Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification
Autor(a) principal: | |
---|---|
Data de Publicação: | 1995 |
Outros Autores: | , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1007/BF01076074 http://hdl.handle.net/11449/224010 |
Resumo: | Alkaline 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 μM, respectively. For pyrophosphate, K0.5 was 128 μM 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 μM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40°C. However, with increasing temperature from 40-60°C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08×10-4 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 μM), manganese (K0.5=5 μM) and cobalt ions (K0.5=10.1 μM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5=653 μM) 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 P1-anchored form of osseous plate alkaline phosphatase is discussed. © 1995 Kluwer Academic Publishers. |
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Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossificationalkaline phosphatasehydrophobic chromatographyosseous platep-nitrophenyl phosphatephosphatidylinositol-specific phospholipase Cphospholipase CAlkaline 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 μM, respectively. For pyrophosphate, K0.5 was 128 μM 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 μM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40°C. However, with increasing temperature from 40-60°C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08×10-4 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 μM), manganese (K0.5=5 μM) and cobalt ions (K0.5=10.1 μM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5=653 μM) 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 P1-anchored form of osseous plate alkaline phosphatase is discussed. © 1995 Kluwer Academic Publishers.Departamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal-UNESP, Av. Bandeirantes 3900, Ribeirão Preto, 14040-901, SPDepartamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto-USP, Av. Bandeirantes 3900, Ribeirão Preto, 14040-901, SPDepartamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal-UNESP, Av. Bandeirantes 3900, Ribeirão Preto, 14040-901, SPUniversidade Estadual Paulista (UNESP)Universidade de São Paulo (USP)Pizauro, João M. [UNESP]Ciancaglini, PietroLeone, Francisco A.2022-04-28T19:54:18Z2022-04-28T19:54:18Z1995-11-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article121-129http://dx.doi.org/10.1007/BF01076074Molecular and Cellular Biochemistry, v. 152, n. 2, p. 121-129, 1995.0300-81771573-4919http://hdl.handle.net/11449/22401010.1007/BF010760742-s2.0-0029558381Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengMolecular and Cellular Biochemistryinfo:eu-repo/semantics/openAccess2022-04-28T19:54:18Zoai:repositorio.unesp.br:11449/224010Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462022-04-28T19:54:18Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification |
title |
Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification |
spellingShingle |
Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification Pizauro, João M. [UNESP] alkaline phosphatase hydrophobic chromatography osseous plate p-nitrophenyl phosphate phosphatidylinositol-specific phospholipase C phospholipase C |
title_short |
Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification |
title_full |
Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification |
title_fullStr |
Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification |
title_full_unstemmed |
Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification |
title_sort |
Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification |
author |
Pizauro, João M. [UNESP] |
author_facet |
Pizauro, João M. [UNESP] Ciancaglini, Pietro Leone, Francisco A. |
author_role |
author |
author2 |
Ciancaglini, Pietro Leone, Francisco A. |
author2_role |
author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (UNESP) Universidade de São Paulo (USP) |
dc.contributor.author.fl_str_mv |
Pizauro, João M. [UNESP] Ciancaglini, Pietro Leone, Francisco A. |
dc.subject.por.fl_str_mv |
alkaline phosphatase hydrophobic chromatography osseous plate p-nitrophenyl phosphate phosphatidylinositol-specific phospholipase C phospholipase C |
topic |
alkaline phosphatase hydrophobic chromatography osseous plate p-nitrophenyl phosphate phosphatidylinositol-specific phospholipase C phospholipase C |
description |
Alkaline 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 μM, respectively. For pyrophosphate, K0.5 was 128 μM 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 μM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40°C. However, with increasing temperature from 40-60°C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08×10-4 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 μM), manganese (K0.5=5 μM) and cobalt ions (K0.5=10.1 μM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5=653 μM) 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 P1-anchored form of osseous plate alkaline phosphatase is discussed. © 1995 Kluwer Academic Publishers. |
publishDate |
1995 |
dc.date.none.fl_str_mv |
1995-11-01 2022-04-28T19:54:18Z 2022-04-28T19:54:18Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1007/BF01076074 Molecular and Cellular Biochemistry, v. 152, n. 2, p. 121-129, 1995. 0300-8177 1573-4919 http://hdl.handle.net/11449/224010 10.1007/BF01076074 2-s2.0-0029558381 |
url |
http://dx.doi.org/10.1007/BF01076074 http://hdl.handle.net/11449/224010 |
identifier_str_mv |
Molecular and Cellular Biochemistry, v. 152, n. 2, p. 121-129, 1995. 0300-8177 1573-4919 10.1007/BF01076074 2-s2.0-0029558381 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Molecular and Cellular Biochemistry |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
121-129 |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
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1797790070231531520 |