weight substances. The slightly disrupted mitochondria sedimented more slowly than did intact mitochondria on a Ficoll gradient. Electron microscopy, both thin section and scanning, showed slightly swollen mitochondria with some disruption of the membranes. Labeling with ferritin-labeled second antibody to citrate synthase antibodies showed again the accessibility of these disrupted mitochondria to the antibody. When either the oxidation of fumarate or the malate dehydrogenase-citrate synthase coupled system are studied, relative kinetic advantages are observed of the gently disrupted systems over the completely solubilized system. These kinetic advantages are more labile to disruption than is the binding of the enzymes to the particle. These results indicate that the Krebs tricarboxylic acid cycle exists as a sequential complex of enzymes, a metabolon, in situ. This study shows that previous studies which showed interactions between sequential enzymes of this pathway and their binding to the inner surface of the inner membrane actually reflected an in vivo organization of this pathway.