AMP hydrolysis in soluble and microsomal rat cardiac cell fractions: kinetic characterization and molecular identification of 5'-nucleotidase.
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.
Pochmann
Daniela
D
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
Innocente
Adrine M
AM
Cotomacci
Guilherme
G
Barreto-Chaves
Maria Luiza M
ML
Sarkis
João J F
JJ
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*
D002412:Cations; D000249:Adenosine Monophosphate; D015720:5'-Nucleotidase; D008274:Magnesium; D000241:Adenosine; D002118:Calcium