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Query: UNIPROT:P06889 (Mol)
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The thermal stability of adenylate kinase from the thermoacidophilic archaeon Sulfolobus acidocaldarius was characterized comprehensively using denaturant-induced unfolding, differential scanning calorimetry, circular dichroism spectroscopy, and enzymological inactivation studies. The thermally induced unfolding of the protein is irreversible due to aggregation, whereas the unfolding induced by guanidinium chloride is reversible. The protein is known to be a homotrimer in its native state and we established that it unfolds upon dissociation in the case of denaturant unfolding. We measured the thermodynamic stability of the protein in a temperature range from 5 to 70 degrees C using denaturant unfolding. The protein has a maximum of stability (intrinsic free energy) of 31 kcal/mol-trimer (130 kJ/mol-trimer) at 32 degrees C (based on the linear extrapolation model). The heat capacity change upon unfolding DeltaCp and the m-value were considered to be constant in this temperature range and calculated to be 2.86 kcal/mol-trimer (11.9 kJ/mol-trimer) and 5.67 kcal/mol-trimer M (23.7 kJ/mol-trimer M), respectively. The influence of trimerization on thermodynamic stability was investigated. The several interrelated aspects of thermal stability such as unfolding kinetics, the temperature-dependence of the free energy, and the concentration and temperature-dependencies of the fraction of denatured protein are described quantitatively. The properties of the Gibbs-Helmholtz function of the adenylate kinase from S. acidocaldarius, in particular, and of oligomeric proteins, in general terms, are discussed and compared with the properties of the analogous function for monomeric proteins. Moreover, we discuss methodological aspects: we obtained the analytical expression of the denaturant-unfolding isotherm for homotrimeric proteins; we include a formula Appendix containing the derivations of the expressions used.
J Mol Biol 1998 Dec 04
PMID:Thermodynamics and kinetics of unfolding of the thermostable trimeric adenylate kinase from the archaeon Sulfolobus acidocaldarius. 982 18

Site-directed mutagenesis of human adenylate kinase (AK) was carried out on residues His36, Lys55, and the C-terminal segment (Val182, V186, and Leu193). Five mutants [(H36T, K55G, V182G, V186S, and L193Stop (deletion of residues 193-194)] were generated and analyzed by steady-state kinetics. H36T, K55G, and L193Stop mutants showed an increase of K(m) values (19.8-, 19.7-, and 11.3-fold) for AMP2- compared to that for the wild-type enzyme, and these residues appeared to interact with AMP2-. V182G showed an increased K(m) value (7.4-fold) for MgATP2-. Therefore, V182 may be essential for interaction with MgATP2-. V186S increased the K(m) value (7.0- and 7.5-fold) for MgATP2- and AMP2-. V186 may thus interact with both substrates. The C-terminal domain of AK appears to be essential for MgATP2- and AMP2- binding.
Biochem Mol Biol Int 1998 Nov
PMID:Site-directed mutagenesis and steady-state kinetic analysis of mutant enzymes of human adenylate kinase. 984 27

Use was made of mitochondria isolated from heart left ventricles of either spontaneously hypertensive or age-matched Wistar-Kyoto rats used as a control to find out whether hypertrophy (5-week-old rats) or hypertrophy/hypertension (24-week-old rats) can cause change in the mechanisms by which ATP is synthesised via ATP synthase and subsequently exported via the ADP/ATP translocator outside mitochondria. To do this, photometric measurements were made of the rate of ATP appearance in the extramitochondrial phase, which occurs as a result of ADP addition to mitochondria. In mitochondria from spontaneously hypertensive rats deficit of ATP production was found dependent on changes in the KmADP and Vmax values of both the ADP/ATP translocator and the ATP synthase. The ADP/ATP translocator was found to determine the rate of ATP production outside mitochondria in all the tested samples. In an initial investigation carried out to ascertain how cell ATP deficit can be counterbalanced, an increase in both adenylate kinase and creatine kinase activities was found in both hypertrophy and hypertrophy/hypertension. A possible increase in anaerobic glycolysis was also suggested by the increased lactate dehydrogenase activity.
Int J Mol Med 1998 Apr
PMID:ATP synthesis and export in heart left ventricle mitochondria from spontaneously hypertensive rat. 985 86

Muscle deconditioning is a common observation in patients with congestive heart failure (CHF), chronic obstructive pulmonary disease, neuromuscular diseases or prolonged bed rest. To gain further insight into metabolic and mechanical properties of deconditioned slow-twitch (soleus) or fast-twitch (EDL) skeletal muscles, we induced experimental muscle deconditioning by hindlimb suspension (HS) in rats for 3 weeks. Cardiac muscle was also studied. Besides profound muscle atrophy, increased proportion of fast type II fibers as well as fast myosin isoenzymes, we found decreased calcium sensitivity of Triton X-100 skinned fiber bundles of soleus muscle directed towards the fast muscle phenotype. Glycolytic enzymes such as hexokinase and pyruvate kinase were increased, and the LDH isoenzyme pattern was clearly shifted from an oxidative to an anaerobic profile. Creatine kinase (CK) and myokinase activities were increased in HS soleus towards EDL values. Moreover, the M-CK mRNA level was greatly increased in soleus, with no change in EDL. However, oxygen consumption rate assessed in situ in saponin skinned fibers (12.5 +/- 0.8 in C and 15.1 +/- 0.9 micromol O2/min/g dw in HS soleus compared to 7.3 +/- 1.3 micromol O2/min/g dw in control EDL), as well as mitochondrial CK (mi-CK) and citrate synthase activities, were preserved in HS soleus. Following deconditioning no change in Km for ADP of mitochondrial respiration, either in the absence (511 +/- 92 in C and 511 +/- 111 microM in HS soleus compared to 9 +/- 4 microM in control EDL) or presence of creatine (88 +/- 10 in C and 95 +/- 16 microM in HS soleus compared to 32 +/- 9 microM in control EDL), was found. The results show that muscle deconditioning induces a biochemical and functional slow to fast phenotype transition in myofibrillar and cytosolic compartments of postural muscle, but not in the mitochondrial compartment, suggesting that these compartments are differently regulated under conditions of decreased activity.
J Mol Cell Cardiol 1998 Nov
PMID:Muscle unloading induces slow to fast transitions in myofibrillar but not mitochondrial properties. Relevance to skeletal muscle abnormalities in heart failure. 992 74

A chromosomal DNA sequence harboring a processed AK2B pseudogene was isolated from a human genomic library. It was a variant of the AK2B gene sequence including several point mutations, deletions, and insertions. The nucleotide sequence of the ORF of the AK2B pseudogene predicted a truncated form of the AK2B mutant suggesting that the processed pseudogene is nonfunctional. A repetitive sequence, AAAAGAGAG, found in the 5' and 3' flanking regions of the pseudogene and the poly(A) tract in the 3' end junction suggest that a mRNA of AK2B may have been converted to the processed pseudogene by retrotransposition events. Previously, it was suggested that an adenylate kinase (AK) 2 related gene on chromosome 2, confirmed by Southern analysis using somatic cell hybrid cell lines, may be a processed pseudogene. It is proposed that the processed pseudogene isolated in this study may be the AK2 related nonfunctional gene localized on human chromosomes 2.
Biochem Mol Biol Int 1999 Jan
PMID:Cloning of the genomic sequence encoding a processed adenylate kinase 2 pseudogene. 1009 43

The catalytic mechanisms of adenylate kinase, guanylate kinase, uridylate kinase, and cytidylate kinase are reviewed in terms of kinetic and structural information that has been obtained in recent years. All four kinases share a highly related tertiary structure, characterized by a central five-stranded parallel beta-sheet with helices on both sides, as well as the three regions designated as the CORE, NMPbind, and LID domains. The catalytic mechanism continues to be refined to higher levels of resolution by iterative structure-function studies, and the strengths and limitations of site-directed mutagenesis are well illustrated in the case of adenylate kinase. The identity and roles of active site residues now appear to be resolved, and this review describes how specific site substitutions with unnatural amino acid side-chains have proven to be a major advance. Likewise, there is mounting evidence that phosphoryl transfer occurs by an associative transition state, based on (a) the stereochemical course of phosphoryl transfer, (b) geometric considerations, (c) examination of likely electronic distributions, (d) the orientation of the phosphoryl acceptor relative to the phosphoryl being transferred, (e) the most likely role of magnesium ion, (f) the lack of restricted access of solvent water, and (g) the results of oxygen-18 kinetic isotope. effect experiments.
Adv Enzymol Relat Areas Mol Biol 1999
PMID:Nucleoside monophosphate kinases: structure, mechanism, and substrate specificity. 1021 7

Cilia of Tetrahymena thermophila possess adenylate kinase [ATP:AMP phosphotransferase, EC 2.7.4.3] activity. More than 95% of the total activity was recovered in the axonemal fraction when cilia were demembranated with 0.2% Nonidet P-40. There was no loss of the specific activity of adenylate kinase when axonemes were thoroughly washed with HMEK solution (10 mM HEPES, 5 mM MgCl2, 0.1 mM EDTA, and 0.1 M KCl, pH 7.4). These results suggest that adenylate kinase is tightly bound to axoneme. Solubilization of adenylate kinase was markedly increased when axonemes were incubated in HME buffer (10 mM HEPES, 1 mM MgCl2, 0.1 mM EDTA, pH 7.4) containing concentrations of NaCl (or KCl) exceeding 1 M. Therefore, routine isolation of adenylate kinase from axonemes involved pre-extracting axonemes with 0.5 M NaCl in HME buffer followed by extraction in HME buffer containing 1.5 M NaCl. Native-gel electrophoresis of the high salt extract revealed two protein bands (band I and band III). An active staining for adenylate kinase showed a single active band corresponding to the position of band III. Two-dimensional gel electrophoresis using native-gel electrophoresis in the first dimension and SDS-PAGE in the second dimension suggests that band III protein contains at least nine polypeptides ranging from 21 to 110 kDa.
Comp Biochem Physiol B Biochem Mol Biol 1999 Oct
PMID:Adenylate kinase is tightly bound to axonemes of Tetrahymena cilia. 1058 2

Coupling of ATP-generating with ATP-consuming processes is an essential component in the cardiac bioenergetics responsible for optimal myocardial function. Although a number of enzymatic systems have been implicated in securing proper intracellular energy communication, their integrative response in a failing myocardium has not been determined so far. Therefore, we measured catalytic activities of enzymes responsible for the communication between ATP-generating and ATP-consuming processes in ventricular samples obtained from normal dogs and dogs with tachycardia-induced heart failure. In the failing myocardium, phosphotransfer activities of creatine kinase, adenylate kinase, 3-phosphoglycerate kinase and pyruvate kinase, which collectively deliver ATP and remove ADP from myofibrillar ATPases, were depressed by 30, 21, 44 and 20%, respectively, when compared to normal controls. The activity of hexokinase, an enzyme which directs phosphoryls into the glycolytic phosphotransfer pathway, was unchanged. Also, the activity of glyceraldehyde-3-phosphate dehydrogenase, which may shuttle inorganic phosphate between ATPases and ATP-synthases, was not affected by heart failure. However, the CO2-hydration activity of carbonic anhydrase, which together with creatine kinase, is presumed responsible for removal of protons from ATPases, was diminished by 21%. As these enzymatic systems are collectively required for adequate delivery of high-energy phosphoryl to, and removal of end-products from, cellular ATPases, the cumulative deficit in their flux capacities may provide a bioenergetic basis for impaired contraction-relaxation in the failing heart.
Mol Cell Biochem 1999 Nov
PMID:Reduced activity of enzymes coupling ATP-generating with ATP-consuming processes in the failing myocardium. 1063 Jun 20

A method for determination of transient (on the millisecond timescale) intramolecular distance distributions (IDDs) by time-resolved dynamic non-radiative excitation energy transfer measurements was developed. The time-course of the development of the IDD between residues 73 and 203 in the CORE domain of Escherichia coli adenylate kinase throughout refolding from the GuHCl-induced denatured state was determined. The mean of the apparent IDD reduced to a value close to its magnitude in the native protein, within 2 ms (the dead-time of the instrument). At that time the width of that distribution was rather large (16+/-2 A). The large width implies that the intramolecular diffusion coefficient of the labeled segment does not exceed 10(-7) cm(2)/second. In a second slower phase of the refolding transition, the width was reduced to its native value (6+/-4 A).
J Mol Biol 2000 Jun 23
PMID:Determination of intramolecular distance distribution during protein folding on the millisecond timescale. 1087 59

Burkholderia cepacia is an emerging opportunistic pathogen that causes fatal infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Various environmental isolates of B. cepacia are, however, capable of degrading environmental pollutants, such as trichloroethylene, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), etc., and are also highly effective in controlling plant diseases caused by nematodes and fungi. Such strains have therefore been proposed for environmental release to clean up toxic dump sites or as biopesticides. Various efforts to distinguish between clinical and environmental isolates of B. cepacia with regard to their virulence characteristics have produced ambiguous results, suggesting that newer methods are needed to test for the presence or absence of pathogenic potential in B. cepacia strains proposed for environmental release. We now report that several clinical strains of B. cepacia secrete cytotoxic factors that allow macrophage and mast cell death in the presence of external ATP. Several environmental strains had reduced activity in this regard. We also demonstrate that, while all the strains secrete enzymes that have nucleoside diphosphate kinase (Ndk), adenylate kinase (Ak) and 5'-nucleotidase activity, the level of secretion of the 5'-nucleotidase (and/or ATPase/phosphatase) appears to be lower in the environmental strains than in the clinical strains. The secretion of these enzymes is specifically activated in the presence of eukaryotic proteins such as alpha2-macroglobulin. As macrophage-or mast cell surface-associated P2Z receptors promote their cell death in the presence of mM concentrations of ATP, and as the secreted ATP-using enzymes generate various phosphorylated or non-phosphorylated adenine nucleotides that may even be better agonists than ATP in activating the P2Z receptors or may act through the activation of additional purinergic receptors, such enzymes may play an important role in allowing B. cepacia to evade host defence.
Mol Microbiol 2000 Jun
PMID:Clinical and environmental isolates of Burkholderia cepacia exhibit differential cytotoxicity towards macrophages and mast cells. 1093 Dec 97


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