title: Alkaline phosphatase from rat osseous plates: purification and biochemical characterization of a soluble form.
abstract: A soluble form of an alkaline phosphatase obtained from rat osseous plates was purified 204-fold with a yield of 24.3%. The purified enzyme showed a single protein band of Mr 80,000 on SDS-PAGE and an apparent molecular weight of 163,000 by gel filtration on Sephacryl S-300 suggesting a dimeric structure for the soluble enzyme. The specific activity of the enzyme at pH 9.4 in the presence of 2 mM MgCl2 was 19,027 U/mg and the hydrolysis of p-nitrophenyl phosphate (K0.5 = 92 microM) showed positive cooperativity (n = 1.5). The purified enzyme showed a broad substrate specificity, however, ATP, bis(p-nitrophenyl) phosphate and pyrophosphate were among the less hydrolyzed substrates assayed. Surprisingly the enzyme was not stimulated by cobalt and manganese ions, in contrast with a 20-25% stimulation observed for magnesium and calcium ions. Zinc ions exerted a strong inhibition on p-nitrophenylphosphatase activity of the enzyme. This paper provides a simple experimental procedure for the isolation of a soluble form of alkaline phosphatase which is induced by demineralized bone matrix during endochondral ossification.
authors.affiliation: Departamento de Química - Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brasil.
authors.forename: J C
authors.initials: JC
authors.lastname: Say
authors.forename: K
authors.initials: K
authors.lastname: Ciuffi
authors.forename: R P
authors.initials: RP
authors.lastname: Furriel
authors.forename: P
authors.initials: P
authors.lastname: Ciancaglini
authors.forename: F A
authors.initials: FA
authors.lastname: Leone
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D001857:Bone Matrix; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006132:Growth Plate / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009596:Nitrophenols / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010012:Osteogenesis; D051381:Rats; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D000469:Alkaline Phosphatase