Isolation and partial characterisation of acid phosphatase isozymes from dormant oilseed of Corylus avellana L.
The acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) complement from dormant hazel (Corylus avellana L.) seeds was found to exhibit significant electrophoretic heterogeneity partially attributable to the presence of distinct molecular forms. In axiferous tissue, total acid phosphatase activity increased in a biphasic fashion during chilling, a treatment necessary to alleviate seed dormancy. Three acid phosphatase isozymes were isolated from cotyledons of dormant hazel seeds by successive ammonium sulphate precipitation, size-exclusion, Concanavalin A affinity, cation- and anion-exchange chromatographies resulting in 75-, 389- and 191-fold purification (APase1, APase2, APase3, respectively). The three glycosylated isoforms were isolated to catalytic homogeneity as determined by electrophoretic, kinetic and heat-inactivation studies. The native acid phosphatase complement of hazel seeds had an apparent Mr of 81.5 +/- 3.5 kDa as estimated by size-exclusion chromatography, while the determined pI values were 5.1 (APase1), 6.9 (APase2) and 7.3 (APase3). The optimum pH for p-nitrophenyl phosphate hydrolysis was pH 3 (APase1), pH 5.6 (APase2) and pH 6 (APase3). The hazel isozymes hydrolysed a variety of phosphorylated substrates in a non-specific manner, exhibiting low Km and the highest specificity constant (Vmax/ Km) for pyrophosphate. They were not primary phytases since they could not initiate phytic acid hydrolysis, while APase2 and APase3 had significant phospho-tyrosine phosphatase activity. Inorganic phosphate was a competitive inhibitor, while activity was significantly impaired in the presence of vanadate and fluoride.
Andriotis
Vasilios M E
VM
School of Plant Sciences, The University of Reading, Whiteknights, Reading, RG6 6AS, UK.
Ross
James D
JD
D000135:Acid Phosphatase / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity; D031211:Corylus / Q000201:enzymology*; D018525:Germination; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010940:Plant Proteins / Q000378:metabolism; D012639:Seeds / Q000201:enzymology / Q000254:growth & development; D013379:Substrate Specificity
D007527:Isoenzymes; D010940:Plant Proteins; D000135:Acid Phosphatase