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Abstract:
Transcarboxylase consists of a central subunit to which two sets of three subunits each are attached at opposite faces. Evidence obtained by various ultracentrifugal techniques has shown that there is an equilibrium between active forms of the enzyme with six, five, four, three, two, and one subunits attached to the central subunit. Since each attached subunit contains two biotins, the biotin content of these forms varies from 12 to 2. By reactive enzyme sedimentation at pH 5.5, it has been shown that the largest form of the enzyme (with 12 biotins and a molecular weight of 1.2 X 10(6) is active and that at pH 6.8 this form of the enzyme dissociates to an enzymatically active form containing three attached subunits. This result is in accord with previous observations (Wood, H.G., Chiao, J.P., and Poto, E.M.(1977), J Biol. Chem. 252, 1490; Wrigley, N.G., Chiao, J.P., and Wood, H.G. (1977), J Biol. Chem. 252, 1500) which showed that dissociation of three of the six attache subunits occurs preferentially from one face of the central subunit and that the remaining three attached subunits are quite firmly bound to the second face of the central subunit. Multiple peaks are obtained in sedimentation velocity experiments at 60 000 rpm because the rate of sedimentation outstrips the rate of equilibration of the different forms of transcarboxylase, whereas at 30 000 rpm a single peak of the multiple form of the enzyme is observed, since equilibration of the different forms keeps abreast of the sedimentation. The rate and extent of dissociation are increased by increase in temperature. It has been shown by centrifugation under oil that the association-dissociation of the large form of the enzymes with six, five, and four attached subunits is not affected by hydrostatic pressure. In contrast, the association-dissociation of the enzyme with three and two attached subunits appears to be affected by pressure. The accumulated results indicate that the native form of the enzyme in the cytosol of the cell consists of a series of enzymatically active forms with one to six attached subunits. The predominant forms in the cytosol will depend on the concentration of the constituent subunits, the pH, the concentration of ions (particularly divalent anions), and the temperature.