Biochemical characterization of a putative calcium influx factor as a diffusible messenger in Jurkat cells, Xenopus oocytes, and yeast

Abstract

Highly purified high performance thin layer chromatography (HPTLC) fractions containing a putative calcium influx factor (CIF) were prepared from the Jurkat cells and Xenopus oocytes in which Ca2+ stores were depleted by thapsigargin treatment and from the yeast in which intracellular Ca2+ stores were also depleted by genetic means. Micro injection of the fractions has been shown to elicit Ca2+‐dependent currents in Xenopus oocytes. The nature of the membrane currents evoked by the putative CIF appeared to be carried by chloride ions since the current was blocked by the selective chloride channel blocker 1 mM niflumic acid and its reversal potential was about ‐24 mV. Injection of the calcium chelator 1,2‐bis(2‐aminophenoxy)ethane‐N, N,N’,N‘‐tetraacetic acid (BAPTA) eradicated the current activities, suggesting the current responses are entirely Ca2+‐dependent. Moreover, the currents were sensitive to the removal of extracellular calcium, indicating the dependence on calcium entry through the plasma membrane calcium entry channels. CIF activities were insensitive to protease, heat, and acid treatments and to Dische‐reaction whereas the activities were sensitive to nucleotide pyrophosphatase and hydrazynolysis. The fraction might have a sugar because it was sensitive to Molisch test and Seliwaniff's resorcinol reaction. From the above results, CIF as a small and stable molecule seems to have pyrimidine, pyrophosphate, and a sugar moiety.