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

As our interest is focused in obtaining a better knowledge of the plant phosphate metabolism and its interactions with Al ion, we used an 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...

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Bibliographic Details
Published in:Asian journal of biochemistry 2007-08, Vol.2 (5), p.302-313
Main Authors: Yereni, M-G, Villanueva, MA, Felipe, V-F, Teresa, H-SSM, Ignacio, I-F
Format: Article
Language:English
Subjects:
Coffea arabica
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Summary:As our interest is focused in obtaining a better knowledge of the plant phosphate metabolism and its interactions with Al ion, we used an 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 rho -nytrophenyl phosphate (p-NPP) could also be used as substrates. Enzymatic activity of the EDTA-inactivated enzyme can be restored by Mg super(2+), as well as other divalent cations such as Fe super(2+), Co super(2+), Cu super(2+), Zn super(2+), Ca super(2+) and Mn super(2+). In contrast, Al super(3+) could only partially reactivate the phosphate hydrolysing activity, which suggests that it is not a cofactor for this enzyme. When Al super(3+) was added to the Mg super(2+)-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 KNO sub(3) was able to decrease the activity suggesting that this enzyme should be related with the V-ATPase protein family or with plant phosphatases.
ISSN:1815-9923
DOI:10.3923/ajb.2007.302.313