Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.
An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286].
Bozzo
Gale G
GG
Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Raghothama
Kashchandra G
KG
Plaxton
William C
WC
D000135:Acid Phosphatase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D000595:Amino Acid Sequence; D002455:Cell Division; D002478:Cells, Cultured; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007700:Kinetics; D018551:Solanum lycopersicum / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D013379:Substrate Specificity
D006023:Glycoproteins; D010710:Phosphates; C091137:purple acid phosphatase; D000135:Acid Phosphatase