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Mitochondrial deoxyribonucleoside salvage enzymes : cloning and characterization of deoxyguanosine kinase and thymidine kinase

Wang, Liya (1997). Mitochondrial deoxyribonucleoside salvage enzymes : cloning and characterization of deoxyguanosine kinase and thymidine kinase. Diss. (sammanfattning/summary) Sveriges lantbruksuniv., Acta Universitatis Agriculturae Sueciae. Veterinaria, 1401-6257
ISBN 91-576-5235-X
[Doctoral thesis]

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Abstract

Mitochondrial deoxyguanosine kinase (dGK) and thymidine kinase (TK2) are responsible for the phosphorylation of deoxyribonucleosides, thus providing DNA precursors for mitochondrial DNA synthesis. Bovine dGK was purified 20,000-fold to apparent homo-geneity. The purified dGK was capable of phosphorylating all the natural occurring purine deoxyribonucleosides, and a number of clinically important anticancer nucleoside analogs, e. g. CdA (2-chloro-2'-deoxyadenosine) and araG (9-p-D-arabinofuranosylguanine). A selective assay method for dGK determination was developed by using araG as substrate.dGK activity levels were elevated in certain brain tumors which may have implications for future anticancer chemotherapy.re anticancer chemotherapy. Amino aicd sequence information from purified bovine dGK was used in RT-PCR to amplify a dGK cDNA fragment. The entire dGK cDNA was subsequently cloned from a human brain cDNA library, and it encoded a 30 kDa protein. The peptide sequences from purified bovine dGK were identified in the deduced amino acid sequence and when expressed in E. coll, the recombinant protein catalysed efficient phosphorylation of dGuo, araG, CdA and dlno similar to purified bovine dGK. Northern blot analysis demonstrated one dominant transcript of 1.35 kb which was found in all tissues at similar levels.A similar approach was used to clone the cDNA for TK2. A TK2 cDNA was cloned from a human brain cDNA library and RACE PCR was used to amplify the cDNA ends from human brain mRNA. All the peptide sequences were found in the deduced amino aicd sequence. Expression of Nterminal truncated TK2 in E. coli yieled a fully active TK2 protein of 25.5 kDa which showed enzymatic properties identical to the purified human and bovine proteins. Northern blot analysis using coding sequence of TK2 cDNA demonstrated several mRNAs, the major ones were 2.2 and 4.0 kb and they were found in most tissues at similar levels.Both dGK and TK2 cDNA sequences show great similarities to the cytosolic deoxycytidine kinase sequence, about 40% at the amino acid level, suggesting that they are evolutionary related and thus established a new enzyme family of mammalian deoxynucleoside kinases.

Authors/Creators:Wang, Liya
Title:Mitochondrial deoxyribonucleoside salvage enzymes : cloning and characterization of deoxyguanosine kinase and thymidine kinase
Series Name/Journal:Acta Universitatis Agriculturae Sueciae. Veterinaria
Year of publishing :1997
Number:7
Number of Pages:54
Publisher:Swedish University of Agricultural Sciences
ISBN for printed version:91-576-5235-X
ISSN:1401-6257
Language:English
Publication Type:Doctoral thesis
Article category:Other scientific
Version:Published version
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 3 Medical and Health Sciences > 301 Basic Medicine > Medicinal Chemistry
(A) Swedish standard research categories 2011 > 4 Agricultural Sciences > 403 Veterinary Science > Medical Bioscience
Keywords:mitochondrial DNA precursor synthesis, deoxyguanosine kinase, mitochondrial thymidine kinase, deoxycytidine kinase, herpes virus thymidine kinase, chemotherapy, nucleoside analogs, araG (9-p-D-arabinofuranosylguanine), CdA (2-chloro-2'-deoxyadenosine), cDNA, sequence motifs
URN:NBN:urn:nbn:se:slu:epsilon-p-117372
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-117372
ID Code:28290
Faculty:VH - Faculty of Veterinary Medicine and Animal Science
Department:(VH) > Department of Veterinary Medical Chemistry
Deposited By: SLUpub Connector
Deposited On:09 Jun 2022 12:30
Metadata Last Modified:13 Jun 2022 09:11

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