Isolation and characterization of mutated mitochondrial GTP:AMP phosphotransferase 1 1Edited by J. Karn

Yamada, Mamoru; Sugahara, Masahiro; Hishitani, Yumiko; Nobumoto, Masaaki; Nakazawa, Atsushi

doi:10.1006/jmbi.1998.1876

Cited by 10 publications

(9 citation statements)

References 31 publications

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“…The genes of AK1 and AK2 have been isolated from a variety of organisms, including bacteria, fungi and mammals (Kohler et al 1999;Miura et al 2001;Schricker et al 2002;Tugarinov et al 2002), but AK3 genes have been isolated from only a few organisms such as Bos taurus, Homo sapiens, Mus musculus, Rattus norvegicus and Trypanosoma cruzi. In addition, very little research on protein characterization of AK3 has been carried out (Schricker et al 1995;Yamada et al 1998). There are no reports at all on the sequence and characterization of AK3 in the trematode, C. sinensis.…”

Section: Introductionmentioning

confidence: 98%

Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis

Guang

et al. 2005

Parasitol Res

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Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in living cells. AK catalyzes reversible high energy phosphoryl transfer reactions between ATP (or GTP) and AMP to generate ADP (or GDP). From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel AK3 isozyme. The 956 bp cDNA encodes a putative protein of 228 amino acids with a predicted molecular mass of 26.2 kDa. The recombinant CsAK3 protein produced in Escherichia coli can be refolded into a functional protein with AK3 activity. The optimum pH and temperature for the enzyme are 8.5 and 40 degrees C, respectively. The calculated activation energy is 56.04 kJ mol-1. The Km of the CsAK3 for AMP and GTP are 118 microM and 359 microM, respectively. CsAK3 is inhibited by Ap5A (>70% inhibition by 2.0 mM AP5A). Ap5A may be a potential lead compound acting on C. sinensis in which AK3 as a drug target.

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Section: Introductionmentioning

confidence: 98%

Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis

Guang

et al. 2005

Parasitol Res

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“…It is known that human tissues contain a cytosolic TMPK1, a uridylate‐cytidylate kinase (UMP‐CMPK) (Van Rompay et al. 1999b), five isozymes of adenylate kinase (AK) (Yamada et al. 1989; Xu et al.…”

Section: Discussionmentioning

confidence: 99%

“…In noncycling cells, the mitochondrial dNTP synthesis is dependent on the salvage pathway by the mitochondrial dGK and TK2 that phosphorylate all four deoxyribonucleotides (Van Rompay et al 2000). It is known that human tissues contain a cytosolic TMPK1, a uridylatecytidylate kinase (UMP-CMPK) (Van Rompay et al 1999b), five isozymes of adenylate kinase (AK) (Yamada et al 1989;Xu et al 1992;Yoneda et al 1998;Van Rompay et al 1999a), and several guanylate kinases (GUK) ( Jamil et al 1975;Brady et al 1996) for the step of conversion of dNMP to dNDP. Among them, only AK2, 3 and 4 are suggested be located in mitochondria for dADP formation, but the enzyme phosphorylating dTMP within the mitochondria remains to be identified.…”

Section: Discussionmentioning

confidence: 99%

Identification of a putative human mitochondrial thymidine monophosphate kinase associated with monocytic/macrophage terminal differentiation

Chen¹,

Lin²,

Chang³

2008

Genes to Cells

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Mitochondrial DNA synthesis requires the supply of thymidine triphosphate (dTTP) independent of nuclear DNA replication. In resting and differentiating cells that withdraw from the cell cycle, mitochondrial thymidine kinase 2 (TK2) mediates thymidine monophosphate (dTMP) formation for the dTTP biosynthesis in mitochondria. However, a thymidine monophosphate kinase (TMPK) that phosphorylates dTMP to form thymidine diphosphate (dTDP) in mitochondria remains undefined. Here, we identified an expressed sequence tag cDNA, which encodes a TMPK with a mitochondrial import sequence at its N‐terminus designated as TMPK2. HeLa cells expressing TMPK2 fused to green fluorescent protein (GFP) displayed green fluorescence in mitochondria. Over‐expression of TMPK2 increased the steady‐state level of cellular dTTP and promoted the conversion of radioactive labeled‐thymidine and ‐dTMP to dTDP and dTTP in mitochondria. TMPK2 RNA was detected in several tissues and erythroblastoma cell lines. We also generated TMPK2 antibody and used it for immunofluorescence staining to demonstrate endogenous expression of TMPK2 in mitochondria of erythroblastoma cells. Finally, we showed that TMPK2 protein expression was upregulated in monocyte/macrophage differentiating cells, suggesting the coordinated regulation of TMPK2 expression with the terminal differentiation program.

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“…In contrast, studies with GTP:AMP phosphotransferases have so far been confined mostly to their mitochondrial localization and substrate specificity and kinetic characterization of various mutants (9,10,12,23,34). The molecular properties of the GTP: AMP phosphotransferase, particularly in the context of protein folding and thermal tolerance, have not been studied.…”

Section: Discussionmentioning

confidence: 99%

A GTP:AMP Phosphotransferase, Adk2p, in Saccharomyces cerevisiae

Gordon

Amutha

et al. 2005

Journal of Biological Chemistry

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Adenylate kinases participate in maintaining the homeostasis of cellular nucleotides. Depending on the yeast strains, the GTP:AMP phosphotransferase is encoded by the nuclear gene ADK2 with or without a single base pair deletion/insertion near the 3 end of the open reading frame, and the corresponding protein exists as either Adk2p (short) or Adk2p (long) in the mitochondrial matrix. These two forms are identical except that the three C-terminal residues of Adk2p (short) are changed in Adk2p (long), and the latter contains an additional nine amino acids at the C terminus of the protein. The short form of Adk2p has so far been considered to be inactive (Schricker, R., Magdolen, V., Strobel, G., Bogengruber, E., Breitenbach, M., and Bandlow, W. (1995) J. Biol. Chem. 270, 31103-31110). Using purified proteins, we show that at the physiological temperature for yeast growth (30°C), both short and long forms of Adk2p are enzymatically active. However, in contrast to the short form, Adk2p (long) is quite resistant to thermal inactivation, urea denaturation, and degradation by trypsin. Unfolding of the long form by high concentrations of urea greatly stimulated its import into isolated mitochondria. Using an integration-based gene-swapping approach, we found that regardless of the yeast strains used, the steady state levels of endogenous Adk2p (long) in mitochondria were 5-10-fold lower compared with those of Adk2p (short). Together, these results suggest that the modified C-terminal domain in Adk2p (long) is not essential for enzyme activity, but it contributes to and strengthens protein folding and/or stability and is particularly important for maintaining enzyme activity under stress conditions. Cellular homeostasis of nucleotides is essential for numerous important biological processes. Among several enzymes that participate in maintaining cellular levels of nucleotides are adenylate kinases, which catalyze the reaction ATP (or GTP) ϩ AMP N ADP (or GDP) ϩ ADP (1-3). These kinases provide ADP for oxidative phosphorylation (4) and also control the intracellular AMP level, which serves as a highly sensitive sensor of cellular energy status. Under stress conditions, increased levels of AMP trigger the AMP-activated protein kinase cascade to switch on catabolic pathways that generate ATP while switching off anabolic processes that consume ATP (5, 6). Adenylate kinases therefore occupy a central position in many cellular functions under normal as well as stress conditions. Although ATP-specific adenylate kinases from different organisms have been extensively studied, not much is known about the GTP-specific isoforms.Adenylate kinase in Escherichia coli is indispensable for growth (7). In mammalian cells, at least three (AK1, AK2, and AK3) isoforms of adenylate kinase exist (8 -11). AK1 and AK2 use Mg 2ϩ ATP as the high energy phosphate donor, whereas AK3 is a GTP:AMP phosphotransferase and uses GTP instead of ATP as a phosphoryl donor. AK1 and AK3 are localized to the cytosol and mitochondrial matrix, respectively. In con...

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Isolation and characterization of mutated mitochondrial GTP:AMP phosphotransferase 1 1Edited by J. Karn

Cited by 10 publications

References 31 publications

Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis

Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis

Identification of a putative human mitochondrial thymidine monophosphate kinase associated with monocytic/macrophage terminal differentiation

A GTP:AMP Phosphotransferase, Adk2p, in Saccharomyces cerevisiae

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