Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification

dc.contributorUniversidade Estadual Paulista (Unesp)
dc.creatorPizauro, J. M. [UNESP]
dc.creatorCiancaglini, P.
dc.creatorLeone, F. A.
dc.date2015-12-07T15:29:25Z
dc.date2015-12-07T15:29:25Z
dc.date1995-11-22
dc.date.accessioned2023-09-12T07:23:44Z
dc.date.available2023-09-12T07:23:44Z
dc.identifierhttp://www.ncbi.nlm.nih.gov/pubmed/8751158
dc.identifierMolecular And Cellular Biochemistry, v. 152, n. 2, p. 121-129, 1995.
dc.identifier0300-8177
dc.identifierhttp://hdl.handle.net/11449/130796
dc.identifier8751158
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8780095
dc.descriptionAlkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released enzyme exhibited 'Michaelian' kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K0.5 = 1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (Ki = 6.2 microM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min-1 to 0.684 min-1. Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K0.5 = 29.5 microM), manganese (K0.5 = 5 microM) and cobalt ions (K0.5 = 10.1 microM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5 = 653 microM) was less effective (only 26%) and occurred with site-site interactions (n = 0.7). Zinc ions had no stimulatory effects. The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed.
dc.descriptionFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.descriptionConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.descriptionFundação para o Desenvolvimento da UNESP (FUNDUNESP)
dc.descriptionFinanciadora de Estudos e Projetos (FINEP)
dc.descriptionDepartamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto-USP
dc.descriptionDepartamento de Tecnologia, Faculdade de Ciências Agrárias e Veterinárias de JaboticabaI-UNESP
dc.descriptionDepartamento de Tecnologia, Faculdade de Ciências Agrárias e Veterinárias de JaboticabaI-UNESP
dc.format121-129
dc.languageeng
dc.publisherKluwer Academic Publishers
dc.relationMolecular And Cellular Biochemistry
dc.relation2.561
dc.relation1,003
dc.rightsAcesso restrito
dc.sourcePubMed
dc.subjectAlkaline phosphatase
dc.subjectP-nitrophenyl phosphate
dc.subjectHydrophobic chromatography
dc.subjectPhosphatidylinositol-specific phospholipase C
dc.subjectOsseous plate
dc.subjectPhospholipase C
dc.titleCharacterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification
dc.typeArtigo


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