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Isolation and Characterization of a Soluble Phosphate Hydrolysing Activity from an in vitro Coffee Cell Line Grown in the Presence of Aluminum

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Author(s): Minero-Garcia Yereni | Villanueva Marco A. | Vazquez-Flota Felipe | Hernandez-Sotomayor S.M. Teresa | Islas-Flores Ignacio

Journal: Asian Journal of Biochemistry
ISSN 1815-9923

Volume: 2;
Issue: 5;
Start page: 302;
Date: 2007;
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Keywords: In vitro coffee cell cultures | phosphate hydrolysing activity | plant protein purification | aluminum stress

ABSTRACT
in vitro coffee cell line (Coffea arabica L.) tolerant to aluminum ion to purify a protein with phosphate hydrolysing activity with the aim to analyze whether the enzyme can be contributing to the Al-tolerance in this coffee cell line. The protein was purified at least 138-fold; silver stain on a 10% SDS-PAGE detected a partially purified 30 kDa polypeptide which showed ability to hydrolyse phosphate either in native gels or soluble assays. The semipurified protein is able to hydrolyse sodium pyrophosphate (PPi-Na) and adenosine triphosphate (ATP) very efficiently, although P-serine (P-ser), P-Threonine (P-thr), P-Tyrosine (P-Tyr), Phytate, D-Myo-Inositol-1P (D-Myo Inos-1P) and the synthetic ?-nytrophenyl phosphate (p-NPP) could also be used as substrates. Enzymatic activity of the EDTA-inactivated enzyme can be restored by Mg2+, as well as other divalent cations such as Fe2+, Co2+, Cu2+, Zn2+, Ca2+ and Mn2+. In contrast, Al3+ could only partially reactivate the phosphate hydrolysing activity, which suggests that it is not a cofactor for this enzyme. When Al3+ was added to the Mg2+-enzyme complex, it strongly inhibited the enzymatic activity either, exerting a negative effect over the enzyme or the substrate. Between a number of phosphohydrolase inhibitors, only KNO3 was able to decrease the activity suggesting that this enzyme should be related with the V-ATPase protein family or with plant phosphatases.]]>
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