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Partial purification and some biochemical properties of nonspecific alkaline phosphatase in germinating chick-pea (Cicer arietinum) seeds
Authors:Trinidad Angosto  Angel Matilla
Institution:Institute) de Biotecnología. Dept. Biologia Vegetal Univ. de Granada, E–18671 Granada, Spain.
Abstract:A procedure for the partial purification of a non-specific alkaline phosphatase (EC 3.1.3.1.) from the embryonic axes of chick-pea seeds is described. Ammonium sulphate precipitation, DEAE-cellulase chromatography, Sephacryl S-200 chroma-tography and polyacrylamide gel electrophoresis are the most important steps. The molecular weight of this non-specific enzyme, as determined by Sephacryl S–200 gel filtration and SDS–polyacrylamide gel electrophoresis, was estimated as being 68 and 78 kDa respectively; the optimum pH for p-nitrophenylphosphate hydrolysis was 7.5, and the Km for this artificial substrate was 0.5 mM. The enzyme catalyzes the hydrolysis of a variety of organic phosphate esters. The best substrates are: phos-phoenolpymvate (Km= 2.4 m M ), NADP+ (Km= 4.0 m M ), 5'-AMP (Km= 4.5 m M ), 5'-ADP (Km= 6.1 m M ) and ribose-5P (Km= 5.8 m M ); but it is unable to hydrolyze 5'-ATP, phosphocreatine and tripolyphosptiate. Phospate was a competitive inhibitor. Zn2+, K+, Hg2+ and Mo6+ were strong inhibitors, whereas F? and Ca2+ inhibited weakly; Co2+ and Ni2+ were activators.
Keywords:Alkaline phosphatase              Cicer arietinum L  cv  Castellana  embryonic axes  germination  seed  seedling growth
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