The effects of N-alkylmaleimides on the activity of rat liver glucose 6-phosphatase.
A series of N-alkylmaleimides has been synthesized and used to investigate the thiol groups that are essential for the activity of rat liver microsomal glucose 6-phosphatase. All of the N-alkylmaleimides inactivated glucose 6-phosphatase when preincubated with microsomes (microsomal fractions) at pH 6.5 and 30 degrees C. When enzyme activity was assayed in intact microsomes, the inactivation was non-linear with respect time, showing an initial rapid phase followed by a slower secondary phase. During the initial rapid phase the inactivation may apparently be completely reversed by disrupting the microsomal membrane with detergent. However, after longer exposure to N-alkylmaleimides the reversal is no longer complete. This observation was explained by the results obtained from studying the inactivation in detergent-disrupted microsomes. In this case glucose 6-phosphatase was also completely inactivated, but much more slowly than was seen in intact microsomes, and the process was linear with respect to time. When assayed in both intact and detergent-disrupted microsomes, glucose 6-phosphatase inactivation was dependent on the number of carbon atoms in the alkyl side chain of the N-alkylmaleimides; this dependence was much more marked in disrupted microsomes. Analysis of the data showed that in neither case was there a saturating effect at high concentrations of maleimide. The data have been interpreted to suggest that there are are least two thiol groups essential for activity located in two separate non-polar regions of the membrane-enzyme system. The conclusions are discussed in the light of the current model for the microsomal glucose 6-phosphatase system.
D000818:Animals; D003840:Deoxycholic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005952:Glucose-6-Phosphatase / Q000037:antagonists & inhibitors*; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000494:pharmacology; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology; D007700:Kinetics; D008297:Male; D008301:Maleimides / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D051381:Rats; D013997:Time Factors
D005958:Glucosephosphates; D008301:Maleimides; D003840:Deoxycholic Acid; D019298:Glucose-6-Phosphate; D005952:Glucose-6-Phosphatase