Some properties of variously activated microsomal glucose-6-phosphatase, inorganic pyrophosphatase and inorganic pyrophosphate-glucose phosphotransferase. Shift in pH optimum

Abstract

1. 1. The activity of microsomal glucose-6-phosphatase (EC 3.1.3.9) and the related enzymatic functions, acid inorganic pyrophosphatase and inorganic pyrophosphate- glucose phosphotransferas is greatly increased by pretreatment of ‘microsomes’ with hydroxyl ion at pH 9.5–9.8. The thermal stability of the preparations so activated is in marked contrast to the great instability observed when a corresponding activation is accomplished with deoxycholate or Triton X-100. 2. 2. It is suggested that comparative levels of these enzyme activities be determined on NH 4OH-treated homogenates or particulate fractions rather than on untreated or detergent-activated preparations. 3. 3. After activation with NH 4OH, deoxycholic acid, or Triton X-100 the optimum pH of all three activities is slufted in the alkaline direction. 4. 4. A similar activation and pH optimum shift is observed with liver mirosomes from fed, fasted and diabetic rats, in which the absolute level of activity varies many-fold. 5. 5. Erroneous results are obtained if activities of untreated microsomes are compared with activated ones at the same pH. This is particularly true for the transferase activity in which the quantitative effect of the pH optimum shift is much greater than for the hydrolase activities. 6. 6. A difference between glucose-6-phosphatase and the related PP i-utilizing enzymatic activites has been noted and studied. Glucose 6-phosphatase hydrolysis by liver microsomes is inhibited by lower concentrations of deoxycholate or Triton X-100 than are required to cause activation. PP i hydrolysis and glucose 6-phosphatase synthesis from PP i, on the other hand, are only activated by these low concentrations of detergents. 7. 7. The results lead to the suggestion that some of the active sites, particularly those for glucose, are on the inner surfaces of the endoplasmic reticulum or are impeded by the spatial conformation of the membrane or by the attachment of ribosomes on the outer surfaces.