Phosphomannoisomerase, an SH-Dependent Metal-Enzyme Complex

Abstract

PHOSPHOMANNOISOMERASE was found in red cells of the pig. The enzyme catalyses the reaction mannose-6-phosphate ⇌ fructose-6-phosphate Enzyme activity was determined by measuring the rate of formation of fructose-6-phosphate. The method was as follows: 1 vol. packed cells + 4 vol. ice-cold distilled water were frozen and thawed several times. 1.0 ml. of this hæmolysate, corresponding to 0.2 ml. original packed cells, 1.0 ml. acetate buffer, 0.1 M, pH 5.6, and 1.0 ml. mannose-6-phosphate (sodium salt 0.03 M, pH 5.9) were kept at 37° C. for the appropriate time and deproteinized by the addition of 3.0 ml. ice-cold 10 per cent perchloric acid. The deproteinizing agent was added to the blank before mannose-6-phosphate. The method of Roe¹ was chosen for the determination of fructose-6-phosphate in the filtrates. Because of the big excess of phosphoglucoisomerase in red cells² the fructose-6-phosphate measured is in equilibrium with glucose-6-phosphate at any stage of the reaction (60 and 40 per cent of glucose-6-phosphate and fructose-6-phosphate respectively at 37° C.) This was confirmed by determinations of glucose-6-phosphate with triphosphopyridine nucleotide and Zwischenferment³ and colorimetric measurements of fructose-6-phosphate in corresponding filtrates. The total original amount of fructose-6-phosphate was calculated by multiplication of that found by 2.5.