Expression level of adenosine kinase in rat tissues. Lack of phosphate effect on the enzyme activity.

Abstract

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.

Molecular characterization of recombinant mouse adenosine kinase and evaluation as a target for protein phosphorylation

“…In this study, we report the cDNA and deduced amino acid sequences for two isoforms of AK expressed in the mouse brain. To date, the existence of AK splice variants has been described in several mammalian species, namely mouse [24,25], rat [26] and human [8,27]. A search for multiple forms of AK in other species is likely to generate similar results.…”

Adenosine Kinase Deficiency Increases Susceptibility to a Carcinogen

“…4,5 Liver is not only the organ with the highest expression levels of ADK in the body, but also 85% of all transmethylation reactions take place in the liver. [7][8][9] Therefore, ADK plays a critical role in the maintenance of transmethylation homeostasis in the liver; consequently homozygous deletion of the ADK gene in mice triggers hepatic steatosis, which is a fatal condition characterized by rapid microvesicular fat infiltration and early postnatal mortality. 4 Similarly, ADK-deficient patients show disruption of the transmethylation pathway resulting in major physiological abnormalities of liver metabolism including microvesicular hepatic steatosis and hepatic encephalopathy.…”

Comparative proteome analysis of the embryo proper and yolk sac membrane of day 11.5 cultured rat embryos

“…Different spots of the same proteins between the embryo proper and yolk sac membrane may indicate the presence of isoforms or posttranslational modification of the proteins. There are at least two isoforms of adenosine kinase with tissue‐dependent expression in rats (Sakowicz et al, 2001). Eukaryotic translation initiation factor 4E is phosphorylated and cleaved upon apoptosis (Scheper and Proud, 2002).…”