In the present study, adenosine kinase (AK) was purified from a number of different sources (e.g. bovine liver, Syrian hamster liver, kidney and heart; Chinese hamster ovary cells and human placenta). The enzyme from bovine liver and Syrian hamster liver was purified to apparent homogeneity by a combination of ion-exchange chromatography, affinity chromatography and gel filtration chromatography. The purified enzyme showed a single band of 40 kDa in SDS-polyacrylamide gel electrophoresis, which was similar to that reported from other sources in the literature. A number of biochemical and enzymatic characteristics of AK were investigated. The reliability and reproducibility of the AK assay as established in previous studies was determined. The apparent Km of partially purified AK from Syrian hamster liver for adenosine was determined to be 0.16 uM, which is consistent with earlier reports. A novel result obtained in these studies is that AK activity was found to be completely dependent upon the presence of phosphate or other pentavalent ions. AK from different sources did not show any activity in the Tris-HCl buffer, pH 7.0, but the activity increased dramatically upon the addition of phosphate and it reached a maximum at 2 mM phosphate. There was no inhibition of AK activity when the phosphate concentration was increased up to 100 mM. AK could also be activated by substituting phosphate with either arsenate or vanadate, which have similar chemical structures to phosphate. The temperature inactivation kinetics of AK showed that AK from human fibroblast cells had higher thermal resistance than AK from Chinese hamster ovary cells at 50°C. The presence of phosphate had no effect on the thermal stability of AK. Antibodies to purified AK from Syrian hamster liver were raised in both rabbits and guinea pigs. Antiserum from the rabbit which gave the strongest response was used for further studies. AK was recognized by and reacted specifically with the antiserum at a dilution of up to 1: 16,000. The AK antibody which was covalently bound to Protein A Sepharose beads immunoprecipitated a 40 kDa protein from radio-labelled Chinese hamster ovary or baby hamster kidney cell extracts. In addition, this antibody preparation immunoprecipitated AK activity from Syrian hamster liver extracts. However, immunoblotting showed that all the antisera could not react with AK from Chinese hamster ovary cells and human cells, suggesting a strong species specificity. A partial protein sequence of AK was obtained by microsequencing of a cyanogen bromide fragment of purified AK from Syrian hamster liver. The sequence was Tyr-Val-Asp-Ser-Leu-Phe-Gly-Ala-Glu-Thr-Glu-Ala-Ala-Leu. Degenerate DNA probes for this sequence can now be made and used for either screening of eDNA libraries or carrying out PCR experiments to facilitate the cloning of the AK gene(s). Finally, conditions for selection of revertants from AK⁻ mutants of Chinese hamster ovary cells have been developed. The AK⁺ revertants from AK⁻ mutants can be obtained using adenosine, alanosine, uridine and erythro-9-(2-hydroxyl-3-nonyl) adenine in the growth medium. Three revertants have been isolated. These revertants regained their AK activities and lost their drug-resistance at same time. This method can also be used in the future to clone the AK gene by transfecting AK⁻ mutants with foreign DNA. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22786 |
| Date | 12 1900 |
| Creators | Hao, Weihua |
| Contributors | Gupta, R. S., Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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