The Specificity of Glucose 6-Phosphatase of Intact Liver Microsomes

Abstract 1. In an attempt to evaluate the significance of the latency, which is characteristic of the various phosphohydrolase and phosphotransferase reactions catalyzed by glucose 6-phosphatase, a detailed study was undertaken of the specificity of the enzyme before and after liver microsomes were disrupted by the addition of detergent or exposure to high pH. Activities with PPi, carbamyl-P, mannose-6-P, ATP, glucosamine-6-P, 2-deoxy-d-glucose-6-P, and glucose-6-P were examined. 2. The following observations were made. (a) With the exception of the activities with PPi and glucose-6-P, all reactions catalyzed by glucose 6-phosphatase of untreated microsomes showed levels of latency in excess of 85%. Mild heating (30°) at pH 5.5 caused some unmasking of the activities and PPi potentiated this effect. Since these are the conditions of assay in untreated microsomes the manifestation of activities with PPi appear to be artifacts and glucose-6-P hydrolysis remains as the only activity with a low level of latency. (b) The Michaelis constant for glucose-6-P was markedly reduced by disruption of microsomes while Michaelis constants for the other substrates were unaffected. (c) Compounds which were good substrates in disrupted preparations (e.g. mannose-6-P, PPi, and carbamyl-P) were weak inhibitors of glucose-6-P hydrolysis by untreated microsomes. In contrast, glucose-6-P was an equally potent inhibitor of mannose-6-P hydrolysis with either treated or untreated microsomal preparations. (d) Disruption of microsomal structure markedly reduced the energy of activation of glucose-6-P phosphohydrolase without altering the value for the activation energy of mannose-6-P phosphohydrolase. 3. These observations can be explained by a model for glucose 6-phosphatase in which the enzyme is positioned within the membrane of the endoplasmic reticulum in an orientation that permits interaction of the catalytic site with glucose-6-P but excludes other phosphate substrates, unless the membrane is disrupted. Fresh, untreated microsomes are considered to consist of two forms: (a) intact structures in which the enzyme is specific for glucose-6-P and (b) disrupted structures in which the enzyme is broadly specific. Exposures to hypotonic media, extremes of pH, detergents, or ultrasound increase the proportion of the disrupted form. 4. We suggest that the physiologically functional form of the enzyme is specific for glucose-6-P.