Mycobacterial adenosine kinase is not a typical adenosine kinase

Adenosine kinase (AK) is only found in eukaryotes. Recently, a Mycobacterium tuberculosis (MTub) protein exhibiting greater sequence similarity to ribokinases (RK) was identified as AK. We have expressed AKs from MTub, human and Chinese hamster (CH) cells in Escherichia coli and also AK from human and MTub in AK-deficient CH cells. While both E. coli and CH cells expressing mammalian AKs efficiently metabolized various adenosine analogs, those expressing MTub-AK were completely inactive. The AK activity of the MTub protein was very low (50-fold lower than E. coli RK) and it was not stimulated by phosphate or inhibited by several AK inhibitors. These results raise questions over MTub protein’s true function and whether it functions as AK in cells.     

Halobacterial adenosine triphosphatases and the adenosine triphosphatase from Halobacterium saccharovorum

Abstract Membranes prepared from various members of the genus Halobacterium contained a Triton X-100 activated adenosine triphosphatase. The enzyme from Halobacterium saccharovorum was unstable in solutions of low ionic strength (< 3 M NaCl) and maximally active in the presence of 3.5 M NaCl. A variety of nucleotide triphosphates was hydrolyzed. MgADP, the product of ATP hydrolysis, was not hydrolyzed and was a competitive inhibitor with respect to MgATP. The enzyme from H. saccharovorum was composed of at least 2 and possibly 4 subunits. The 83-kDa and 60-kDa subunits represented about 90% of total protein. The 60-kDa subunit reacted with dicyclohexylcarbodiimide (DCCD) when inhibition was carried out in an acidic medium. The significance of the two minor components (28 kDa and 12 kDa) is not established. The enzyme from H. saccharovorum , which differs from previously described halobacterial ATPases, possesses properties of an F₁F₀ as well as an E₁E₂ ATPase.     

Modification of sarcoplasmic reticulum adenosine triphosphatase by adenosine triphosphate magnesium

Lineweaver-Burk plots of Ca²⁺-activated adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum have been determined for a wide range of substrate concentrations. The plots measured at constant Mg²⁺ concentrations are normally nonlinear, but approach linearity either as the sarcoplasmic reticulum ages, or when small quantities of Triton-X100 are added. Titration with N-ethylmaleimide has the same effect on the activity of the ATPase measured either at high or low substrate concentrations. Lineweaver-Burk plots measured under conditions where the Mg²⁺ concentration is varied so as to be always equal to the ATP concentration are linear. These results have been interpreted as evidence that the adenosine triphosphatase has a single active site which uses MgATP as its substrate and which can be modified by free Mg²⁺.     

Cosignaling of adenosine and adenosine triphosphate in hypobaric hypoxia-induced hypothermia

Purines, that is, adenosine and ATP, are not only products of metabolism but are also neurotransmitters. Indeed, purinergic neurotransmission is involved in thermoregulatory processes that occur during normoxia. Exposure to severe hypoxia elicits a sharp decrease in body core temperature (T(CO)), and adenosinergic mechanisms have been suspected to be responsible for this hypothermia. Because ATP per se and its metabolite adenosine could have complex interactions in some neural networks, we hypothesize that both adenosine and ATP are involved in the central mechanism of hypoxia-induced hypothermia. Their role in the thermoregulatory process was therefore investigated in a 24-h hypobaric hypoxia (Fi(O2) = 10%), using CGS-15943, a nonselective antagonist of adenosine receptors, and suramin, an ATP receptor antagonist. T(CO) and spontaneous activity (A(S)) were monitored by telemetry in conscious rats, receiving CGS-15943 (10 mg/kg ip), suramin (7 nmol icv), or both. The same treatments were done in normoxia to evaluate the specificity of their thermoregulatory action observed in hypoxia. Suramin/CGS-15943 treatment blunted the profound hypothermia observed in control rats throughout the hypoxia exposure, whereas CGS-15943 treatment blunted hypothermia during only 3 h, and suramin treatment had no effect. These results suggest that suramin potentiates the CGS-15943 effects and consequently that adenosine and ATP signaling act in synergy. In normoxia, suramin/CGS-15943 induced an increase in T(CO) but to a far lesser extent than observed in hypoxia. Thus it might be suggested that the suramin/CGS-15943 blunting of hypoxia-induced hypothermia would be specific to hypoxia-induced mechanisms.     

Probing the adenosine receptor with adenosine and xanthine biotin conjugates

Biotin-containing analogs of a potent agonist (N6-phenyladenosine) and a potent antagonist (1,3-dipropyl-8-phenylxanthine) of adenosine receptor activity have been synthesized. A spacer chain to the biotin moiety is attached in both cases to the para-position of the phenyl ring. Two biotin conjugates of N6-phenyladenosine differing only in the length of the spacer chain bind to the adenosine receptor and to avidin simultaneously. The shorter-chain derivative was more potent in inhibiting binding of N6-[3H]cyclohexyladenosine to rat cerebral cortical membranes (Ki of 11 nM in the absence of avidin, 36 nM for the avidin complex). Three biotin conjugates of 1,3-dipropyl-8-phenylxanthine bound competitively to the adenosine receptor, but only in the absence of avidin. The results are interpreted in terms of the possible orientation of the ligands at the receptor binding site.     

A putative adenosine kinase family protein possesses adenosine diphosphatase activity

Adenosine kinase is a potential target for development of new types of drugs. The COG1839 family has been defined as “adenosine-specific kinase” family based on structural analysis and the adenosine-binding ability of a family member, PAE2307. However, there has been no experimental evidence with regard to the enzymatic function of this protein family. Here we measured the enzymatic activity of TTHA1091, a COG1839 family protein from Thermus thermophilus HB8. The phosphorylation of adenosine by TTHA1091 was undetectable when ATP or ADP were used as phosphate donor. However, the degradation of ADP to AMP was detected, indicating that this protein possessed adenosine diphosphatase (ADPase) activity. The (ADPase) activity was inhibited by divalent cations and was specific to ADP and CDP. Thus, this study provides the first experimental evidence for the enzymatic function of the “adenosine-specific kinase” family and suggests a need to reexamine its functional annotation.     

Influences of adenosine receptor antagonism on vasodilator responses to adenosine and exercise in adenosine responders and nonresponders.

We previously demonstrated a bimodal distribution of vasodilator responsiveness to adenosine (Ado) infusion in human subjects, despite similar responses to exercise between subgroups [subjects responsive to Ado infusion (Ado responders) and subjects with blunted vasodilator responses to Ado infusion (Ado nonresponders]). (Martin EA, Nicholson WT, Eisenach JH, Charkoudian N, and Joyner MJ. J Appl Physiol 101: 492-499, 2006). A component of this difference was attributed to a larger nitric oxide component of Ado-mediated vasodilation in responders. However, there may also be differences in Ado receptors between these subgroups. We hypothesized that Ado receptor antagonism would reduce vasodilator responsiveness to Ado and exercise only in Ado responders. To test this hypothesis, we compared forearm vasodilation induced by intra-arterial infusion of three doses of Ado to vasodilation during three workloads of forearm handgrip exercise before and after Ado receptor antagonism with aminophylline (Aph) in 19 subjects. In Ado responders, the change in forearm vascular conductance above baseline for the low, medium, and high doses of Ado, respectively, was 93 +/- 16, 140 +/- 14, 194 +/- 18 before Aph and 27 +/- 12, 71 +/- 19, and 134 +/- 34 ml.min(-1).100 mmHg(-1) after Aph (P < 0.05 for low and medium dose before vs. after Aph). For nonresponders, these values were 30 +/- 5, 39 +/- 6, and 78 +/- 9 ml.min(-1).100 mmHg(-1) before Aph (P < 0.05 vs. responders), with no difference after Aph (P > 0.05). We found that Ado receptor blockade significantly inhibited exercise hyperemia only at high workloads in both responders and nonresponders (P < 0.05 before vs. after Aph). We conclude that there may be reduced Ado receptor responsiveness or sensitivity in nonresponders. Furthermore, Ado may play a limited role exercise hyperemia in both subgroups.     

The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation

The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.     

Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles.

The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase.     

Localization of adenosine deaminase and adenosine deaminase complexing protein in rabbit brain

Adenosine deaminase and adenosine deaminase complexing protein have been localized in rabbit brain. Brains fixed in paraformaldehyde or in Clarke's solution were blocked coronally. Blocks from brains fixed in paraformaldehyde were either frozen in liquid nitrogen or embedded in paraffin. Tissue fixed in Clarke's solution was embedded in paraffin. Sections from each block were stained by the peroxidase-antiperoxidase method for adenosine deaminase or complexing protein using affinity-purified goat antibodies. Adenosine deaminase and complexing protein did not co-localize. Adenosine deaminase was detected in oligodendroglia and in endothelial cells lining blood vessels, whereas complexing protein was concentrated in neurons. The subcellular location and appearance of the peroxidase reaction product associated with individual cells was also quite distinctive. The cell bodies of adenosine deaminase-positive oligodendroglia were filled with intense deposits of peroxidase reaction product. In contrast to oligodendroglia, the reaction product associated with most neurons stained for complexing protein was concentrated in granular-appearing cytoplasmic deposits. In some instances, these deposits were clustered about the nuclear membrane. Staining of neurons in the granular layer of cerebellum was an exception. Granule cells were lightly outlined by peroxidase reaction product. Cerebellar islands, also referred to as glomeruli, were stained an intense uniform brown. These results raise the possibility that oligodendroglia and blood vessel endothelia, through the action of adenosine deaminase, might play a role in controlling the concentration of extracellular adenosine in brain. They do not, however, support the suggestion that complexing protein aids in adenosine metabolism by positioning adenosine deaminase on the plasma membrane.     

Radioenzymatic determination of adenosine

Adenosine has been measured at the nanomolar level by an enzymatic radioactive assay. The nucleoside is converted into [U-14C]ribose-labeled inosine via the following reactions: adenosine + H2O----adenine + ribose (adenosine nucleosidase); adenine + [U-14C]ribose 1-phosphate in equilibrium with T[U-14C]ribose-adenosine + Pi (adenosine phosphorylase); [U-14C]ribose-adenosine + H2O----[U-14C]ribose-inosine + NH3 (adenosine deaminase). The radioactivity of inosine, separated by thin-layer chromatography, is a measure of the adenosine initially present.