The presence of a large central vacuole in plant cells has greatly hindered histochemical and biochemical investigations, because, when a homogenate is prepared, the vacuolar sap, which frequently includes large quantities of the substrate and product of an enzyme reaction and in certain cases enzyme inhibitors, becomes intimately mixed with the somewhat limited amount of protoplasm present. Bentley (1952), for instance, has shown that malonic acid is widely distributed in plants; fluoroacetic acid occurs in
Dichapetalum cymosum
(Marais 1944), and transaconitic acid may be present in certain members of the Ranunculaceae. Though these facts pose interesting problems in the permeability relations of cell components, they greatly complicate the analysis of homogenates. Dialysis provides one method of isolating active enzyme material from the homogenate, but the concomitant loss of cofactors has limited its application. A partial solution to the problem has been obtained by isolating particulate material by differential centrifugation. This method has made possible investigations of metabolism not amenable to study by the classical methods of enzymology and simultaneously has provided an approach to the study of the spatial relations of enzymes. The pioneer work was performed by Hill & Bhagvat (1939), who demonstrated the association of cytochrome oxidase with particulate material. Several workers (Meeuse 1950; Bhagvat & Hill 1951; Stafford 1951) have demonstrated the association of succinoxidase with particulate material isolated from homogenates prepared in dilute phosphate buffer, but cytochrome
c
was necessary for activity. Meeuse (1950) also found that malic oxidase was associated with particulate material, but DPN and cytochrome
c
were required for activity. Millerd, Bonner, Axelrod & Bandurski (1951), Stafford (1953), Price & Thimann (1953) and Davies (1953) have isolated particulate material from seedlings which was active in the oxidation of Krebs-cycle intermediates. Millerd
et al
. (1951) were also able to demonstrate that phosphorylation accompanied the oxidation of Krebs-cycle intermediates.
REGULATORY MECHANISMS OF CELLULAR RESPIRATION
