title: AMP hydrolysis in soluble and microsomal rat cardiac cell fractions: kinetic characterization and molecular identification of 5'-nucleotidase.
abstract: 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 K(m) (Michaelis constant) values calculated from the Eadie-Hofstee plot were 59.7+/-10.4 microM and 134.8+/-32.1 microM, with V(max) values of 6.7+/-0.4 and 143.8+/-23.8 nmol P(i)/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 Ca(2+) 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.
authors.affiliation: 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. danipochmann@hotmail.com
authors.forename: Daniela
authors.initials: D
authors.lastname: Pochmann
authors.forename: Adrine M
authors.initials: AM
authors.lastname: Innocente
authors.forename: Guilherme
authors.initials: G
authors.lastname: Cotomacci
authors.forename: Maria Luiza M
authors.initials: ML
authors.lastname: Barreto-Chaves
authors.forename: João J F
authors.initials: JJ
authors.lastname: Sarkis
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism*; D000241:Adenosine / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D002412:Cations; D000249:Adenosine Monophosphate; D015720:5'-Nucleotidase; D008274:Magnesium; D000241:Adenosine; D002118:Calcium