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 recombi nant 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 molecul ar mass of these bands was 48 and 38 kDa in rat kidney, liver, spleen, brai n, 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 relat ive AK mRNA level in rat tissues we used the multiplex PCR technique with b eta -actin mRNA as a reference. We found the highest level of AK mRNA in th e liver, which decreased in the order kidney > spleen > lung > heart > brai n > muscle. Measurement of AR activity in cytosolic fractions of rat tissue s showed the highest activity in the liver (0.58 U/g), which decreased in t he order kidney > spleen > lung > brain > heart > skeletal muscle. Kinetic studies on recombinant AR as well as on AK in the cytosolic fraction of var ious rat tissues showed that this enzyme is not affected by phosphate ions. The data presented indicate that in the rat tissues investigated at least t wo isoforms of adenosine kinase are expressed, and that the expression of t he AK gene appears to have some degree of tissue specificity.