Adenosine kinase (AK) is a key enzyme responsible for maintaining the intracellular concentrations of adenosine (Ado), which shows potent cardioprotective and neuromodulatory activities. Many different types of mutants affected in AK have been isolated in my lab. The AK gene in mammalian cells is very large and the ratio of non-coding to coding sequence for this gene (>500) is highest of all known genes in mammalian cells. The AK gene in mammalian cells is also linked in a head-to-head manner with the gene for the mu3A adaptor protein, which is affected in the Herman-Pudlak syndrome. Both these genes are transcribed from a single bi-directional promoter. Many of the AK mutants that we have isolated contain large deletions in the AK gene some of which extends into the neighboring mu3A gene. Other mutants involve specific point mutations affecting the biochemical properties of the enzyme.

Another novel property of AK that we have discovered, which it shares with a few other enzymes, is that its activity in different organisms is completely dependent upon the presence of pentavalent ions. Our work has led to identification of many novel compounds, which compete with pentavalent ion and act as either activators or inhibitors of AK. The inhibitors of AK are of great interest due to their potential cardioprotective and neuromodulatory effect.

A variety of studies on AK and related enzymes have been carried out to understand its mechanism of action and to identify potent inhibitors of the enzyme with therapeutic potential. These studies include:

1. Relationship of AK-mu3A gene alterations to Herman-Pudlak Syndrome
2. Molecular characterization of human cell mutants affected in AK
3. Understanding the molecular mechanism by which phosphorus and other pentavalent ions stimulate AK activity. Structural studies on AK have also been carried out to determine the binding site of pentavalent ions.
4. Screening of various chemical libraries to identify different novel inhibitors of AK, and to characterize the mechanism of action of these inhibitors
5. Biochemical studies on other enzymes that are similar to AK also show pentavalent ion dependency

The amino acid sequences of these two isoforms in human, mouse and rat are identical except for the sequence at the N-terminus region (NTS). The NTS of adenosine kinase (AdK)-long in these species is highly conserved with complete conservation of the central basic cluster (PKPKKLK).

About 40 μg of total protein from various cell lines: Chinese hamster ovary (CHO), a human epithelial cell line derived from cervix (HeLa), a human epithelial cell line from connective tissue (HT-1080) and a fibroblastic cell line of connective tissue (LMTK) was loaded onto 12 per cent sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE gels).

After electrophoresis and transfer of proteins to nitrocellulose membrane, the blot was reacted with 1:1000 dilution of rabbit polyclonal antibody to adenosine kinase. The blot was treated with a secondary antibody conjugated to horseradish peroxidase and the cross-reactive bands were visualized by chemiluminescence.

Immunofluorescence localization of adenosine kinase in the (a) HT-1080, (b) HeLa, (c) LM (TK−) and (d) CHO cell lines using the AdK-antibody. In HeLa and CHO cells, which contain only the long isoform of AdK, immunofluorescence labeling was mainly observed in the nucleus.

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