William J. Arion

[111] Glucose-6-phosphatase

Publisher Summary This chapter describes the assays for the determination of glucose-5-phosphatase and a modified method for measuring glucose-6-P hydrolysis. The methods discussed are glucose-6-P hydrolysis, PPi hydrolysis, and phosphotransferase activities. Sodium cacodylate buffers are employed in all assay systems because this compound buffers effectively over the range pH 5–7.4 and exerts no specific effect on the reactions. Sodium acetate buffer also may be employed in the phosphotransferase and inorganic pyrophosphatase assay systems; however it is not suitable for the glucose-6-P hydrolysis assay. Citrate recently has been found to inhibit competitively with respect to phosphate substrates, below pH 6. Glucose-6-phosphatase is a microsomal enzyme which has resisted attempts to obtain a satisfactory purification. A number of significant efforts to solubilize and purify this enzyme recently have been carried out. Moderate enrichments have been obtained by differential centrifugation of microsomal preparations after treatment with detergents or proteolytic enzymes, and by fractional precipitation of detergent-dispersed microsomal preparations with acetone in the presence of added magnesium++ (Mg++).

Measurement of intactness of rat liver endoplasmic reticulum.

Publisher Summary This chapter discusses the measurement of the intactness of rat liver endoplasmic reticulum. The uniform distribution of the glucose-6-phosphatase system throughout the parenchymal ER and the contrast between the broadly specific luminal enzyme and the glucose-6-P-specific transporter presented the basis for a method to quantify the integrity of the permeability barrier defined by the ER membranes. The principal source of error in determining the latency of mannose-6-phosphatase is the quantitative assay of the phosphohydrolase activity in fully disrupted membranes. The latency of mannose-6-phosphatase can be used to estimate ER membrane intactness in whole liver homogenates and isolated hepatic nuclei. However, the investigator should be aware that a lower concentration of detergent is required to effect the complete disruption of the nuclear envelope and that the transformation of mannose-6-P to glucose-6-P catalyzed by the sequential actions of phospho-mannose isomerase (mannose-6-phosphate isomerase) and phospho-glucose isomerase (glucose-6-phosphate isomerase) in the supernatant may confound the assessment of intactness in whole liver homogenates.