Phosphatidylinositol (PtdIns) synthase in microsomal fractions derived from Tetrahymena vorax was studied to determine its activity requirements. The suitability of inositol isomers as substrates for the synthase and in headgroup exchange reactions also was investigated. Tetrahymena PtdIn synthase activity was optimum in the presence of 2 mM MgCl2 plus 2 mM MnCl2, a pH of 7.8, and a temperature of 30 degrees C. The enzyme retained approximately 80% of its activity after incubation at 70 degrees C for 10 min. PtdIns headgroup exchange activity was maximal in the presence of cytidine monophosphate. By following either the accumulation of radiolabeled reaction products or the loss of radiolabel from precursors, each of the inositol isomers tested appeared to serve as substrates for both the PtdIns synthase and PtdIns:inositol phosphatidyl transferase activities. In each case, myo-inositol and scyllo-inositol were the preferred substrates. The data suggest two routes for the formation of phosphatidyl-non-myo-inositols in Tetrahymena and the potential for the production of novel, non-myo-inositol-containing second messengers.

Properties of phosphatidylinositol synthase (PtdIns‐synthase) from the ciliate Tetrahymena were investigated using microsomal fractions enriched for the enzyme. Optimum conditions for PtdIns‐synthase activity as assayed in a Triton X‐100/CDP‐DAG mixed micelle system with [3H] myo‐Ins as co‐substrate were a pH of 8.0 using 50 mM Tris‐HCl buffer and a temperature of 30°C. Incubation of PtdIns‐synthase at pre‐selected temperatures prior to assay at 30°C showed that the enzyme was stable for at least 20 min at 40°C. A sharp decline in activity was seen after incubation at 50°C for 20 min. Activity was stimulated by 2 mM Mg2+, and stimulated to a slightly lesser degree by 1 mM Mn2+; however, these two ions in combination were synergistic. Other metal cations (Ca, Co, Cu, Fe, Mo, Zn, and Li) at a concentration of 2 mM had no appreciable effect on activity. Under optimum assay conditions, Tetrahymena PtdIns‐synthase activity was linear over at least 20 min and was linear with increasing concentrations of the enzyme. The enzyme was capable of utilizing both [3H]scyllo‐and [3H] (1D) chiro‐Ins as substrate to form phosphatidyl‐[3H]scyllo‐and phosphatidyl‐[3H](1D) chiro‐inositol, respectively, with (1D)chiro‐inositol being the least preferred substrate. The in vitro synthesized myo‐and scyllo‐inositol‐containing phosphoinositides were susceptible to hydrolysis by Bacillus cereus phosphatidylinositol‐specific phospholipase C, but the (1D) chiro‐inositol phosphoinositide was resistant. We hypothesize that putative DAG and scyllo‐IP3 second messengers produced by the cleavage of polyphospho‐scyllo‐inositides may function in novel transmembrane signaling cascades.