Expression level of adenosine kinase in rat tissues. Lack of phosphate effect on the enzyme activity.
In this report we describe cloning and expression of rat adenosine kinase (AK) in Esccherichaia coli cells as a fusion protein with 6xHis. The recombinant protein was purified and polyclonal antibodies to AK were generated in rabbits. Immunoblot analysis of extracts obtained from various rat tissues revealed two protein bands reactive with anti-AK IgG. The apparent molecular mass of these bands was 48 and 38 kDa in rat kidney, liver, spleen, brain, and lung. In heart and muscle the proteins that react with AK antibodies have the molecular masses of 48 and 40.5 kDa. In order to assess the relative AK mRNA level in rat tissues we used the multiplex PCR technique with beta-actin mRNA as a reference. We found the highest level of AK mRNA in the liver, which decreased in the order kidney > spleen > lung > heart > brain > muscle. Measurement of AK activity in cytosolic fractions of rat tissues showed the highest activity in the liver (0.58 U/g), which decreased in the order kidney > spleen > lung > brain > heart > skeletal muscle. Kinetic studies on recombinant AK as well as on AK in the cytosolic fraction of various rat tissues showed that this enzyme is not affected by phosphate ions. The data presented indicate that in the rat tissues investigated at least two isoforms of adenosine kinase are expressed, and that the expression of the AK gene appears to have some degree of tissue specificity.
Sakowicz
M
M
Department of Molecular Medicine, Medical University of Gdańsk, Poland.
Grdeń
M
M
Pawełczyk
T
T
D000248:Adenosine Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D003001:Cloning, Molecular; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D003920:Diabetes Mellitus / Q000201:enzymology; D020869:Gene Expression Profiling; D007700:Kinetics; D009928:Organ Specificity; D010710:Phosphates / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats
D010710:Phosphates; D012333:RNA, Messenger; D000248:Adenosine Kinase