Mitochondrial GTP‐AMP Phosphotransferase

Tomasselli, Alfredo G.; Schirmer, R. Heiner; Noda, Lafayette

doi:10.1111/j.1432-1033.1979.tb12818.x

Cited by 69 publications

(36 citation statements)

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“…AK1 and AK3 are localized to the cytosol and mitochondrial matrix, respectively. In contrast, AK2 resides in the cytosol and mitochondrial intermembrane space (2)(3)(4)(12)(13)(14). Basic cellular and mitochondrial functions are highly conserved from yeast to human (15), and in agreement with this notion, the yeast Saccharomyces cerevisiae also contains three isoforms of adenylate kinase localized to the cytosol and/or mitochondria.…”

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confidence: 58%

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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confidence: 58%

A GTP:AMP Phosphotransferase, Adk2p, in Saccharomyces cerevisiae

Gordon

Amutha

et al. 2005

Journal of Biological Chemistry

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“…Other chemicals were of analyticaI grade or the highest purity available. GTP : AMP phosphotransferase was prepared as previously described [2], except that in the present preparation the enzyme was not eluted from the Affi-Gel Blue column with GTP but instead with a gradient of NaCl (0.18-2.0 M in 20 mM Hepes buffer pH 8.0, total of eight column volumes). Protein concentrations were determined by the biuret procedure [3] using bovine serum albumin as a standard.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Protein concentrations were determined by the biuret procedure [3] using bovine serum albumin as a standard. The chromatographic assay and equilibrium dialysis binding studies were carried out as previously described [4].Conversion of the indole chromophore of Trp to oxindole, which absorbs much less at 280 nm, has been performed as described by Spande and Witkop [5]. In a typical experiment 1 .O ml of an exactly determined amount of protein in 0.075 M CHSCOONa buffer pH 4.0 or 70% CH3COOH was reacted in a 1 .O-cm quartz cell provided with a stirring bar.…”

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Fluorescence and chemical modification of tryptophan as probes of structure of GTP:AMP phosphotransferase from beef heart mitochondria

Tomasselli

Dougherty

Noda

1983

European Journal of Biochemistry

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Selective modification of the two Trp residues of GTP : AMP phosphotransferase from beef heart mitochondria (M, 26000; MgGTP + AMP S MgGDP + ADP) has been attained by treatment of the enzyme with Nbromosuccinimide at pH 4.0. Almost complete loss of activity is observed when one Trp is oxidized. Fluorescence -emission spectra (Aexc 295 nm) were recorded over the pH range 1.9 -12.2. Quenching constants, K , with acrylamide were 4.9, 3.4, 3.1, 2.4, 9.2 and 9.4M-' at respective pH values of 11.1, 7.5, 5.5, 4.0, 1.9 and 7.5 with 6 M guanidine/HCl. Over the pH range 8.0-5.5 the fluorescence peak has a constant height with maximum at 333-334 nm, which can be segregated by acrylamide quenching into a peak with maximum at 338 nm and another with maximum at 330 nm. Dropping the pH from 5.5 to 4.0 results in the fluorescence at 338 nm decreasing to 335 nm (indicative of less exposure of the Trp) while that at 330 nm remains constant. Thus the limitation of reactivity to Nbromosuccinimide to pH 4.0 or lower cannot be accounted for by increased exposure of the Trp residues but rather must be explained by a change in the microenvironment of each Trp. As shown by K values above, at pH 2.0 Trp residues are exposed to the solvent, as in the case of treatment with 6 M guanidine hydrochloride.In raising the pH from 8.0 to 12.0 a number of changes occur: (a) the I,,, of emission shifts from 333 -334 nm to 343 nm; (b) residue(s) become(s) more available to acrylamide quenching; (c) fluorescence decreases and enzymatic activity increases, both with a midpoint at about 10.6; (d) absorption difference spectra show a maximum at 295 nm typical of Tyr ionization. These data are consistent with conformational change as the pH becomes more alkaline making the Trp residue(s) more exposed to the solvent and/or to non-radiative energy transfer to .tyrosinate.The bulky aromatic hydrophobic residue of Trp may serve an important role in maintaining the integrity of the tertiary structure of a protein or polypeptide by hydrophobic interactions with surrounding amino acids and bv steric be strengthened by testing against an X-ray atomic model that will be completed in the future [I].hindrance. Hence the relativel; small number of Trp residues in proteins and the availability of peculiar chemical and physical probes sensitive to Trp make possible the study of this amino acid to obtain insights about dynamic conformational changes of proteins. GTP : AMP phosphotransferase from beef heart mitochondria has two Trp residues, no -SH or -S-Sgroups and a M , of about 26000. Two kinds of investigations on its Trp residues have been done in the present studies. a) A chemical conversion of the hydrophobic indole ring into the more hydrophilic oxindole has been performed with N-bromosuccinimide (SucNBr) to obtain a relationship between Trp oxidation and loss of enzymatic activity as a preliminary evaluation of the importance of these residues to the catalytic function. b) Fluorescence in the presence and absence of acrylamide with the intact and SucNBr-modifie...

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“…The solution was subjected to 500 V for 27 h. Sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis was run according to Laemmli [12]. The equilibrium dialysis technique [8] was used to study the interaction of the enzyme with its substrates.…”

Section: Bacteriamentioning

confidence: 99%

ATP‐AMP phosphotransferase from Paracoccus denitrificans

Yeh

Tomasselli

Noda

1983

European Journal of Biochemistry

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Adenylate kinase has been extracted from Paracoccus denitrificans by toluene treatment and purified 370-fold (overall yield of 44%, 554 U/mg, M , 22000, PI 4.7; pH optimum 7.5-8.0) by using successive column chromatography on DEAE-cellulose DE-52, Matrex-Blue A and Sephadex G-75. The enzyme was homogeneous by dodecylsulfate gel electrophoresis and constancy of specific activity across the single peak found in Sephadex G-75 chromatography. Crystals in the form of heavy needles have been obtained in poly(ethy1ene glycol)-400. The enzyme is specific for adenine (deoxyadenine) nucleotides with K,,, values for ATP, ADP and AMP of 340 pm, 980 pM and 93 pM respectively and dissociation constants, for ATP, MgATP and AMP of 7.3 IM, 7.1 pM and 6.9 pM respectively. The enzyme was noted to have no disulfide bond, two free sulfhydryl groups and a total of 208 residues. Aboe 50 mM KCl the enzyme activity drops off gradually. The enzyme is stable at neutral pH and below a temperature of 44 "C. Adenylate kinase from P. denitrificans resembles adenylate kinases from other sources with respect to such properties as the dependence on specific divalent cations, maximal activities at a ratio of Mg2+ : ATP of about 1 : 1 and the ratio of Mg2+ :ADP of 1 : 2 , in having MgATP (or MgADP) binding site and another substrate binding site for AMP (or ADP), and in requiring an intact histidine and one or more arginine residues for enzymatic activity.Adenylate kinase from P. denirrificans appears to resemble the so-called muscle-type (cytoplasmic) rather than the mitochondrial-type adenylate kinase by comparisons of the following properties: low molecular weight, having -SH groups and no disulfide bonds, pH-optimum of 8.0 for formation of ADP, positive cross-reaction by ELISA test and in requiring a low concentration of Ap,A to inhibit 95 % activity.Adenylate kinases are ubiquitous small monomeric intracellular enzymes ( M , 20000-32000) which catalyze the interconversion of adenine nucleotides according to the equation, MgATP + AMP + MgADP + ADP. The enzyme has been purified and characterized from a number of sources [l] and primary and tertiary structures of porcine and human adenylate kinases have been published [2-41. The catalytic mechanism of adenylate kinase and the detailed role(s) of this enzyme in vivo have not yet been elucidated. One approach to these problems is studying adenylate kinase (and functionally related enzymes) from different sources in order to take advantage of specific isozymes carrying special feature(s). In this paper we report the purification and characterization of adenylate kinase from Paracoccus denitrijkans and compare it with several other adenylate kinases. The microorganism is a simple bacterium having many mitochondria-like characteristics which are otherwise more randomly distributed among bacteria [5]. Adenylate kinase from P. denitrificans is of further interest because of its similarity with the enzyme from muscle (cytoplasm) rather than with the enzyme from mitochondria.Abbreviations. ApsA, adenosi...

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Mitochondrial GTP‐AMP Phosphotransferase

Cited by 69 publications

References 30 publications

A GTP:AMP Phosphotransferase, Adk2p, in Saccharomyces cerevisiae

A GTP:AMP Phosphotransferase, Adk2p, in Saccharomyces cerevisiae

Fluorescence and chemical modification of tryptophan as probes of structure of GTP:AMP phosphotransferase from beef heart mitochondria

ATP‐AMP phosphotransferase from Paracoccus denitrificans

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