Abstract
Publisher Summary This chapter describes an assay method and the purification procedure for fructose-l,6-bisphosphatase from mouse and rabbit intestinal mucosa. The purified enzyme from intestinal mucosa has similar properties to those of the known liver, kidney, and muscle fructose-l,6-bisphosphatases, such as molecular and subunit molecular weights, adenosine monophosphate (AMP) inhibition, pH optimum, and the metal requirement. The production of fructose 6-phosphate is measured spectrophotometrically by following the reduction of NADP+ with glucose-6-phosphate isomerase and glucose-6-phosphate dehydrogenase. Purification of the enzyme from mouse small intestinal mucosa involves extraction, heat treatment, phosphocellulose treatment, phosphocellulose chromatography, and Blue Sepharose treatment. Intestinal homogenates contain a proteolytic activity that catalyzes the conversion of fructose-1,6-bisphosphatase to a form having the increased activity at alkaline pH. The heat treatment inactivates the proteolytic activity and there is no evidence that the enzyme is modified after this step. Rabbit intestinal enzyme is well adsorbed on Blue Sepharose gel and is completely eluted at high purity from the gel with the low concentration of AMP, while mouse intestinal enzyme is poorly adsorbed on Blue Sepharose gel around pH 6 under the present conditions.
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