In order to further characterize the acid phosphatase present in the eggs of A. gemmatalis, we proceeded to obtain an enriched fraction of acid phosphatases from 24-h-old egg homogenates by HPLC chromatography. After gel filtration, a major peak with PNPPase activity with an approximate molecular mass of 45 kDa was enriched by a factor of forty times ( Fig. 1C). Samples eluting adjacently to this major fraction www.selleckchem.com/products/icg-001.html were pooled, labeled as agAP, and further characterized. AgAP maximum activity was observed at pH 4.0 at 37 °C (data not shown) resulting in a km and Vmax of 0.32 mM and 6.13 nM pNP/s,
respectively. Tyrosine dephosphorylation of a major 80-kDa yolk protein was observed in vitro, suggesting that agAP targets yolk proteins during embryo development ( Fig. 2A). GSK 3 inhibitor Under the improved conditions, agAP was shown to preferentially hydrolyze phosphotyrosine (Ptyr) (300.5 nmols Pi × mg ptn−1 × min−1) as compared against phosphoserine and phosphothreonine (20. 00 and 0.901 nmols Pi × mg ptn−1 × min−1, respectively) ( Fig. 2B). Like other egg phosphatases (Fialho et al., 2002), agAP was strongly modulated by classical inhibitors of lysosomal acid phosphatases such as Na+/K+ tartrate and NaF (Table 1). The assayed inhibitors are used to classify these enzymes, at millimolar and submillimolar
concentration levels. Ammonium molybdate and sodium orthovanadate (modulators of tyrosine phosphatases) also inhibited agAP. Other phosphatase modulators such as CuSO4 or Pi were moderately effective against agAP Alanine-glyoxylate transaminase activity. The alkaline phosphatase inhibitors (levamisole and tetramisole) or phosphoesterase inhibitor (caffeine) had no effect on agAP, confirming the acidic nature of agAP. In order to evaluate the subcellular compartmentalization of agAP in yolk granules suspensions, the β-glycerophosphate-CeCl3 assays
for cytochemical detection of acid phosphatase activity was used (Hulstaert et al., 1983). After 24 h of oviposition, acid phosphatase activity was mainly restricted to a smaller population of vesicles sized 200–600 nm separated from larger yolk granules (Fig. 3A–D). Tracing of cerium by X-ray microanalysis was used to confirm CePO4 precipitation by endogenous agAP (Fig. 3E). During the assay, acid phosphatase is stained electron-dense by the deposition of insoluble CePO4 from usage of CeCl3 as a released phosphate capture agent. Negative controls were performed by avoiding addition of phosphatase substrate to the reaction medium, and no precipitates were found under those conditions (data not shown). PolyP is an ubiquitous biological polymer that plays a role in the regulation of several physiological processes. While specific PolyPases have been described from a few eukaryotic models (Lichko et al., 2006 and Tammenkoski et al., 2008), reports suggested that general phosphatases could also hydrolyze PolyP, thus regulating cellular levels of the polymer.
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