Abstract
The adenosine kinase enzymes from arabinofuranosyladenine-resistant (araAr) mutants of the baby hamster kidney cell line were analyzed using adenosine kinase-specific antibody probes purified by adenosine kinase-Sepharose column chromatography. Wild-type baby hamster kidney cells were shown to produce two adenosine kinase polypeptides of Mr 43,000 and 40,000. The class I araAr mutants that have no detectable adenosine kinase activity are completely deficient in the two adenosine kinase polypeptides. As expected, the class II araAr mutants, which had been shown to have an altered ribonucleotide diphosphate reductase activity, produce a wild-type level of the two adenosine kinase polypeptides. The five class III araAr mutants which are adenosine-sensitive (AdoS) have various levels of adenosine kinase activity and produced two adenosine kinase polypeptides with similar Mr as that of wild-type cells. The adenosine kinase proteins synthesized by two of the AdoS mutants, ara-19a and ara-74b, differed from wild type in their isoelectric points. These results plus the observations that the AdoS mutants produce adenosine kinase enzymes with altered kinetic properties suggest a point mutation in the adenosine kinase gene. An araAr mutant, ara-60a, with intermediate adenosine sensitivity, was shown to have two truncated adenosine kinase polypeptides. This observation strongly supports the genetic data which suggests that there is only one functional adenosine kinase allele in baby hamster kidney cells and that the two adenosine kinase polypeptides are due to posttranscriptional modification.
Highlights
Which are adenosine-sensitive (Ado")have variouslev- cations stimulated the ara-SlOd but not the wild-type adenels of adenosine kinaseactivityand produced two osine kinase activity [3]
74b, differed fromwild type intheir isoelectric points. the Ado" phenotype of all class 111cells. These results plus the observations that the Ado" mu- In this study, we describe the analysis of the adenosine tants produce adenosine kinase enzymes with altered kinase protein inthe mutant anwd ild-type cells using specific kinetic propertiesuggest a point mutation inthe aden- adenosine kinase antibody probes
An araA' mutant, ara-60a, with ducetwo adenosine kinase polypeptides with molecular intermediate adenosine sensitivity, wasshown to have weights of 43,000 and 40,000
Summary
Thiswas followed by similar injections 1 and 4 weeks later Purification of Hamster Liver Adenosine Kinase-The BHK of 140 and 125 pg of adenosinekinase, respectively, in Freund's tissueculture cell line was originallyderivedfrom Syrian incompleteadjuvant. The adenosinekinaseantibodytiter of the serum was monitored weekly using the Bio-Rad dot immunoassay kit In this assay, 1 pg of pure adenosine kinase was used as the test antigen,thepreimmuneserum served as a control,andBio-Rad horseradish peroxidase-conjugated goat anti-rabbit IgG was used as hamster kidney [13].Since relatively largequantities of adenosine kinase were needed to immunize the rabbit,we purified the adenosine kinasefrom Syrian hamster tissue rather than the BHK cell line. Low and high affinty anti-adenosine gave rise to a single CoomassieBlue-stained protein as deterkinase antibodies were isolated essentially according to the method described by Tan-Wilson et al [8].This procedurewas carried out a t. Fractions were dialized against 10 mM K,HPO,, pH 7.0, buffer and we decided to purify specificanti-adenosine kinase stored a t 4 "C in the presence of 0.1% sodium azide
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