Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase from tomato (Lycopersicon esculentum cv. Lukullus)--purification, biochemical properties and behaviour during stress.
Dinucleoside 5',5"'-P1,P4-tetraphosphate hydrolase (EC 3.6.1.17) has been purified to homogeneity from tomato (Lycopersicon esculentum) cells grown in suspension. The purification procedure comprised ammonium sulphate fractionation following five standard chromatography steps and a final chromatography on Ap4A-Sepharose. The homogeneous hydrolase has a molecular mass of 20 kDa and an isoelectric point of 4.5. The enzyme hydrolyses diadenosine tetraphosphate (Ap4A) asymmetrically to AMP and ATP. Among other naturally occurring dinucleoside oligophosphates, Ap5A and Ap6A are substrates whereas Ap3A is not. Of various phosphonate analogues tested, the Ap5A analogue, AppCH2pCH2ppA, was not cleaved and the Ap3A analogue, ApCH2CH2ppA, was a very poor substrate. Enzyme activity is stimulated by 5 mM Mg2+ and inhibited by fluoride anion; I50 = 6.25 microM. The K(m) value for Ap4A is 0.8 microM. The enzyme exhibits a broad pH optimum from pH 6.5 to 9.0. In order to analyze the protein at the molecular level an internal peptide sequence from the homogeneous enzyme was identified. Within the sequence of 17 amino acids a kinase II motif as a general part of a conserved sequence of nucleotide binding sites was found. Against the internal peptide sequence a polyclonal antiserum was raised. By investigating the intracellular level of Ap4A hydrolase under different kinds of environmental stress, no changes occurred in response to heat shock. But, heavy metal stress and phosphate deprivation lead to a decrease in Ap4A hydrolase.
Feussner
K
K
Institut für Pflanzenbiochemie, Halle, Halle/Saale, Bundesrepublik Deutschland.
Guranowski
A
A
Kostka
S
S
Wasternack
C
C
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine
D000906:Antibodies; D010446:Peptide Fragments; D017886:Durapatite; D017766:Acid Anhydride Hydrolases; C020023:bis(5'-nucleosyl)tetraphosphatase (asymmetrical)