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.
D000818:Animals; D003902:Detergents / Q000494:pharmacology*; D005952:Glucose-6-Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D008297:Male; D008626:Mercuribenzoates / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000201:enzymology*; D017830:Octoxynol; D009941:Organomercury Compounds / Q000494:pharmacology*; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011487:Protein Conformation; D051381:Rats; D011919:Rats, Inbred Strains; D012685:Sepharose; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology*; D013816:Thermodynamics
C043407:4-mercuribenzoate; D003902:Detergents; D008626:Mercuribenzoates; D009941:Organomercury Compounds; D013501:Surface-Active Agents; D011092:Polyethylene Glycols; D017830:Octoxynol; D012685:Sepharose; C010615:Nonidet P-40; D005952:Glucose-6-Phosphatase