Investigations on the possible involvement of phospholipids in the glucose-6-phosphate transport system of rat-liver microsomal glucose-6-phosphatase.
Interrelationships between the catalytic behavior of glucose-6-phosphatase and the structure of rat-liver microsomal membranes were investigated. 2. Rabbit anti-microsomal serum completely inhibited glucose-6-phosphate hydrolysis in detergent-modified microsomes but showed no inhibitory effect on the enzyme activity of intact or mechanically disrupted vesicles. 2. Controlled proteolysis of intact microsomes using carboxypeptidase A and/or aminopeptidase M largely denatured enzymes situated on the outer surface of the microsomal vesicles such as monodehydroascorbate reductase and cytochrome c reductase. However, it did not affect the glucose-6-phosphatase activity at all, which remained in a latent state within the membrane. 3. Temperature studies on glucose-6-phosphatase have revealed that only the enzyme activity of intact microsomes exhibited a nonlinear Arrhenius plot, whereas detergent-modified microsomes showed a linear temperature response. 4. Treatment of microsomes with phospholipase C and toluene-2,4-diisocyanate resulted in an apparent loss of about 65% and 85% of the original glucose-6-phosphatase activity and was closely correlated with hydrolysis and chemical modification of phosphatidylethanolamine, respectively. These apparent inactivations could be reversed by addition of Triton X-114 alone without any phospholipid supplementation. These observations indicate that glucose-6-phosphatase is buried within the microsomal membrane, not exposed on either side. They also suggest that phospholipids are involved in the glucose-6-phosphate transport mechanism.
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005952:Glucose-6-Phosphatase / Q000502:physiology*; D005958:Glucosephosphates / Q000502:physiology*; D007106:Immune Sera / Q000494:pharmacology; D007122:Immunoelectrophoresis; D008862:Microsomes, Liver / Q000201:enzymology*; D010743:Phospholipids / Q000502:physiology*; D051381:Rats; D013696:Temperature; D014051:Toluene 2,4-Diisocyanate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005958:Glucosephosphates; D007106:Immune Sera; D010743:Phospholipids; D014051:Toluene 2,4-Diisocyanate; D005952:Glucose-6-Phosphatase; D010738:Type C Phospholipases