PARTIAL PURIFICATION AND CHARACTERIZATION OF A CALCIUM-DEPENDENT ALKALINE PHOSPHATASE FROM THE CYANOBACTERIUM ARTHROSPIRA PLATENSIS?

In the present study, Triton X-114 (TX-114) is used to extract and partially purify alkaline phosphatase (ALP) from a membranous fraction of Arthrospira platensis Gomont containing cell wall, plasma membrane, thylakoids, and sheath. TX-114 has a double effect: solubilizing cell components to liberate the enzyme and, after phase partitioning, removing chl and other pigments present in the crude extract. The recovery of the enzyme in the aqueous phase suggests the overall hydrophilic character of this enzyme. ALP was kinetically characterized at pH 11.0 using p-nitrophenyl phosphate as substrate, giving a Km value of 1.7 mM. Orthovanadate was seen to be a competitive inhibitor of ALP, with a Ki of 0.8 mM. The enzyme was almost completely inactivated in the presence of 70 mu M EDTA, although the addition of Ca2+ reverted this inactivation; these results indicate that ALP from A. platensis is a calcium-dependent metalloenzyme. When the effect of Ca2+ was investigated in detail, a value of 0.067 mu M-1 for the affinity constant was obtained. The enzyme was histochemically localized in the cytoplasm, cell wall, and sheath using the enzyme-labeled fluorescent substrate (ELF) method. It is assumed that the same enzyme is either soluble in the cytoplasm and in some way trapped in the cell wall or in the sheath. ALP localization within the sheath and the subsequent release of phosphorus (P) may benefit the neighboring cells surrounding this layer.