Properties of a 5′-AMP specific nucleotidase which accumulates in one cell type during development of Dictyostelium discoideum

Abstract

5′-AMP nucleotidase activity accumulates during the culmination stage of development in a thin layer of cells at the prestalk-prespore interface of Dictyostelium discoideum. In this report we characterize a highly purified preparation of this enzyme in an attempt to determine the physiological significance of the accumulation and localization of the activity during cellular differentiation. A pH optimum of 9.5 was determined using nine different buffer systems tested over a range of pH from 3 to 13.5. The Michaelis constants for p-nitrophenylphosphate (NPP) and 5′-AMP were 1.8 and 1.2 m m, respectively. Substrate concentrations of 5′-AMP in excess of 2.5 m m were found to inhibit the activity. Little or no effect on the activity of the enzyme was observed in the presence of EDTA, Mg 2+, Mn 2+, Ca 2+, Fe 2+, or Zn 2+ ions. However, the enzyme appears to be a zinc metalloprotein as evidenced by its inhibition with 1,10-phenanthroline and recovery of activity in the presence of zinc. Other inhibitors of enzymatic activity include dithiothreitol and imidazole. The enzyme was bound by calcium phosphate, but could not be immobilized on matricies containing other substrate or product analogs, including 5′-AMP, cyclic AMP, ATP, phenylalanine, blue dextran, and Procion Red HE3B. The hydrophobicity of 5′-AMP nucleotidase was demonstrated by its strong affinity for immobilized alkyl and ω-amino alkyl ligands, as well as phenyl Sepharose. Isoelectric focusing of the enzyme in granulated gel required both the presence of detergent to prevent aggregate formation and precipitation of the enzyme, and the addition of zinc after focusing to reverse Ampholine inhibition. Apparently, Ampholine chelates zinc away from the enzyme much like 1,10-phenanthroline. Using this method, the isoelectric point of 5′-AMP nucleotidase was found to be 4.5–4.9, with a 30% recovery of the applied activity.