Evidence for changes in the conformational status of rat liver microsomal glucose-6-phosphate:phosphohydrolase during detergent-dependent membrane modification. Effect of p-mercuribenzoate and organomercurial agarose gel on glucose-6-phosphatase of native and detergent-modified microsomes.

Comparative studies investigating influences of temperature and time of preincubation on the interactions of an organomercurial agarose gel and p-mercuribenzoate with glucose-6-phosphatase of native and Triton X-114-modified rat liver microsomes were carried out. The effect of p-mercuribenzoate on glucose 6-phosphate hydrolysis is a result of two processes, a moderate membrane perturbation connected with release of some latency and temperature- and time-dependent inhibition of the catalytic activity. Short-term preincubation with both organic mercurials at 37 degrees C is a necessary condition for the entire inhibition of the enzyme activity of native as well as of Triton X-114-modified microsomes. A binding site of the phosphohydrolase itself is accessible to p-mercuribenzoate and the phenyl mercury residue of the affinity gel from the cytoplasmic surface even in native microsomes. Kinetic analyses reveal a formally competitive mechanism of inhibition using native microsomes, but the kinetic picture changes to a noncompetitive pattern of Lineweaver-Burk plots when the inhibitor-loaded microsomes are modified optimally by Triton X-114. This behavior can be evaluated as the first convincing evidence for drastic changes of the conformational status of the phosphohydrolase during the membrane modification process. A combined conformational flexibility-substrate transport model characterizing the microsomal glucose-6-phosphatase as an integral channel-protein embedded within the hydrophobic interior of the membrane is proposed.

Latency studies on rat liver microsomal glucose-6-phosphatase. Correlation of membrane modification and solubilization by Triton X-114 with the enzymatic activity.

Interrelationships between the catalytic properties of glucose-6-phosphatase and the membrane structure of rat liver microsomes were investigated. Membrane modification and solubilization employing the nonionic surfactant Triton X-114 were standardized and analysed by ultracentrifugation, surface tension- and turbidity measurements. The effect of Triton X-114 on the glucose-6-phosphatase activity was studied systematically and the whole magnitude of time- and temperature-dependent inactivation of this enzyme has been demonstrated. The results show that the activity measured is always a resultant of two processes, the beginning of inactivation and the release of latency. Maximal activation of about 600% (83% of apparent latency) was obtained at 0 degree C. A correlation between membrane modification and solubilization and the conditions under preincubation and test incubation reveals that studies on detergent-disrupted microsomes are performed on structures reassembled from solubilizates and this implies a modified microenvironment in the reconstitutes. Kinetic analyses suggest interrelationships between Triton X-114 and the permeability barrier of the glucose-6-phosphatase system. At 0 degree C 2-propanol and ethanol are more potent tools for membrane modification than Triton X-114 and release 88% and 86% latent activity corresponding to an activation of the glucose-6-phosphatase of about 850% and 700%, respectively. These observations suggest that detergent treatment of microsomes could not preserve the functional integrity of the glucose-6-phosphate phosphohydrolase, which is one dogma of the substrate-transport hypothesis developed by Arion and his co-workers (Arion, W.J., et al. (1975) Mol. Cell. Biochem. 6, 75-83).