Cloning of human adenosine kinase cDNA: sequence similarity to microbial ribokinases and fructokinases.

Abstract

Adenosine kinase catalyzes the phosphorylation of adenosine to AMP and hence is a potentially important regulator of extracellular adenosine concentrations. Despite extensive characterization of the kinetic properties of the enzyme, its primary structure has never been elucidated. Full-length cDNA clones encoding catalytically active adenosine kinase were obtained from lymphocyte, placental, and liver cDNA libraries. Corresponding mRNA species of 1.3 and 1.8 kb were noted on Northern blots of all tissues examined and were attributable to alternative polyadenylylation sites at the 3' end of the gene. The encoding protein consists of 345 amino acids with a calculated molecular size of 38.7 kDa and does not contain any sequence similarities to other wellcharacterized mammalian nucleoside kinases, setting it apart from this family of structurally and functionally related proteins. In contrast, two regions were identified with significant sequence identity to microbial ribokinase and fructokinases and a bacterial inosine/guanosine kinase. Thus, adenosine kinase is a structurally distinct mammalian nucleoside kinase that appears to be akin to sugar kinases of microbial origin.Adenosine kinase (AK; ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) is an abundant enzyme in mammalian tissues that catalyzes the transfer of the y-phosphate from ATP to adenosine, thereby serving as a potentially important regulator of concentrations of both extracellular adenosine and intracellular adenine nucleotides. Adenosine has widespread effects on the cardiovascular, nervous, respiratory, and immune systems (1) and it has been postulated that inhibitors of AK could play an important pharmacologic role in increasing intravascular adenosine concentrations and acting as antiinflammatory agents (2, 3). In addition, this enzyme is responsible for the phosphorylation and consequent clinical activity of several therapeutically useful nucleosides, including the antiviral drug ribavirin and the immunosuppressive drug mizoribine (4, 5). AK has been purified from a number of sources including rabbit liver (6), human placenta (7) and liver (5), murine leukemia cells (8), and Leischmania donovani (9) RNA was purified by the acid guanidine isothiocyanate procedure (1 1).AK Purification. AK was purified from the human Tlymphoblast cell line Molt-4 by a modification of the procedure previously described (7). Cytosol was passed over a 5'-AMPSepharose column equilibrated with 20 mM imidazole HCl, pH 7.6, containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM EGTA. The column was eluted with buffer containing 5 mM adenosine and the peak fractions were pooled in 20% (vol/vol) glycerol. Several 2-ml fractions were loaded onto a Superose 12 column (50 x 1.6 cm) previously equilibrated with 50 mM imidazolevHCl, pH 7.2/50 mM KCI/5 mM dithiothreitol (DTT)/1 mM ATP/1 mM MgCl2/5% glycerol. Fractions containing AK activity, assayed as described (7), were pooled, diluted into buffer containing 1 mM PMSF, and applied to a DE52 column. Fractions we...

The adenosine kinase hypothesis of epileptogenesis

“…The generation of mice with moderately reduced levels of ADK in brain rather than complete ADK deficiency seems to be essential, since several lines of evidence suggest that only small changes in ADK levels are permissible: (i) The homozygous disruption of ADK is lethal (Boison et al, 2002b); (ii) ADK is highly conserved in evolution (Spychala et al, 1996), suggesting that mutations are not easily tolerated; (iii) No mutations of human ADK are known in man (OMIM, Online Mendelian Inheritance in Man, Victor A. McKusick et al, Johns Hopkins University) indicating that most mutations are lethal; (iv) Excessive levels of ADK lead to severe deficits in A 1 R activation and to lethal status epilepticus or cell death after otherwise non-lethal triggers Kochanek et al, 2006;Li et al, 2007a;Pignataro et al, 2007b); (v) Inadequate levels of ADK in brain are expected to lead to a rise in adenosine to unacceptably high levels, with likely lethal consequences, e.g. central apnea and perinatal mortality induced by elevated adenosine (Montandon et al, 2006).…”

Structure–activity relationship for adenosine kinase from Mycobacterium tuberculosis

“…Substrates for human Ado kinase were assayed similarly with the following changes: assay conditions consisted of 50 mM HEPES (pH 6.0), 40 mM KCl, 1 mM MgCl 2 , 1 mM ATP, 0.1% BSA, 10 μM deoxycoformycin, and 100 μM of the appropriate test compound. Human Ado kinase was prepared as previously described from clone 20-1 (a generous gift from Dr. Jozef Spychala, UNC Chapel Hill, Chapel Hill, NC) [1,31].…”

“…Ado kinase belongs to the PfkB family of carbohydrate and nucleoside kinases, a group of proteins that catalyze the transfer of the γphosphate of ATP to their substrates. This family includes ribokinase, hexokinases, and phosphofructokinase among its members [1]. Ado kinase activity is found in most eukaryotes, fungi, plants, and parasites, but it is seldom found in bacteria.…”

Structure and alternative splicing of the ketohexokinase gene

“…It is most similar in sequence to prokaryotic ribokinases and furanose sugar kinases (~20 % peptide sequence identity to Escherichia coli ribokinase; [2,10]), a family which also includes human adenosine kinase [11].…”