BACKGROUND
adenosine kinase  is a major purine salvage pathway enzyme belonging to the ribokinase family of proteins  <cit> .
it plays a central role in regulating the intracellular and interstitial concentrations of the purine nucleoside adenosine , which exhibits potent cardioprotective and neuroprotective activity  <cit> .
during ischemia, the compromised regeneration of atp causes an increase in the intracellular concentration of ado, which results in its net efflux into extracellular space where it binds to gi/o-coupled ado receptors: a <dig>  a2a, a2b, and a <dig>  to modulates a variety of physiological responses to reduce tissue damage from ischemic injury  <cit> .
the expression of adk undergoes rapid coordinated changes in the brain following epileptic seizures or stroke, resulting in an acute surge of ado, which serves to minimize damage to the brain  <cit> .
strong evidence in support of the protective role of ado has been obtained from studies where transient down regulation of adk after acute brain injury protected brain from seizures and cell death, whereas its overexpression as in epilepsy caused seizure aggravation and promoted cell death  <cit> .
adk, in addition to its central role in purine salvage and atp catabolism, also plays a critical role in the maintenance of methylation reactions.
in the s-adenosylmethionine  dependent methylation pathway, ado and homocysteine  are produced as a result of hydrolysis of s-adenosyl-homocysteine , which is the common end product of all methylation reactions  <cit> .
the hydrolysis reaction, which is catalyzed by the enzyme sah-hydrolase, is reversible and the equilibrium constant of this reaction favors sah formation.
hence, unless the hydrolysis product, ado and hcy are rapidly removed, it will lead to the buildup of sah, which is a potent inhibitor of transmethylation reactions  <cit> .
in the guinea-pig heart, the transmethylation pathway has been shown to be an important intracellular source of ado under normal conditions and the ado produced by this mechanism is mainly salvaged by adk  <cit> .
studies with the adk knockout mouse, which causes liver failure and early postnatal death  <cit> , indicate that the effects of adk deficiency on transmethylation reactions are the main underlying causes for its lethal effect  <cit> .
the deficiency of adk due to its pivotal role in the maintenance of transmethylation reaction also causes developmental abnormalities and reduced salt stress in plants  <cit> .
two isoforms of adk are present in mammalian species  <cit> .
these isoforms differ from each other only in their n-termini.
the long isoform  of adk contains an extra 20- <dig> amino acids in place of the first four amino acids of the adk-short  isoform  <cit> .
studies with the recombinant adk-l and adk-s proteins have revealed no differences in their biochemical or kinetic properties  <cit> .
however, we recently showed that the n-terminal extension in the adk-l functions as a nuclear localization signal  <cit> .
thus, of the two adk isoforms, adk-l is targeted to the nucleus whereas adk-s is localized in the cytoplasm  <cit> .
the differential subcellular localization of these two adk isoforms suggests that they carry out different physiological functions.
however, there is no information available at present regarding the unique cellular functions of these isoforms or how their expression is regulated.
unlike the lethal phenotype of adk-/- mice, adk deficient mutants can be readily obtained in cultured cells by selecting in the presence of toxic concentrations of the ado analogs  <cit> .
most of such mutants lack adk activity and some mutants that have been studied in detail contained large deletions within the adk gene  <cit> .
our recent work shows that in contrast to the chinese hamster ovary  cells that expresses only the adk-l isoform, in the ch v <dig> cell line established from embryonic lung  <cit>  both adk-l and adk-s isoforms are expressed.
hence, to gain further insights into the cellular functions of adk, in the present work we report the isolation and characterization of mutants resistant to ado analogs from v <dig> and other ch cell lines.
our results show that these mutants exhibit interesting differences in their cross-resistance pattern towards the n- and c- ado analogs and also in the expression profiles of the two adk isoforms.
.
several of these mutants contained novel molecular alterations in adk affecting its activity/function.
one of these mutants that exhibited increased resistance to only c-ado analogs, and whose drug-resistance phenotype was dominantly expressed in cell hybrids formed with adk+ cells, has been characterized in detail  <cit> .
we showed that a single point mutation in adk is responsible for its novel genetic and biochemical characteristics.
these mutants provide important insights and novel tools for understanding the cellular functions and regulation of the adk isoforms in mammalian cells.
RESULTS
the adk-l and adk-s isoforms differ in their first exons and the promoter sequences
the adk genes in human as well as rodent species are unusually large  and they consist of  <dig> exons that range in length from  <dig> to  <dig> nt  <cit> .
we have previously shown that the promoter for the adk-l isoform in human, ch and other mammals is bidirectional and it is linked in a head-to-head fashion with the gene encoding the clathrin adaptor mu3a protein  <cit> .
the first exon of the adk-l isoform contains the sequence information distinguishing it from the adk-s isoform .
the first exon and the promoter for the adk-s isoform have not yet been identified.
our blast searches on the human genome with the nucleotide sequence specific for the adk-s isoform and its upstream non-coding region  <cit>  have revealed that it matches perfectly with a sequence within the first intron of the adk-l isoform.
this sequence region encodes for all amino acids that are distinctive of the adk-s isoform .
the analysis of the 3- <dig> kb region upstream of the adk-s coding sequence using the emboss program  <cit>  indicates the presence of a promoter about  <dig> bp upstream of the initiator codon.
this predicted promoter is located within a cpg island and several transcription factor binding sites are present in its proximity .
studies to determine the promoter activity of this region and the significance of various regulatory elements present in its vicinity will be undertaken in future.
because the adk-l and adk-s isoforms are identical except for the amino acids encoded by their first exons, the observed structural organization of the adk gene indicates that the two adk isoforms arise by differential splicing of their unique first exons with the other adk exons.
the fact that each of these exons has their own promoters strongly suggests that the expression of the two adk isoforms is independently regulated at the transcriptional level.
expression of the two isoforms differ in various tissues and cell lines
the expression profile of adk in rat tissues was examined by western blotting .
in immunoblots of different tissue extracts, the antibody to adk detected only two closely related protein species of molecular masses , which correspond to the two isoforms of adk.
in contrast to an earlier study, where three isoforms of adk were reported  <cit> , no third isoform was detected in our study.
the expression of the two adk isoforms varied markedly among different tissues.
in liver, kidney, lung and pancreas both isoforms were expressed in comparable amounts .
in contrast, in heart, thymus and skeletal muscle, expression of mainly the adk-l isoform was observed.
of the different tissues examined, brain was the only tissue where the short isoform was predominantly expressed   <cit> .
the expression of the two adk isoforms was also examined in a number of other mammalian cell lines.
in contrast to the cho lines that expressed mainly the adk-l isoform, in the ch v <dig> and gm <dig> cell lines derived from embryonic lung  <cit> , both adk-l as well as adk-s isoforms were expressed .
the expression of the two isoforms was comparable in the gm7s cells, whereas in v <dig> cell line the adk-l isoform showed slightly higher expression.
in our earlier work, the human hela cells also showed expression of only the adk-l isoform, whereas in human ht- <dig> cell line and mouse lm  cells, both adk isoforms were expressed in comparable amounts  <cit> .
isolation and characterization of mutants resistant to adenosine analogs from v <dig> cells
to gain insights into the cellular functions of adk and the regulation of its isoforms, mutants resistant to the ado analogs tubercidin and formycin a  were selected from v <dig> cells.
of these analogs, tubercidin  similar to adenosine is an n-nucleoside, whereas foa is a c-nucleoside analog.
in our earlier work, interesting differences have been observed in the cross-resistance patterns of adk mutants towards the n- and c- ado analogs  <cit> .
under the conditions employed , the resistant mutants were obtained at a frequency of ~  <dig>  × 10- <dig>
the degree of resistance of the mutants towards foa and tubercidin was determined in comparison to the parental v <dig> cells.
in the presence of foa, the colony forming ability of wt v <dig> or cho cells decreased sharply at drug concentrations between 5- <dig> ng/ml and no colonies were obtained at  <dig> ng/ml.
for tubercidin, the colony forming ability of the wt cells decreased sharply between 2- <dig> ng/ml and no colonies were observed at  <dig> ng/ml.
based on these studies, the d <dig> values of the wt v <dig> cells for foa and tubercidin were approximated to be  <dig> ng/ml and  <dig> ng/ml, respectively .
the degree of resistance of the mutant cell lines in comparison to the parental sensitive cell line was determined based on the ratios of their d <dig> values  <cit> .
based on their relative resistance to foa and tubercidin, the different mutants that we have isolated could be divided into two groups .
all four tubr mutants  and ten fomr mutants viz.
vf2- <dig>  vf <dig>  vf <dig>  and vf <dig>  which exhibited >50-fold resistance to both foa and tubercidin formed the first group.
the resistance levels of these mutants to these analogs is similar to that for the toyocamycin and tubercidin resistant mutant of cho cells  isolated previously  <cit> .
in contrast to these mutants, ten other fomr mutants  were between 10- <dig> fold resistant to foa, but only showed minimal  resistance to tubercidin.
the drug-resistance profile of these mutants  is similar to that of the fomr- <dig> mutant of cho cell isolated in earlier work  <cit> .
the degree of resistance of various cell lines towards formycin a and tubercidin was determined as described in materials and methods.
assuming the d <dig> values of these drugs for wt v <dig> cells as  <dig>  the relative resistance of the mutant cell lines was calculated.
and  refer to the two class of mutants of v <dig> cells.
to determine if these mutants were affected in the expression of the adk isoforms, western blot analysis was performed .
two closely migrating protein bands with molecular masses of ~ 40- <dig> kda corresponding to the two adk isoforms were detected in the wt v <dig> cells and a number of mutants .
however, large differences were observed in various mutants with regard to expression level of the two isoforms.
vf <dig> mutant expresses only the adk-s isoform, whereas in vf1- <dig> and vf10- <dig> mutants only the adk-l isoform was found.
in contrast to these mutants, several other mutants such as vt <dig>  vt <dig>  vf <dig>  vf <dig> and vf <dig> showed no expression of either of the two adk isoforms.
some of the other mutants including vf <dig>  vf <dig>  vf <dig> and vf <dig>  also showed higher expression of the adk-s isoform.
western blot analysis was also performed on some of the mutants of cho cells that are studied in this work .
in the three mutants isolated from the cho cells, which express only the adk-l isoform, no cross-reactive protein was observed in the toyr- <dig> mutant, whereas the drtoyr- <dig> and fomr- <dig> mutants showed similar expression of the adk-l isoform as the wt cells.
the most common mechanism for resistance to ado analogs involves deficiency or loss of adk activity.
hence, adk activity in the cell extracts of various mutants was measured and normalized with respect to both protein and adk activity in the wt v <dig> cells.
a summary of these results is presented in figure  <dig>  the cell extracts from twelve mutants  contain negligible  adk activity.
the cell extracts from eleven other mutants  contain low , but significant level of adk activity .
in contrast to these mutants that contain either no or very low level of adk activity, two other mutants vf <dig> and vf <dig> contain significantly higher amounts of adk activity than in the wt v <dig> cells.
the adk activity was also examined in the three adk mutants of cho cells.
all three of these mutants i.e.
toyr- <dig>  drtoyr- <dig> and fomr- <dig> contain only background activity .
of these mutants, the toyr- <dig> contains a large deletion in the adk gene.
hence, this level of background activity seen in the cell extracts of many mutants  is not due to adk but it is possibly due to activities of other enzymes in cell extracts  <cit> .
molecular characterization of the mutants affected in adk
the mutants of cho cells lacking in adk activity that have been previously studied contained large deletions in the adk gene  <cit>  and they provided no useful information regarding structure-activity aspects of adk.
with the availability of a good antibody to adk, the mutants lacking adk antibody cross-reactive bands can now be readily identified and excluded from further analyses.
these studies have revealed that the mutants drtoyr- <dig> and fomr- <dig> isolated in our earlier work both contained normal amounts of the adk antibody cross-reactive bands .
hence, these mutants and two other mutants vf <dig> and vf <dig> obtained in this work were further characterized.
of these mutants, the mutant fomr- <dig> is of particular interest.
this mutant is highly resistant to foa and other c-ado analogs, but shows very little or no resistance to various n-ado analogs including toyocamycin and tubercidin  <cit> .
interestingly, although this mutant was able to phosphorylate adenosine and various n-ado analogs in vivo, the cell extracts from this mutant showed no adk activity and all efforts to detect/recover adk activity in its cell extracts have been unsuccessful  <cit> .
another novel aspect of this mutant is that its drug resistance phenotype expresses dominantly in cell hybrids formed with the wt cho cells  <cit> , which is in contrast to the recessive behaviour of all other adk- mutants that have been studied  <cit> .
hence, it was of much interest to determine the nature of the molecular alteration in this mutant.
to characterize the molecular alterations in this and other mutants, full-length adk sequences from the mutant cdna were pcr amplified and sequenced.
of the four independent clones from the fomr- <dig> mutant that were sequenced, all were found to contain a single c → t mutation that changed a conserved ser residue at position  <dig> into phenylalanine.
the changed amino acid corresponds to position  <dig> in the human adk structure   <cit> .
to confirm the significance of this molecular alteration for the biochemical and drug-resistance phenotype of the fomr- <dig> mutant, the mutation causing s <dig> → f alteration was introduced by site-directed mutagenesis into the wt ch  adk in the mammalian expression vector pcdna <dig> .
the wt cho cells and an adk- mutant of cho cell  were transformed with this plasmid dna and stable transformants were selected in presence of the neomycin analog g <dig>  the degree of resistance of these transformants as well as the wt cho, fomr- <dig> and toyr- <dig> cell lines towards tubercidin and foa was determined.
the results of these experiments are shown in figure  <dig>  as expected, the wt cho cells were sensitive to both tubercidin and foa, whereas the toyr- <dig> mutant containing a deletion in the adk gene was highly resistant to both of them.
further, as reported in our earlier work  <cit> , the fomr- <dig> mutant was highly resistant to foa but it displayed negligible resistance to tubercidin.
the transformants obtained upon transfection of wt cells with the adk plasmid were interesting since they also exhibited resistant to foa but were sensitive to tubercidin.
because wt cho cells contained normal adk, the foa resistance of these transformants indicated that the mutant adk was able to confer resistance despite the presence of wild-type adk.
the results obtained upon transformation of the toyr- <dig> mutant with the adk plasmid were equally interesting.
although the toyr- <dig> mutant is highly resistant to both tubercidin and foa, the transformants obtained in this case were found to be only marginally resistant to tubercidin and they displayed similar level of resistance to foa as the fomr- <dig> mutant.
these results indicate that the expression of the adk plasmid has made the toyr- <dig> cells sensitive towards tubercidin, while maintaining resistance towards formycin a. these results provided strong evidence that the s191f mutation in the adk was behaving dominantly and it was responsible for the novel genetic and biochemical properties of the fomr- <dig> mutant.
the drtoyr- <dig> mutant is highly resistant to both n- and c- adenosine analogs.
the sequencing of adk cdna from this mutant revealed that it contained a single point mutation , which changes a conserved glu <dig> residue in the nxxe motif to lys .
earlier studies indicate that the conserved asn and glu residues in the nxxe motif  are involved in the binding of activating phosphate ion, indicating that this motif is essential for adk function  <cit> .
we have also introduced the e242k mutation in cho adk cdna in pet-15b expression vector  <cit> .
upon expression, the resulting protein showed no adk activity  confirming that this mutation leads to the inactivation of the enzyme and is responsible for the drug resistance phenotype of the drtoyr <dig> mutant.
these results provide strong evidence that the nxxe motif is essential for adk function in vivo.
the adk cdna was also pcr sequenced from the vf <dig> and vf <dig> mutants.
for vf <dig> mutant, of the four clones sequenced,  <dig> contained a single point mutation changing the leu <dig> into phe.
the remaining two clones showed no change in the adk sequence.
similarly, of the  <dig> clones that were sequenced from the vf <dig> mutant,  <dig> contained the same mutation  as observed in the vf <dig> mutant.
however, these clones in addition also contained a second mutation changing phe <dig> into leu.
the mutant vf <dig> displays a higher degree of resistance to foa in comparison to the vf <dig>  thus, it is possible that these two mutations synergistically reduce the binding of foa to adk.
the locations of various amino acids that are altered in these mutants i.e.
ser191phe, leu188phe, phe221leu and glu242lys in the human adk structure are shown in figure  <dig> b and 5c.
discussion and
CONCLUSIONS
this paper presents information regarding several novel characteristics of adk from mammalian cell lines.
we show that the two isoforms of adk differ from each other only in their first exons.
because each of these isoforms contains its own promoter, the transcription of these two isoforms should be regulated independently at the genetic level.
the expression of the two isoforms also differs greatly in various rat tissues.
whereas in liver, kidney, lung and pancreas both isoforms were expressed at comparable levels, in other tissues such as heart, thymus, skeletal muscle and brain, either the adk-l or adk-s isoform is predominantly expressed.
large differences in the expression of these two isoforms have also been observed in earlier studies  <cit> .
importantly, our studies also showed that the expression of the two isoforms also differed greatly in established cell lines.
whereas the cho and hela cells expressed only the adk-l isoform, in two other cell lines v <dig> and gm7s from the same species, both adk isoforms were expressed in comparable amounts.
it is of interest that the v <dig> and gm <dig> cell lines, which are derived from lung, expresses both isoforms.
therefore, it is possible that the expression of these isoforms in cultured cells reflect their expression profiles in corresponding tissues.
however, the main focus of this work was on isolation and characterization of mutant ch cell lines that are affected in adk.
from ch v <dig> cell line expressing both adk isoforms,  <dig> mutants resistant to foa and tubercidin were isolated.
about half of these mutants were highly resistant to both tubercidin and foa, whereas the remaining, although they were highly resistant to foa, exhibited only marginal resistance to tubercidin.
biochemical studies have revealed that all of these mutants except vf <dig> and vf <dig> contained either no or very low level of adk activity.
this accounts for their resistance to the ado analogs, which are converted into their toxic forms by adk.
the expression of the two adk isoforms in different mutants also exhibited important differences.
whereas the mutants vf <dig>  vf <dig>  vt <dig> and vt <dig> containing only background adk activity lacked both isoforms, many other mutants with similar level of adk activity expressed either the adk-s isoform viz.
vf <dig>  vf <dig> and vf <dig> or the adk-l isoform viz.
the mutants vf1- <dig> and vf <dig>  surprisingly, the mutants vf <dig>  vf <dig>  vf <dig> and vt <dig> that show negligible or greatly reduced level of adk activity expressed both adk isoforms at a level comparable to the wt v <dig> cells.
although, the molecular alterations in most of these mutants remain to be identified, it is quite likely that in mutants that do not express either of these isoforms, mutations or deletions affecting one or more of the common adk exons  have occurred.
these mutants could be similar to the mutants cho cells that have been previously characterized, which contained large deletions leading to loss of several introns and exons  <cit> .
in contrast to these mutants, the mutants where expression of either the adk-l or adk-s isoform is preferentially affected are most likely to contain mutations in the promoter regions for these isoforms.
thus, further molecular characterization of these mutants, such as the methylation status, should provide useful insights concerning the functional significance of the two isoforms and how their expression may be regulated in mammalian cells.
the molecular alterations in a number of mutants resistant to ado analogs were also identified in the present studies.
one of these mutants, drtoyr- <dig> was specifically altered in the conserved nxxe motif, which has been indicated to be important in the binding of activating phosphate ion to adk as well as other pfkb family of proteins  <cit> .
the complete loss of adk activity in this mutant, both in vivo and in vitro, now provides direct evidence that this motif, which is in close proximity to the binding sites for mg2+ ion as well the substrate adenosine , is essential for adk function in vivo.
in this work, we have also identified the molecular alteration in the fomr- <dig> mutant, whose genetic and biochemical characteristics have remained puzzling for > <dig> years.
we show that this mutant contains a single base substitution mutation that changes a conserved ser <dig> into phe.
this mutation when introduced into either ch or human adk  was sufficient to confer a similar genetic and biochemical phenotype as observed for the fomr- <dig> mutant.
the ser191phe mutation is present in the adk structure  <cit>  on the periphery of the protein near the entrance of the substrate-binding pocket .
the c- ado analogs, to whom the fomr- <dig> mutant exhibits selective resistance exist predominantly in syn conformation in contrast to the anti-conformation for ado and various n-nucleoside analogs  <cit> .
hence, it is likely that this molecular alteration selectively prevents the binding of foa and other c-nucleosides to the mutant enzyme.
however, there are two other puzzling aspects of the fomr- <dig> mutant that remain to be addressed.
first, the cell extracts from this mutant show no adk activity despite its containing adk activity in vivo.
second, the drug-resistance phenotype of this mutant is dominant in cell hybrids formed with wt cells  <cit> .
the first observation suggests that the reaction product of adk i.e.
amp is not released from the mutant enzyme under in vitro conditions, but in the cellular milieu it is likely directly transferred to the next enzyme amp-kinase  in the pathway.
the ampk, which carries out the reaction amp + atp ↔  <dig> adp, plays a key role in maintaining the equilibrium concentrations of all three adenine nucleotides  <cit> .
the subsequent phosphorylation of adp into atp is carried out by the enzyme nucleoside diphosphate kinase  <cit> .
because the toxicity of ado analogs  requires their phosphorylation into higher phosphorylated forms , which interfere with different metabolic pathways  <cit> , if the mutation in adk prevents the conversion of foa or foa-po <dig> into higher phosphorylated forms then their toxicity will be averted.
in view of these consideration, to account for the lack of adk activity in the fomr- <dig> extracts and the dominant expression of this mutation, we postulate that the mutant adk and the enzyme ampk exist as a complex in cells such that the amp formed from adk reaction is not released but directly transferred to ampk for conversion into adp .
further, although this complex binds adenosine, n-ado analogs and the monophosphates of n-adenosine analogs  normally, it is unable to bind foa or foa-mp as a result of the ser191phe mutation.
the formation of a complex between these two enzymes will explain both the lack of adk activity in the mutant cell extracts  and also the dominant expression of foa resistance in this mutant.
in cell hybrids formed between wt and fomr- <dig>  although the wt adk can phosphorylate foa, the foa-mp formed, due to its non-recognition by the adk-ampk complex, is not further metabolized to higher phosphorylated forms to cause toxic effect.
thus, these cells display resistance to foa.
it should be noted that the ser191phe mutation in the fomr- <dig> mutant is present on the surface of adk  and it replaces a hydrophilic amino acid with a hydrophobic residue thus creating a surface hydrophobic patch  that could be important in its complex formation with the ampk.
although, this model is speculative, based upon our results, it is the only model that can account for the different characteristics of the fomr- <dig> mutant, which has remained an enigma for > <dig> years.
however, this model makes a number of predictions, which will be experimentally tested, providing further insights into the cellular function of this enzyme.
lastly, we have also identified the molecular alterations in two of the v <dig> cell mutants, v <dig> and vf <dig>  which similar to the fomr- <dig> mutant also exhibit preferential resistance to the c-ado analogs.
the observed molecular alterations in these two mutants are located on the same face of adk as the mutation in the fomr- <dig> mutant .
thus, it is likely that the genetic changes in these mutants, leu188phe and phe221leu, also selectively affect the binding of foa to the mutant adk.
however, it is of interest that of the four clones that were sequenced for these mutants, only two contained mutational alterations, whereas the other two showed no change.
this is in contrast to the fomr- <dig> mutant, where all four sequenced clones contained the ser191phe mutation.
earlier work on cho and v <dig> cells has provided evidence that the cho cell line is functional hemizygous for many genetic loci including the adk gene, whereas in v <dig> cells two functional copies of these genes were inferred  <cit> .
in this context, our observations that in the fomr- <dig> cells only the mutated form of adk was found, whereas in mutant v <dig> cells both wt and the mutated forms of adk were present support to this inference.
the fact that the vf <dig> and vf <dig> mutants are resistant to foa, despite their containing normal levels of adk activity, indicates that the mutations in them are also expressing codominantly.
the mutants vf <dig> and vf <dig> are also of much interest, because in comparison to all other adk mutants, they are the only mutants exhibiting enhanced adk activity relative to the parental v <dig> cells.
these mutants also show higher expression of the adk-s isoform in comparison to the adk-l isoform.
these observations indicate that these mutants, in addition to the molecular changes that we have identified in this work, also contain additional genetic changes affecting the expression of the two isoforms.
to understand the functional significance of different molecular alterations in these mutants, it is of much interest to further characterize adk from these mutants at genetic, molecular and biochemical levels.

 adenosine kinase is a major purine salvage pathway enzyme belonging to the ribokinase family of proteins. it plays a central role in regulating the intracellular and interstitial concentrations of the purine nucleoside adenosine, which exhibits potent cardioprotective and neuroprotective activity. during ischemia, the compromised regeneration of atp causes an increase in the intracellular concentration of ado, which results in its net efflux into extracellular space where it binds to gi/o-coupled ado receptors: a a2a, a2b, and a to modulates a variety of physiological responses to reduce tissue damage from ischemic injury. the expression of adk undergoes rapid coordinated changes in the brain following epileptic seizures or stroke, resulting in an acute surge of ado, which serves to minimize damage to the brain. strong evidence in support of the protective role of ado has been obtained from studies where transient down regulation of adk after acute brain injury protected brain from seizures and cell death, whereas its overexpression as in epilepsy caused seizure aggravation and promoted cell death. adk, in addition to its central role in purine salvage and atp catabolism, also plays a critical role in the maintenance of methylation reactions. in the s-adenosylmethionine dependent methylation pathway, ado and homocysteine are produced as a result of hydrolysis of s-adenosyl-homocysteine, which is the common end product of all methylation reactions. the hydrolysis reaction, which is catalyzed by the enzyme sah-hydrolase, is reversible and the equilibrium constant of this reaction favors sah formation. hence, unless the hydrolysis product, ado and hcy are rapidly removed, it will lead to the buildup of sah, which is a potent inhibitor of transmethylation reactions. in the guinea-pig heart, the transmethylation pathway has been shown to be an important intracellular source of ado under normal conditions and the ado produced by this mechanism is mainly salvaged by adk. studies with the adk knockout mouse, which causes liver failure and early postnatal death, indicate that the effects of adk deficiency on transmethylation reactions are the main underlying causes for its lethal effect. the deficiency of adk due to its pivotal role in the maintenance of transmethylation reaction also causes developmental abnormalities and reduced salt stress in plants. two isoforms of adk are present in mammalian species. these isoforms differ from each other only in their n-termini. the long isoform of adk contains an extra 20- amino acids in place of the first four amino acids of the adk-short isoform. studies with the recombinant adk-l and adk-s proteins have revealed no differences in their biochemical or kinetic properties. however, we recently showed that the n-terminal extension in the adk-l functions as a nuclear localization signal. thus, of the two adk isoforms, adk-l is targeted to the nucleus whereas adk-s is localized in the cytoplasm. the differential subcellular localization of these two adk isoforms suggests that they carry out different physiological functions. however, there is no information available at present regarding the unique cellular functions of these isoforms or how their expression is regulated. unlike the lethal phenotype of adk-/- mice, adk deficient mutants can be readily obtained in cultured cells by selecting in the presence of toxic concentrations of the ado analogs. most of such mutants lack adk activity and some mutants that have been studied in detail contained large deletions within the adk gene. our recent work shows that in contrast to the chinese hamster ovary cells that expresses only the adk-l isoform, in the ch v cell line established from embryonic lung both adk-l and adk-s isoforms are expressed. hence, to gain further insights into the cellular functions of adk, in the present work we report the isolation and characterization of mutants resistant to ado analogs from v and other ch cell lines. our results show that these mutants exhibit interesting differences in their cross-resistance pattern towards the n- and c- ado analogs and also in the expression profiles of the two adk isoforms.. several of these mutants contained novel molecular alterations in adk affecting its activity/function. one of these mutants that exhibited increased resistance to only c-ado analogs, and whose drug-resistance phenotype was dominantly expressed in cell hybrids formed with adk+ cells, has been characterized in detail. we showed that a single point mutation in adk is responsible for its novel genetic and biochemical characteristics. these mutants provide important insights and novel tools for understanding the cellular functions and regulation of the adk isoforms in mammalian cells.  the adk-l and adk-s isoforms differ in their first exons and the promoter sequences the adk genes in human as well as rodent species are unusually large and they consist of exons that range in length from to nt. we have previously shown that the promoter for the adk-l isoform in human, ch and other mammals is bidirectional and it is linked in a head-to-head fashion with the gene encoding the clathrin adaptor mu3a protein. the first exon of the adk-l isoform contains the sequence information distinguishing it from the adk-s isoform. the first exon and the promoter for the adk-s isoform have not yet been identified. our blast searches on the human genome with the nucleotide sequence specific for the adk-s isoform and its upstream non-coding region have revealed that it matches perfectly with a sequence within the first intron of the adk-l isoform. this sequence region encodes for all amino acids that are distinctive of the adk-s isoform. the analysis of the 3- kb region upstream of the adk-s coding sequence using the emboss program indicates the presence of a promoter about bp upstream of the initiator codon. this predicted promoter is located within a cpg island and several transcription factor binding sites are present in its proximity. studies to determine the promoter activity of this region and the significance of various regulatory elements present in its vicinity will be undertaken in future. because the adk-l and adk-s isoforms are identical except for the amino acids encoded by their first exons, the observed structural organization of the adk gene indicates that the two adk isoforms arise by differential splicing of their unique first exons with the other adk exons. the fact that each of these exons has their own promoters strongly suggests that the expression of the two adk isoforms is independently regulated at the transcriptional level. expression of the two isoforms differ in various tissues and cell lines the expression profile of adk in rat tissues was examined by western blotting. in immunoblots of different tissue extracts, the antibody to adk detected only two closely related protein species of molecular masses, which correspond to the two isoforms of adk. in contrast to an earlier study, where three isoforms of adk were reported, no third isoform was detected in our study. the expression of the two adk isoforms varied markedly among different tissues. in liver, kidney, lung and pancreas both isoforms were expressed in comparable amounts. in contrast, in heart, thymus and skeletal muscle, expression of mainly the adk-l isoform was observed. of the different tissues examined, brain was the only tissue where the short isoform was predominantly expressed. the expression of the two adk isoforms was also examined in a number of other mammalian cell lines. in contrast to the cho lines that expressed mainly the adk-l isoform, in the ch v and gm cell lines derived from embryonic lung, both adk-l as well as adk-s isoforms were expressed. the expression of the two isoforms was comparable in the gm7s cells, whereas in v cell line the adk-l isoform showed slightly higher expression. in our earlier work, the human hela cells also showed expression of only the adk-l isoform, whereas in human ht- cell line and mouse lm cells, both adk isoforms were expressed in comparable amounts. isolation and characterization of mutants resistant to adenosine analogs from v cells to gain insights into the cellular functions of adk and the regulation of its isoforms, mutants resistant to the ado analogs tubercidin and formycin a were selected from v cells. of these analogs, tubercidin similar to adenosine is an n-nucleoside, whereas foa is a c-nucleoside analog. in our earlier work, interesting differences have been observed in the cross-resistance patterns of adk mutants towards the n- and c- ado analogs. under the conditions employed, the resistant mutants were obtained at a frequency of ~ × 10- the degree of resistance of the mutants towards foa and tubercidin was determined in comparison to the parental v cells. in the presence of foa, the colony forming ability of wt v or cho cells decreased sharply at drug concentrations between 5- ng/ml and no colonies were obtained at ng/ml. for tubercidin, the colony forming ability of the wt cells decreased sharply between 2- ng/ml and no colonies were observed at ng/ml. based on these studies, the d values of the wt v cells for foa and tubercidin were approximated to be ng/ml and ng/ml, respectively. the degree of resistance of the mutant cell lines in comparison to the parental sensitive cell line was determined based on the ratios of their d values. based on their relative resistance to foa and tubercidin, the different mutants that we have isolated could be divided into two groups. all four tubr mutants and ten fomr mutants viz. vf2- vf vf and vf which exhibited >50-fold resistance to both foa and tubercidin formed the first group. the resistance levels of these mutants to these analogs is similar to that for the toyocamycin and tubercidin resistant mutant of cho cells isolated previously. in contrast to these mutants, ten other fomr mutants were between 10- fold resistant to foa, but only showed minimal resistance to tubercidin. the drug-resistance profile of these mutants is similar to that of the fomr- mutant of cho cell isolated in earlier work. the degree of resistance of various cell lines towards formycin a and tubercidin was determined as described in materials and methods. assuming the d values of these drugs for wt v cells as the relative resistance of the mutant cell lines was calculated. and refer to the two class of mutants of v cells. to determine if these mutants were affected in the expression of the adk isoforms, western blot analysis was performed. two closely migrating protein bands with molecular masses of ~ 40- kda corresponding to the two adk isoforms were detected in the wt v cells and a number of mutants. however, large differences were observed in various mutants with regard to expression level of the two isoforms. vf mutant expresses only the adk-s isoform, whereas in vf1- and vf10- mutants only the adk-l isoform was found. in contrast to these mutants, several other mutants such as vt vt vf vf and vf showed no expression of either of the two adk isoforms. some of the other mutants including vf vf vf and vf also showed higher expression of the adk-s isoform. western blot analysis was also performed on some of the mutants of cho cells that are studied in this work. in the three mutants isolated from the cho cells, which express only the adk-l isoform, no cross-reactive protein was observed in the toyr- mutant, whereas the drtoyr- and fomr- mutants showed similar expression of the adk-l isoform as the wt cells. the most common mechanism for resistance to ado analogs involves deficiency or loss of adk activity. hence, adk activity in the cell extracts of various mutants was measured and normalized with respect to both protein and adk activity in the wt v cells. a summary of these results is presented in figure the cell extracts from twelve mutants contain negligible adk activity. the cell extracts from eleven other mutants contain low, but significant level of adk activity. in contrast to these mutants that contain either no or very low level of adk activity, two other mutants vf and vf contain significantly higher amounts of adk activity than in the wt v cells. the adk activity was also examined in the three adk mutants of cho cells. all three of these mutants i.e. toyr- drtoyr- and fomr- contain only background activity. of these mutants, the toyr- contains a large deletion in the adk gene. hence, this level of background activity seen in the cell extracts of many mutants is not due to adk but it is possibly due to activities of other enzymes in cell extracts. molecular characterization of the mutants affected in adk the mutants of cho cells lacking in adk activity that have been previously studied contained large deletions in the adk gene and they provided no useful information regarding structure-activity aspects of adk. with the availability of a good antibody to adk, the mutants lacking adk antibody cross-reactive bands can now be readily identified and excluded from further analyses. these studies have revealed that the mutants drtoyr- and fomr- isolated in our earlier work both contained normal amounts of the adk antibody cross-reactive bands. hence, these mutants and two other mutants vf and vf obtained in this work were further characterized. of these mutants, the mutant fomr- is of particular interest. this mutant is highly resistant to foa and other c-ado analogs, but shows very little or no resistance to various n-ado analogs including toyocamycin and tubercidin. interestingly, although this mutant was able to phosphorylate adenosine and various n-ado analogs in vivo, the cell extracts from this mutant showed no adk activity and all efforts to detect/recover adk activity in its cell extracts have been unsuccessful. another novel aspect of this mutant is that its drug resistance phenotype expresses dominantly in cell hybrids formed with the wt cho cells, which is in contrast to the recessive behaviour of all other adk- mutants that have been studied. hence, it was of much interest to determine the nature of the molecular alteration in this mutant. to characterize the molecular alterations in this and other mutants, full-length adk sequences from the mutant cdna were pcr amplified and sequenced. of the four independent clones from the fomr- mutant that were sequenced, all were found to contain a single c → t mutation that changed a conserved ser residue at position into phenylalanine. the changed amino acid corresponds to position in the human adk structure. to confirm the significance of this molecular alteration for the biochemical and drug-resistance phenotype of the fomr- mutant, the mutation causing s → f alteration was introduced by site-directed mutagenesis into the wt ch adk in the mammalian expression vector pcdna. the wt cho cells and an adk- mutant of cho cell were transformed with this plasmid dna and stable transformants were selected in presence of the neomycin analog g the degree of resistance of these transformants as well as the wt cho, fomr- and toyr- cell lines towards tubercidin and foa was determined. the results of these experiments are shown in figure as expected, the wt cho cells were sensitive to both tubercidin and foa, whereas the toyr- mutant containing a deletion in the adk gene was highly resistant to both of them. further, as reported in our earlier work, the fomr- mutant was highly resistant to foa but it displayed negligible resistance to tubercidin. the transformants obtained upon transfection of wt cells with the adk plasmid were interesting since they also exhibited resistant to foa but were sensitive to tubercidin. because wt cho cells contained normal adk, the foa resistance of these transformants indicated that the mutant adk was able to confer resistance despite the presence of wild-type adk. the results obtained upon transformation of the toyr- mutant with the adk plasmid were equally interesting. although the toyr- mutant is highly resistant to both tubercidin and foa, the transformants obtained in this case were found to be only marginally resistant to tubercidin and they displayed similar level of resistance to foa as the fomr- mutant. these results indicate that the expression of the adk plasmid has made the toyr- cells sensitive towards tubercidin, while maintaining resistance towards formycin a. these results provided strong evidence that the s191f mutation in the adk was behaving dominantly and it was responsible for the novel genetic and biochemical properties of the fomr- mutant. the drtoyr- mutant is highly resistant to both n- and c- adenosine analogs. the sequencing of adk cdna from this mutant revealed that it contained a single point mutation, which changes a conserved glu residue in the nxxe motif to lys. earlier studies indicate that the conserved asn and glu residues in the nxxe motif are involved in the binding of activating phosphate ion, indicating that this motif is essential for adk function. we have also introduced the e242k mutation in cho adk cdna in pet-15b expression vector. upon expression, the resulting protein showed no adk activity confirming that this mutation leads to the inactivation of the enzyme and is responsible for the drug resistance phenotype of the drtoyr mutant. these results provide strong evidence that the nxxe motif is essential for adk function in vivo. the adk cdna was also pcr sequenced from the vf and vf mutants. for vf mutant, of the four clones sequenced, contained a single point mutation changing the leu into phe. the remaining two clones showed no change in the adk sequence. similarly, of the clones that were sequenced from the vf mutant, contained the same mutation as observed in the vf mutant. however, these clones in addition also contained a second mutation changing phe into leu. the mutant vf displays a higher degree of resistance to foa in comparison to the vf thus, it is possible that these two mutations synergistically reduce the binding of foa to adk. the locations of various amino acids that are altered in these mutants i.e. ser191phe, leu188phe, phe221leu and glu242lys in the human adk structure are shown in figure b and 5c. discussion and  this paper presents information regarding several novel characteristics of adk from mammalian cell lines. we show that the two isoforms of adk differ from each other only in their first exons. because each of these isoforms contains its own promoter, the transcription of these two isoforms should be regulated independently at the genetic level. the expression of the two isoforms also differs greatly in various rat tissues. whereas in liver, kidney, lung and pancreas both isoforms were expressed at comparable levels, in other tissues such as heart, thymus, skeletal muscle and brain, either the adk-l or adk-s isoform is predominantly expressed. large differences in the expression of these two isoforms have also been observed in earlier studies. importantly, our studies also showed that the expression of the two isoforms also differed greatly in established cell lines. whereas the cho and hela cells expressed only the adk-l isoform, in two other cell lines v and gm7s from the same species, both adk isoforms were expressed in comparable amounts. it is of interest that the v and gm cell lines, which are derived from lung, expresses both isoforms. therefore, it is possible that the expression of these isoforms in cultured cells reflect their expression profiles in corresponding tissues. however, the main focus of this work was on isolation and characterization of mutant ch cell lines that are affected in adk. from ch v cell line expressing both adk isoforms, mutants resistant to foa and tubercidin were isolated. about half of these mutants were highly resistant to both tubercidin and foa, whereas the remaining, although they were highly resistant to foa, exhibited only marginal resistance to tubercidin. biochemical studies have revealed that all of these mutants except vf and vf contained either no or very low level of adk activity. this accounts for their resistance to the ado analogs, which are converted into their toxic forms by adk. the expression of the two adk isoforms in different mutants also exhibited important differences. whereas the mutants vf vf vt and vt containing only background adk activity lacked both isoforms, many other mutants with similar level of adk activity expressed either the adk-s isoform viz. vf vf and vf or the adk-l isoform viz. the mutants vf1- and vf surprisingly, the mutants vf vf vf and vt that show negligible or greatly reduced level of adk activity expressed both adk isoforms at a level comparable to the wt v cells. although, the molecular alterations in most of these mutants remain to be identified, it is quite likely that in mutants that do not express either of these isoforms, mutations or deletions affecting one or more of the common adk exons have occurred. these mutants could be similar to the mutants cho cells that have been previously characterized, which contained large deletions leading to loss of several introns and exons. in contrast to these mutants, the mutants where expression of either the adk-l or adk-s isoform is preferentially affected are most likely to contain mutations in the promoter regions for these isoforms. thus, further molecular characterization of these mutants, such as the methylation status, should provide useful insights concerning the functional significance of the two isoforms and how their expression may be regulated in mammalian cells. the molecular alterations in a number of mutants resistant to ado analogs were also identified in the present studies. one of these mutants, drtoyr- was specifically altered in the conserved nxxe motif, which has been indicated to be important in the binding of activating phosphate ion to adk as well as other pfkb family of proteins. the complete loss of adk activity in this mutant, both in vivo and in vitro, now provides direct evidence that this motif, which is in close proximity to the binding sites for mg2+ ion as well the substrate adenosine, is essential for adk function in vivo. in this work, we have also identified the molecular alteration in the fomr- mutant, whose genetic and biochemical characteristics have remained puzzling for > years. we show that this mutant contains a single base substitution mutation that changes a conserved ser into phe. this mutation when introduced into either ch or human adk was sufficient to confer a similar genetic and biochemical phenotype as observed for the fomr- mutant. the ser191phe mutation is present in the adk structure on the periphery of the protein near the entrance of the substrate-binding pocket. the c- ado analogs, to whom the fomr- mutant exhibits selective resistance exist predominantly in syn conformation in contrast to the anti-conformation for ado and various n-nucleoside analogs. hence, it is likely that this molecular alteration selectively prevents the binding of foa and other c-nucleosides to the mutant enzyme. however, there are two other puzzling aspects of the fomr- mutant that remain to be addressed. first, the cell extracts from this mutant show no adk activity despite its containing adk activity in vivo. second, the drug-resistance phenotype of this mutant is dominant in cell hybrids formed with wt cells. the first observation suggests that the reaction product of adk i.e. amp is not released from the mutant enzyme under in vitro conditions, but in the cellular milieu it is likely directly transferred to the next enzyme amp-kinase in the pathway. the ampk, which carries out the reaction amp + atp ↔ adp, plays a key role in maintaining the equilibrium concentrations of all three adenine nucleotides. the subsequent phosphorylation of adp into atp is carried out by the enzyme nucleoside diphosphate kinase. because the toxicity of ado analogs requires their phosphorylation into higher phosphorylated forms, which interfere with different metabolic pathways, if the mutation in adk prevents the conversion of foa or foa-po into higher phosphorylated forms then their toxicity will be averted. in view of these consideration, to account for the lack of adk activity in the fomr- extracts and the dominant expression of this mutation, we postulate that the mutant adk and the enzyme ampk exist as a complex in cells such that the amp formed from adk reaction is not released but directly transferred to ampk for conversion into adp. further, although this complex binds adenosine, n-ado analogs and the monophosphates of n-adenosine analogs normally, it is unable to bind foa or foa-mp as a result of the ser191phe mutation. the formation of a complex between these two enzymes will explain both the lack of adk activity in the mutant cell extracts and also the dominant expression of foa resistance in this mutant. in cell hybrids formed between wt and fomr- although the wt adk can phosphorylate foa, the foa-mp formed, due to its non-recognition by the adk-ampk complex, is not further metabolized to higher phosphorylated forms to cause toxic effect. thus, these cells display resistance to foa. it should be noted that the ser191phe mutation in the fomr- mutant is present on the surface of adk and it replaces a hydrophilic amino acid with a hydrophobic residue thus creating a surface hydrophobic patch that could be important in its complex formation with the ampk. although, this model is speculative, based upon our results, it is the only model that can account for the different characteristics of the fomr- mutant, which has remained an enigma for > years. however, this model makes a number of predictions, which will be experimentally tested, providing further insights into the cellular function of this enzyme. lastly, we have also identified the molecular alterations in two of the v cell mutants, v and vf which similar to the fomr- mutant also exhibit preferential resistance to the c-ado analogs. the observed molecular alterations in these two mutants are located on the same face of adk as the mutation in the fomr- mutant. thus, it is likely that the genetic changes in these mutants, leu188phe and phe221leu, also selectively affect the binding of foa to the mutant adk. however, it is of interest that of the four clones that were sequenced for these mutants, only two contained mutational alterations, whereas the other two showed no change. this is in contrast to the fomr- mutant, where all four sequenced clones contained the ser191phe mutation. earlier work on cho and v cells has provided evidence that the cho cell line is functional hemizygous for many genetic loci including the adk gene, whereas in v cells two functional copies of these genes were inferred. in this context, our observations that in the fomr- cells only the mutated form of adk was found, whereas in mutant v cells both wt and the mutated forms of adk were present support to this inference. the fact that the vf and vf mutants are resistant to foa, despite their containing normal levels of adk activity, indicates that the mutations in them are also expressing codominantly. the mutants vf and vf are also of much interest, because in comparison to all other adk mutants, they are the only mutants exhibiting enhanced adk activity relative to the parental v cells. these mutants also show higher expression of the adk-s isoform in comparison to the adk-l isoform. these observations indicate that these mutants, in addition to the molecular changes that we have identified in this work, also contain additional genetic changes affecting the expression of the two isoforms. to understand the functional significance of different molecular alterations in these mutants, it is of much interest to further characterize adk from these mutants at genetic, molecular and biochemical levels. 
several other adk mutants were altered in surface residues that likely affect its binding to the adenosine analogs and its interaction with other cellular proteins. these adk mutants provide important insights as well as novel tools for understanding the cellular functions of the two isoforms and their regulation in mammalian cells. to gain insights into cellular functions of these isoforms, mutants resistant to toxic adenosine analogs formycin a and tubercidin were selected from chinese hamster cell lines expressing either one or both isoforms. another mutant, fomr- exhibiting increased resistance to only c-adenosine analogs and whose resistance was expressed dominantly in cell-hybrids contained a single mutation leading to ser191phe alteration in adk. we show that the two adk isoforms differ only in their first exons and the promoter regions; hence they arise via differential splicing of their first exons with the other exons common to both isoforms. we demonstrate that this mutation in adk is sufficient to confer the novel genetic and biochemical characteristics of this mutant. the expression of these isoforms also varied greatly in different rat tissues and cell lines with some tissues expressing both isoforms and others expressing only one of the isoforms. we have characterized the molecular alterations in a number of these mutants. one of these mutants lacking adk activity was affected in the conserved nxxe motif thereby providing evidence that this motif involved in the binding of mg2+ and phosphate ions is essential for adk function. the adk activity in most of these mutants was reduced to <5% of wild-type cells and they also showed large differences in the expression of the two isoforms. two isoforms of the enzyme adenosine kinase, which differ at their n-terminal ends, are found in mammalian cells. the unusual genetic and biochemical characteristics of the fomr- mutant suggest that adk in this mutant might be complexed with the enzyme amp-kinase.
 two isoforms of the enzyme adenosine kinase, which differ at their n-terminal ends, are found in mammalian cells. however, there is no information available regarding the unique functional aspects or regulation of these isoforms.  we show that the two adk isoforms differ only in their first exons and the promoter regions; hence they arise via differential splicing of their first exons with the other exons common to both isoforms. the expression of these isoforms also varied greatly in different rat tissues and cell lines with some tissues expressing both isoforms and others expressing only one of the isoforms. to gain insights into cellular functions of these isoforms, mutants resistant to toxic adenosine analogs formycin a and tubercidin were selected from chinese hamster cell lines expressing either one or both isoforms. the adk activity in most of these mutants was reduced to <5% of wild-type cells and they also showed large differences in the expression of the two isoforms. thus, the genetic alterations in these mutants likely affected both regulatory and structural regions of adk. we have characterized the molecular alterations in a number of these mutants. one of these mutants lacking adk activity was affected in the conserved nxxe motif thereby providing evidence that this motif involved in the binding of mg2+ and phosphate ions is essential for adk function. another mutant, fomr- exhibiting increased resistance to only c-adenosine analogs and whose resistance was expressed dominantly in cell-hybrids contained a single mutation leading to ser191phe alteration in adk. we demonstrate that this mutation in adk is sufficient to confer the novel genetic and biochemical characteristics of this mutant. the unusual genetic and biochemical characteristics of the fomr- mutant suggest that adk in this mutant might be complexed with the enzyme amp-kinase. several other adk mutants were altered in surface residues that likely affect its binding to the adenosine analogs and its interaction with other cellular proteins.  these adk mutants provide important insights as well as novel tools for understanding the cellular functions of the two isoforms and their regulation in mammalian cells. 