Extracellular alkaline phosphatase is a sensitive marker for cellular stimulation and exocytosis in heterotroph cell cultures of Chenopodium rubrum

We investigated the response of extracellular phosphatase to heat shock in heterotrophic Chenopodium rubrum L. cell cultures. Surprisingly, in contrast to the generally used acid phosphatase, an extracellular alkaline phosphatase showed the most sensitive response. This phosphatase was characterized as a marker for cellular stimulation by its high correlations with induced changes of extracellular pH: 10 μM nigericin (correlation coefficient r=0.91), 100 μM salicylic acid (r=0.84), heat shock 5 min 37 °C (r=0.79), and heat shock after pre-treatment with 5 μM fusicoccin (r=0.92) or 0.5% ethanol (r=0.90). Cellular stimulation was estimated with concentrations of acids and bases, yielding similar levels of pH change (0.5 pH) in cell-free supernatant: salicylic acid (200 μM), benzoic acid (600 μM), HCl (140 μM), NaOH (100 μM), and KOH (100 μM). The Golgi apparatus inhibitor Brefeldin A (200 μM) reduced the heat-shock-induced phosphatase (−33%). The pH optimum of heat-shock-induced phosphatase was 3; however, there the proportion of constitutive phosphatase was higher than at pH 8–9.5, indicating different pH dependence of constitutive and induced activity. Thus, heat-shock-induced phosphatase was characterized by alkaline activity with inhibitors (10 μM molybdate: −52%, 2.5 mM phosphate: −64%, 10 μM ZnCl2: −82%), substrates (2.5 mM, tyrosine phosphate: 255 pkat g−1, p-nitrophenyl phosphate: 92 pkat g−1, serine phosphate: 0, threonine phosphate: 0), Hill coefficient (nH=1.4) indicating two binding sites, and the extent of heat-shock stimulation (p-nitrophenyl phosphate: +190%, tyrosine phosphate: +180%). SDS-PAGE showed a correlation of alkaline phosphatase with the heat-shock-induced release of highly N-glycosylated 53 kDa protein, detected by peroxidase-labeled concanavalin A affinoblotting after endoglycosidase H treatment. The 53 kDa protein showed no in-gel phosphatase activity after SDS-PAGE and regeneration treatment, in contrast to a putative dimer (105 kDa).