title: Rat osseous plate alkaline phosphatase: effect of neutral protease digestion on the hydrolysis of pyrophosphate and nitrophenylphosphate.
abstract: Collagenase treatment, commonly used to prepare alkaline phosphatase-rich matrix vesicles from epiphyseal cartilage growth plates, seems to affect the integrity of this membrane-bound enzyme. Alkaline phosphatase-rich rat osseous plates were incubated with 1,000 U/mL collagenase for 3 h, at 37 degrees C and after purification on Sepharose 4B, kinetic studies were performed using nitrophenylphosphate and pyrophosphate as substrates. The optimum apparent pH for the hydrolysis of p-nitrophenylphosphate and pyrophosphate increased from 9.4 to 10.25 and from 8.0 to 9.0, respectively, as a consequence ofcollagenase treatment. In the absence of Mg2+ ions, the enzyme hydrolyzed PNPP with KM = 322.5 +/- 15.3 microM and V = 965.2 +/- 45.8 U/mg, while in the presence of 2 mM Mg2+ ions, V increased 66%. Cobalt (K0.5 = 5.3 +/- 0.3 microM) and manganese (K0.5 = 0.72 +/- 0.03 microM) ions stimulated the PNPPase activity of the collagenase-treated enzyme, but with a lower apparent affinity when compared with that of not-treated enzyme. In the absence of Mg2+ ions pyrophosphate was hydrolyzed according to Michaelis-Menten kinetics (KM = 105.1 +/- 6.3 microM and V = 64.9 +/- 3.9 U/mg), but site-site interactions (nH = 1.2) were observed in the presence of 2 mM Mg2+ ions (V = 110.8 +/- 5.5 U/mg; K0.5 = 42.7 +/- 2.0 microM). To our knowledge this is the first report showing significant alterations on phosphohydrolytic activity and metal binding properties of bone alkaline phosphatase due to associated neutral proteases in collagenase preparations often used for the isolation of matrix vesicles.
authors.affiliation: Departamento de Química e Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
authors.forename: Rúbia R
authors.initials: RR
authors.lastname: Gonçalves
authors.forename: Rosa P M
authors.initials: RP
authors.lastname: Furriel
authors.forename: João A
authors.initials: JA
authors.lastname: Jorge
authors.forename: Francisco A
authors.initials: FA
authors.lastname: Leone
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D011756:Diphosphates / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D006132:Growth Plate / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D051381:Rats
chemical_list: D011756:Diphosphates; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D000469:Alkaline Phosphatase; D010450:Endopeptidases