Further purification of adenosine kinase from rat heart using affinity and ion-exchange chromatography

Jong, Jan Willem de; Uitendaal, M.P.; Harmsen, Eef

doi:10.1016/0003-2697(80)90206-7

Cited by 20 publications

(8 citation statements)

References 15 publications

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“…Protein concentrations were determined by using the Bio-Rad protein assay and normalized for all enzymatic determinations. All enzyme assays were conducted according to the radiometric methods described previously (17,18). AK reactions were performed in a 50-l volume at 37°C in TMD buffer with 1 mM ATP, 5 mM NaF (to inhibit phosphatase activity), 25 Ci of [ 3 H]adenosine (48 Ci mmol Ϫ1 ), and appropriate volumes of crude parasite lysate.…”

Section: Methodsmentioning

confidence: 99%

Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii

Chaudhary

Darling

Fohl

et al. 2004

Journal of Biological Chemistry

106

104

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We have exploited a variety of molecular genetic, biochemical, and genomic techniques to investigate the roles of purine salvage enzymes in the protozoan parasite Toxoplasma gondii. The ability to generate defined genetic knockouts and target transgenes to specific loci demonstrates that T. gondii uses two (and only two) pathways for purine salvage, defined by the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK). Both HXGPRT and AK are single-copy genes, and either one can be deleted, indicating that either one of these pathways is sufficient to meet parasite purine requirements. Fitness defects suggest both pathways are important for the parasite, however, and that the salvage of adenosine is more important than salvage of hypoxanthine and other purine nucleobases. HXGPRT and AK cannot be deleted simultaneously unless one of these enzymes is provided in trans, indicating that alternative routes of functionally significant purine salvage are lacking. Despite previous reports to the contrary, we found no evidence of adenine phosphoribosyltransferase (APRT) activity when parasites were propagated in APRT-deficient host cells, and no APRT ortholog is evident in the T. gondii genome. Expression of Leishmania donovani APRT in transgenic T. gondii parasites yielded low levels of activity but did not permit genetic deletion of both HXGPRT and AK. A detailed comparative genomic study of the purine salvage pathway in various apicomplexan species highlights important differences among these parasites.Like all parasitic protozoa, the obligate intracellular parasite Toxoplasma gondii lacks the ability to synthesize the purine ring de novo, and thus relies entirely on the salvage of purines from the host cell to meet its nutritional needs (1-3). This requirement, coupled with the shortcomings of conventional therapies for treating congenital toxoplasmosis and opportunistic infections associated with AIDS and other immunosuppressive conditions (4 -8), makes purine salvage an attractive target for chemotherapy.The purine metabolism of T. gondii has previously been examined biochemically, resulting in the identification of various activities capable of assimilating nucleosides and nucleobases from the host cell into the purine nucleotide pools of the parasite (2, 3). (See "Discussion" for a model of the purine salvage pathway in Toxoplasma and other apicomplexan parasites.) Reported salvage activities include the phosphoribosylation of adenine, guanine, hypoxanthine, and xanthine, and the phosphorylation of adenosine. The latter seems to contribute most significantly to parasite purine economy, as adenosine is incorporated into nucleotide pools at a considerably higher rate than any purine nucleobase (2, 3).Most of the reported salvage activities can be accounted for by two enzymes: hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) 1 and adenosine kinase (AK). The genes for both have been cloned and expressed in bacterial systems, and the purified proteins have be...

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Section: Methodsmentioning

confidence: 99%

Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii

Chaudhary

Darling

Fohl

et al. 2004

Journal of Biological Chemistry

106

104

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“…Purification of adenosine kinase Rat heart adenosine kinase was partially purified by a combination of gel filtration (Namm & Leader, 1974) and affinity chromatography (De Jong et al, 1980). During preliminary work it was observed that considerable loss of enzyme activity occurred when adenosine kinase was present in a solution containing a low protein concentration.…”

Section: Materials and Methods Animals Chemicals And Enzymesmentioning

confidence: 99%

“…During the elution of the heart extract from the gel-filtration column, the passing of the void volume was monitored by the activity of an ATPase that was found to be eluted with the excluded protein fraction. The included protein fraction of the column, which contained the activity of adenosine kinase, was then passed directly from the gelfiltration column on to the affinity column (1 cm x 7cm) containing 5'-AMP-Sepharose 4B (De Jong et al, 1980). After the elution of the included protein fraction through the coupled column system, the affinity column was disconnected, and unbound protein present in the column was washed out.…”

Section: Materials and Methods Animals Chemicals And Enzymesmentioning

confidence: 99%

Properties of rat heart adenosine kinase

Fisher¹,

Newsholme²

1984

Biochemical Journal

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Adenosine kinase was purified 870-fold from rat heart by a combination of gel filtration and affinity chromatography. The preparation was free of purine-metabolizing enzymes that could interfere in the assay of the kinase. A study of the properties of the purified enzyme showed that it is activated by Na+ and K+, it possesses a broad pH optimum between 6 and 8, MgATP is the nucleotide substrate, free Mg2+ is an inhibitor with respect to both MgATP and adenosine, and the enzyme is subject to substrate inhibition by adenosine. The severity of this inhibition increases as the concentration of free Mg2+ increase. The Km for MgATP was calculated to be 0.8 mM and that for adenosine, at likely physiological concentrations of MgATP and free MgCl2, was about 0.2 microM. In vivo the enzyme is likely to be saturated with both MgATP and adenosine. Indeed, the adenosine concentration in rat heart in vivo is probably sufficient to cause substrate inhibition, and this would be increased by an increase in free Mg2+ concentration. Changes in the concentrations of adenosine and free Mg2+ may play a role in modifying the activity of the enzyme in vivo.

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“…The activity of AK was measured with the radiometric method described by De Jong et al [13]. There was no difference in results obtained with fresh and frozen (for < 2 days) supernatant fluids.…”

Section: Determination Of Enzyme Kineticsmentioning

confidence: 99%

Kinetics of adenylate metabolism in human and rat myocardium

Tavenier

Składanowski

Abreu

et al. 1995

Biochimica et Biophysica Acta (BBA) - General Subjects

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A bstractPathways producing and converting adenosine have hardly been investigated in human heart, contrasting work in other species. We compared the kinetics of enzymes associated with purine degradation and salvage in human and rat heart cytoplasm assaying for adenosine deaminase, nucleoside phosphorylase, xanthine oxidoreductase, AMP deaminase, AMP-and IMP-specific 5'-nucleotidases, adenosine kinase and hypoxanthine guanine phosphoribosyltransferase (HGPRT). Xanthine oxidoreductase was not detectable in human heart. The /Tm-values of the AMP-catabolizing enzymes were 2-5 times higher in human heart; the substrate affinity of the other enzymes was in the same order of magnitude in both species. The maximal activity (Vmax) of adenosine kinase was the same in both species, but HGPRT in man was only 12% of that in the rat. For human heart the VLax-values of adenosine deaminase, nucleoside phosphorylase, AMP-and IMP-specific 5'-nucleotidases, and AMP deaminase were 25-50% of those for rat heart. We conclude that human heart is less geared to purine catabolism than rat heart as is evident from the lower activities of the catabolic enzymes. Maintenance of the nucleotide pool may thus play a more important role in human heart.

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Further purification of adenosine kinase from rat heart using affinity and ion-exchange chromatography

Cited by 20 publications

References 15 publications

Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii

Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii

Properties of rat heart adenosine kinase

Kinetics of adenylate metabolism in human and rat myocardium

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