Isolation and partial characterisation of acid phosphatase isozymes from dormant oilseed of Corylus avellana L.

Abstract

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.

Phytic acid mobilization is an early response to chilling of the embryonic axes from dormant oilseed of hazel (Corylus avellana)

“…The initial increase in phytase activity during chilling occurred before the seeds had realized their full germination potential and not in those warm-imbibed for the same period. Thus, the initial rise is linked to dormancy relief, while the subsequent increase is attributed to the initiation of germinative growth and is not part of the dormancy relieving process, similar to the total APase in hazel (Andriotis and Ross, 2004) and chilled apple seeds (Rychter et al, 1972). Nevertheless, phytase was extracted from seeds held at 5 8C before activity measurements were conducted at 37 8C.…”

“…Kent cobnut) fruits were commercially obtained, the cupules removed and the seed lot was air-dried at room (ambient) temperature until no further change in weight was observed. Dry hazelnuts were either dry-stored in airtight containers at 5 8C for no more than a year or used for stratification and warmincubation (control) treatments as in Andriotis and Ross (2004). Drydormant samples (week 0 samples) were prepared from nuts removed directly from dry storage.…”

Revisiting histidine-dependent acid phosphatases: a distinct group of tyrosine phosphatases

“…In particular, its protein levels negatively correlate with the phosphorylation of a 14-kDa pTyr protein, a putative intracellular substrate for this AcP [49]. Similarly, AcPs exhibiting significant phosphatase activity towards pTyr substrates have also been reported in dormant hazel ( Corylus avellana ) seeds [50]. Further studies will be required to elucidate the biological significance of these AcPs in plant signaling pathways.…”

Rice Bean (Vigna umbellata Thunb.) Acid Phosphatase: Effect of Metal Ions and Metabolites

“…Three oxygen are in the ionic form. Inorganic phosphate (Pi), a product of APase catalyzed hydrolysis, whatever the substrate may be, is a potent competitive inhibitor (Kaneko et al, 1990;Biswas & Cundiff, 1991;Miernyk, 1992;Granjeiro et al, 1999;Roknabadi et al, 1999;Turner & Plaxton, 2001;Cirkovic et al, 2002;Coello, 2002;Greiner & Jany, 2003;Andriotis & Ross, 2004;Hoehamer et al, 2005;Nadir et al, 2012). It is agreed upon that Pi could play a physiological role in sustaining a stable phosphate level in the cell through feedback regulation of enzyme activity.…”

“…It is agreed upon that Pi could play a physiological role in sustaining a stable phosphate level in the cell through feedback regulation of enzyme activity. Vanadate is also one of the anions that show inhibition in most cases (Gellatly et al, 1994;Granjeiro et al, 1999;Roknabadi et al, 1999;Turner & Plaxton, 2001;Coello, 2002;Greiner & Jany, 2003;Andriotis & Ross, 2004;Senna et al, 2006;Nadir et al, 2012).…”