In:
Zeitschrift für Naturforschung C, Walter de Gruyter GmbH, Vol. 51, No. 7-8 ( 1996-8-1), p. 477-486
Abstract:
Dinucleoside 5′,5‴-P 1 ,P 4 -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 chroma tography steps and a final chromatography on Ap 4 A-Sepharose. The homogeneous hydrolase has a molecular mass of 20 kD a and an isoelectric point of 4.5. The enzyme hydrolyses diadenosine tetraphosphate (Ap 4 A) asymmetrically to AMP and ATP. Among other naturally occurring dinucleoside oligophosphates, Ap 5 A and Ap 6 A are substrates whereas Ap 3 A is not. Of various phosphonate analogues tested, the Ap 5 A analogue, AppCH 2 pCH 2 ppA, was not cleaved and the Ap 3 A analogue, ApCH 2 CH 2 ppA. was a very poor substrate. Enzyme activity is stimulated by 5mм Mg 2+ and inhibited by fluoride anion; I 50 = 6.25 μм . The K m value for Ap 4 A is 0.8 μм . 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 Ap 4 A 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 Ap 4 A hydrolase.
Type of Medium:
Online Resource
ISSN:
1865-7125
,
0939-5075
DOI:
10.1515/znc-1996-7-805
Language:
English
Publisher:
Walter de Gruyter GmbH
Publication Date:
1996
detail.hit.zdb_id:
2078107-6
SSG:
12
