Identification and characterization of phospholipase D in a unicellular red alga (Porphyridium cruentum)

Abstract

Axenically cultured cells of Porphyridium cruentum were assayed for enzymes catalyzing the hydrolysis of lecithin. The sonicated cells showed virtually exclusive and complete conversion of 14C-choline-labelled lecithin to 14C-choline, with no significant formation of glycerylphosphorylcholine or phosphorylcholine. The enzymatic activity showed a sharp pH optimum at 7.0, and was strongly inhibited by chelating agents (two types), sulfhydryl-group binding reagents (three types), and surface-active agents (anionic and non-ionic). The inhibition from ethylenediamine tetraacetate (EDTA) was reversed by Ca2+ or Sr2+ but not by Mg2+ or Ba2+, and that from —SH binding reagents was reversed by dithiothreitol. Heavy metal ions (Zn2+, Cu2+, Ba2+, Co2+, Mn2+, Fe2+) inhibited the activity, Ca2+ caused stimulation, while Mg2+ and Sr2+ had little effect. The results indicate the occurrence of a pH and heavy-metal ion sensitive, Ca2+-(or Sr2+-) requiring phospholipase D in the red alga, and the involvement of sulfhydryl groups in the expression of its activity. The algal enzyme resembles those from higher plants in its Ca2+ requirement and sensitivity to EDTA and organomercurial sulfhydryl binding reagent, but differs markedly in its optimum pH and several other properties.