66] Glucose-6-phosphatase from cerebrum

Abstract

Publisher Summary This chapter describes an assay method and the purification procedure for the enzyme glucose-6-phosphatase from cerebrum of the brain. Glucose-6-phosphatase from brain and liver are known to act as phosphotransferases under appropriate conditions and as an inorganic pyrophosphatase. The well-known method of Zakim and Vessey for measuring glucose-6-phosphatase is employed successfully in the case of brain. The major amount of enzyme is obtained in the microsomal (endoplasmic reticulum vesicle) fraction upon cellular fractionation of brain and involves preparation of crude brain microsomes and extraction and purification of enzyme from rat brain. The strategy involves homogenization, delipidation by removing the myelin fraction by flotation, recovery of the membrane pellet, extraction of the latter with a buffered deoxycholate solution, and purification by gel filtration in the presence of deoxycholate. Rat brain microsomal glucose-6-phosphatase has a latency with respect to saturating concentrations (20 mM) of glucose 6-phosphate and mannose 6-phosphate of about 60–70%. Brain microsome preparations do not exhibit sensitivity to diisothiocyanostilbene disulfonate (DIDS) under conditions that inhibit the glucose-6-phosphatase of intact liver microsomes.