Isolation and characterization of pigeon breast muscle cytosolic 5'-nucleotidase-I (cN-I).
5'-Nucleotidase specific towards dCMP and AMP was isolated from avian breast muscle and characterized. It was found to be similar to a type-I form (cN-I) identified earlier as the AMP-selective 5'-nucleotidase responsible for adenosine formation during ATP breakdown in transfected COS-7 cells. Expression pattern of the cN-I gene in pigeon tissues indicated breast muscle as a rich source of the transcript. We purified the enzyme from this source using two-step chromatography and obtained an active homogenous preparation, free of ecto-5'-nucleotidase activity. The tissue content of the activity was calculated at 0.09 U/g wet weight. The specific activity of the enzyme preparation was 4.33 U/mg protein and it preferred dCMP and AMP to dAMP and IMP as a substrate. Its kinetic properties were very similar to those of the enzyme purified earlier from heart tissue. It was strongly activated by ADP. Inhibition by inorganic phosphate was more pronounced than in heart-isolated cN-I. Despite this difference, a similar physiological function is suggested for cN-I in both types of muscle.
Tkacz-Stachowska
Kinga
K
Department of Molecular Enzymology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdańsk, Gdańsk, Poland.
Lechward
Katarzyna
K
Skladanowski
Andrzej C
AC
D015720:5'-Nucleotidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D010856:Columbidae; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D015720:5'-Nucleotidase