Adenosine kinase from bovine adrenal medulla

Abstract

Adenosine kinase from bovine adrenal medulla was purified 1600-fold by using ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration yielded a relative molecular mass around 42 000 and Michaelis constants were 0.2 pM for adenosine and 20 pM for MgATP. The enzyme showed a broad specificity for purine nucleoside triphosphate as phosphate donors. Both free MgZ + and ATP were inhibitors. AMP was a competitive inhibitor with regard to adenosine and a non-competitive inhibitor versus MgATP, while ADP was a uncompetitive inhibitor with regard to adenosine and a non-competitive inhibitor versus MgATP.Adenosine kinase was strongly inhibited by the bis(adenyly1) polyphosphates Ap,A and Ap,A. These compounds inhibited the enzyme competitively versus MgATP (Ki = 0.06 pM for Ap4A and 0.4 pM for Ap,A) and uncompetitively with regard to adenosine. The results of the kinetic analysis suggest an ordered bi-bi mechanism, adenosine being the first substrate. The phosphorylation of adenosine was unaffected in the presence of vanadate ions.The catalysis of adenosine phosphorylation to AMP by adenosine kinase was first described in yeast [l] but its presence has been firmly established in a great variety of mammaiian cells [2 -51. The use of affinity Chromatography on AMP linked to agarose or Sepharose beads allowed highly purified mammalian adenosine kinase preparations, including human, to be obtained [6 -Adenosine kinase plays a key role in the regulation of intracellular levels of adenosine [12]. This enzyme can use a number of adenosine analogues as substrates and this property has been exploited in the field of antineoplasic chemotherapy [4, 131; in fact, the observed resistance of some tumoural cell types to these drugs are correlated with decreased levels of adenosine kinase [14, 151. Nevertheless, other adenosine analogues, such as those found in some marine organisms [16] or chemically synthesized [17] are potent inhibitors of kinase activity. All these compounds might be useful tools in furthering our understanding of the roles of adenosine and adenosine kinase in normal and certain pathological states, as well as in the therapeutic approach.The chromaffin cells from adrenal medulla store, in their secretory granules, catecholamines and ATP at high concen- catecholamines and the remaining soluble intragranular content in response to cholinergic stimulation of the adrenal medulla. Evidence has been obtained that intragranular ATP may be labelled by exogenous radioactive adenosine in experiments of perfusion of whole adrenal gland [21, 221. On the other hand, isolated or cultured chromaffin cells readily incorporate extracellular adenosine into their nucleotide pools [23, 241. These findings suggest the involvement of adenosine kinase in the mechanisms of purine ring replenishment in the chromaffin cells, but to our knowledge no studies on adenosine kinase have been performed in the adrenal medulla. In this paper, purification and properties of adenosine kinase from bovine adrenal medull...

Structure of Human Adenosine Kinase at 1.5 Å Resolution,

“…Since the adenosine 1 binding site is significantly more buried and further from the solvent interface than adenosine 2, it seems reasonable to postulate that, without invoking a protein conformational change, adenosine binds prior to ATP. This is consistent with most of the earlier kinetic studies (21)(22)(23)(24) but not all (20).…”

“…Studies with partially purified adenosine kinase from Ehrlich ascites tumor cells identified ATP as the first substrate to bind and AMP as the last product to be released (20). Other studies, however, predicted adenosine to be the first substrate to bind (21)(22)(23)(24) while still others predicted ADP to be the last substrate to leave (15).…”

Identification and Characterization of a Unique Adenosine Kinase from Mycobacterium tuberculosis

“…Therefore, the loss of activity appears to be a specific effect of Mg 2ϩ and not an effect of ionic strength. The ATP/Mg ratio plays a critical role in regulating activity for AKs from various sources (21,23,25,34), and M. tuberculosis AK assays were typically performed with an ATP/Mg ratio of 1:2.…”

Adenosine‐AMP exchange activity is an integral part of the mammalian adenosine kinase

“…In 1972 Henderson et al, (1) proposed an order Bi Bi mechanism for AK in which ATP was the first substrate to bind and AMP the last product to be released. Later studies with purified enzyme from a variety of sources, corroborated an ordered Bi Bi mechanism, but they suggested adenosine as the first substrate to bind and AMP the last product to dissociate (8,9,11). In contrast to the above studies, Chang et al, (10) (12) have suggested that the catalytic mechanism of AK involves an ordered Bi Bi reaction in which ATP binds first to the enzyme and ADP is released last.…”

“…1.20) catalyzes the phosphorylation of adenosine and numerous other purine nucleoside analogs into corresponding monophosphates, using either ATP or GTP as the preferential phosphoryl group donors (see [1][2][3][4][5]. Although AK has been purified from a variety of sources (see [1][2][3][4][5][6][7][8][9][10], its kinetic mechanism remains controversial (1,8,(10)(11)(12) and has not yet been firmly established.…”