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

Abstract

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: 10microM nigericin (correlation coefficient r=0.91), 100microM salicylic acid (r=0.84), heat shock 5min 37 degrees C (r=0.79), and heat shock after pre-treatment with 5microM 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 (200microM), benzoic acid (600microM), HCl (140microM), NaOH (100microM), and KOH (100microM). The Golgi apparatus inhibitor Brefeldin A (200microM) 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 (10microM molybdate: -52%, 2.5mM phosphate: -64%, 10microM ZnCl(2): -82%), substrates (2.5mM, tyrosine phosphate: 255pkat g(-1), p-nitrophenyl phosphate: 92pkat 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 53kDa protein, detected by peroxidase-labeled concanavalin A affinoblotting after endoglycosidase H treatment. The 53kDa protein showed no in-gel phosphatase activity after SDS-PAGE and regeneration treatment, in contrast to a putative dimer (105kDa).

• Authors: Anchalee Chaidee, Chatchawal Wongchai, Wolfgang Pfeiffer
• Journal: Journal of plant physiology (J Plant Physiol) Vol. 165 Issue 16 Pg. 1655-66 (Nov 01 2008) ISSN: 1618-1328 [Electronic] Germany
• PMID: 18433930 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Biomarkers
• Plant Proteins
• Brefeldin A
• Alkaline Phosphatase
• Salicylic Acid
• Nigericin

Topics

• Alkaline Phosphatase (metabolism)
• Biomarkers (metabolism)
• Brefeldin A (pharmacology)
• Cells, Cultured
• Chenopodium (cytology, drug effects, enzymology)
• Electrophoresis, Polyacrylamide Gel
• Exocytosis
• Heat-Shock Response (drug effects)
• Hot Temperature
• Hydrogen-Ion Concentration
• Kinetics
• Nigericin (pharmacology)
• Plant Proteins (metabolism)
• Salicylic Acid (pharmacology)