The present study describes the enzymatic properties and molecular identification of 5′-nucleotidase in soluble and microsomal fractions from rat cardiac ventricles. Using AMP as a substrate, the results showed that the cation and the concentration required for maximal activity in the two fractions was magnesium at a final concentration of 1 mM. The pH optimum for both fractions was 9.5. The apparent Km (Michaelis constant) values calculated from the Eadie–Hofstee plot were 59.7±10.4 μM and 134.8±32.1 μM, with Vmax values of 6.7±0.4 and 143.8±23.8 nmol Pi/min/mg of protein (means±S.D., n=4) from soluble and microsomal fractions respectively. Western blotting analysis of ecto-5′-nucleotidase revealed a 70 kDa protein in both fractions, with the major proportion present in the microsomal fraction. The presence of these enzymes in the heart probably has a physiological function in adenosine signalling. Furthermore, the presence of ecto-5′-nucleotidase in the microsomal fraction could have a role in the modulation of the excitation–contraction-coupling process through involvement of the Ca2+ influx into the sarcoplasmic reticulum. The measurement of maximal enzyme activities in the two fractions highlights the potential capacity of the different pathways of purine metabolism in the heart.
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September 24 2008
AMP hydrolysis in soluble and microsomal rat cardiac cell fractions: kinetic characterization and molecular identification of 5′-nucleotidase
Daniela Pochmann;
Daniela Pochmann
1
*Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rua Ramiro Barcelos, 2600 – ANEXO, 90035-003, Porto Alegre, RS, Brazil
1To whom correspondence should be addressed (email [email protected]).
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Adrine M. Innocente;
Adrine M. Innocente
*Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rua Ramiro Barcelos, 2600 – ANEXO, 90035-003, Porto Alegre, RS, Brazil
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Guilherme Cotomacci;
Guilherme Cotomacci
†Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 2145, 05508-900, São Paulo, SP, Brazil
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Maria Luiza M. Barreto-Chaves;
Maria Luiza M. Barreto-Chaves
†Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 2145, 05508-900, São Paulo, SP, Brazil
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João J. F. Sarkis
João J. F. Sarkis
*Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rua Ramiro Barcelos, 2600 – ANEXO, 90035-003, Porto Alegre, RS, Brazil
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Publisher: Portland Press Ltd
Received:
August 15 2007
Revision Received:
August 01 2008
Accepted:
August 06 2008
Accepted Manuscript online:
August 06 2008
Online ISSN: 1573-4935
Print ISSN: 0144-8463
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biosci Rep (2008) 28 (5): 267–273.
Article history
Received:
August 15 2007
Revision Received:
August 01 2008
Accepted:
August 06 2008
Accepted Manuscript online:
August 06 2008
Citation
Daniela Pochmann, Adrine M. Innocente, Guilherme Cotomacci, Maria Luiza M. Barreto-Chaves, João J. F. Sarkis; AMP hydrolysis in soluble and microsomal rat cardiac cell fractions: kinetic characterization and molecular identification of 5′-nucleotidase. Biosci Rep 1 October 2008; 28 (5): 267–273. doi: https://doi.org/10.1042/BSR20070039
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