PARTIAL-PURIFICATION OF HUMAN INTESTINAL ALKALINE-PHOSPHATASE WITH AFFINITY CHROMATOGRAPHY - SOME PROPERTIES AND INTERACTION OF CONCANAVALIN A WITH ALKALINE-PHOSPHATASE

Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from human intestine was purified with concanavalin A-Sepharose and tyraminyl derivative-Sepharose affinity chromatography. The enzyme obtained with these techniques had a specific activity of approximately 513.2 .mu.mol p-nitrophenylphosphate hydrolyzed/min per mg of protein at pH 10.0. The highly purified enzyme showed 1 major enzymatically active band and a possible minor enzymatically active band on acrylamide gel and cellogel electrophoresis; the 2 fractions showed identical antigenicty. The highly purified intestinal enzyme was compared with the purified hepatic enzyme: the saccharide content of each showed a marked difference. The interaction of alkaline phosphatase with concancavalin A, a carbohydrate-binding protein, was studied. Concanavalin A showed an organ-specific behavior to alkaline phosphatase isoenzyme, i.e., the effect on the enzyme activity and the optimum pH of the activity. The concanavalin A and alkaline phosphatase complex showed a protective effect against heat denaturation and inactivation of proteinase [calf intestine] digestion. There was no difference in stability between the intestinal enzyme and the hepatic enzyme. Alkaline phosphatase preparations from human intestine and liver can bind with concanavalin A; these interactions of concanavalin A with the enzyme occurred reversibly when .alpha.-methyl-D-mannoside was added. The double reciprocal plots of 1/v vs. 1/s at higher concentrations of concanavalin A showed that the mechanism of inhibition was mixed type. From the results of Dixon plots, the inhibition constant (Ki) was calculated to the 0.025 .mu.M for human intestinal enzyme. The effect of concanavalin A on L-phenylalanine inhibition of the intestinal alkaline phosphatase indicates that concanavalin A does not interfere with L-phenylalaine binding, but its effect on L-homoarginine inhibition of the hepatic enzyme seems to show that concanavalin A interfered with L-homoarginine binding.