Specific Assays of some Phosphatases in Subcellular Fractions of Small Intestinal Mucosa

Abstract

IN the mucosa of the small intestine, alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1) is known to be mainly localized in the brush border region^1,2 while glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) is a predominantly microsomal enzyme³. Thus these two enzymes may be used as markers for the assessment of the purity of subcellular fractions obtained in fractionation studies. However, as pointed out by Ginsburg and Hers³ and Triantaphyllopoulos and Tuba⁴, the assay of glucose-6-phosphatase is complicated by the fact that glucose-6-phosphate may be hydrolysed by more than one enzyme. Similarly, acid phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.2) and alkaline phosphatase of the intestinal mucosa have a rather broad pH optimum. These two enzymes are therefore difficult to assay accurately by merely choosing buffers of different pH values. These difficulties can be overcome by the use of specific inhibitors. In the following, procedures are described for the assay of these three phosphatases which will give a more accurate determination of their respective activities than was possible with the assay systems so far known.