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- Brodelius, Peter, et al.
(författare)
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The Synthesis of Three AMP-Analogues: N6-(6-Amino-hexyl)-Adenosine 5'-Monophosphate, N6-(6-Aminohexyl)-Adenosine 2',5'-Bisphosphate, and N6-(6-Aminohexyl)-Adenosine 3',5'-Bisphosphate and Their Application as General Ligands in Biospecific Affinity Chromatography
- 1974
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 47, s. 81-89
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Tidskriftsartikel (refereegranskat)abstract
- Phosphorylation of 6-chloropurine riboside with phosphorus oxychloride and phosphorus trichloride gave a mixture of the two isomers, 6-chloropurine-riboside 2’,5‘-bisphosphate and 6-chloropurine-riboside 3‘,5‘-bisphosphate. Reaction with Iy6-diaminohexane followed by resolution of the isomeric mixture on a Dowex 1-X2 column yielded N6-(6-aminohexyl)-adenosine 2’,5’-bisphosphate and N6-(6-aminohexyl)-adenosine 3’,5‘-bisphosphate.The inhibition of several NADP+-dependent and NAD+-dependent dehydrogenases by N6-(6-aminohexyl)-adenosine 2’,5‘-bisphosphate, N6-(6-aminohexyl)-adenosine 3’,5’-bisphosphate and N6-(6-aminohexyl)-adenosine 5’-monophosphate was examined.These three AMP-analogues were attached to Sepharose 4B by the cyanogen bromide method and the binding of several NAD(P)+-dependent enzymes were investigated. NADP+-dependent enzymes were bound to Sepharose substituted with N6-(6-aminohexyl)-adenosine 2’,5‘-bisphosphate, whereas NAD+-dependent enzymes were not bound under the same conditions. Conversely, when N6-(6-aminohexy1)-adenosine 5‘-monophosphate was used as the immobilised ligand only the NAD+- dependent enzymes were bound, as well as glucose-6-phosphate dehydrogenase showing weak affinity. These observations strongly suggest that these two immobilised analogues represent true biospecific and group-specific adsorbents. The enzymes were eluted with their complementary nucleotides, NAD(H) and NADP(H). These techniques were utilised to purify several NADPf-dependent enzymes from a crude Candida utilis extract by chromatography on the new biospecific adsorbent.
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- Lindberg, Margareta, et al.
(författare)
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A New Immobilized NAD+ Analogue, Its Application in Affinity Chromatography and as a Functioning Coenzyme
- 1973
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Ingår i: Eur J Biochem. - : Wiley. - 0014-2956. ; 40:1, s. 187-193
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Tidskriftsartikel (refereegranskat)abstract
- Alkylation of NAD+ with iodoacetic acid followed by alkaline rearrangement gave N6-carboxymethyl-NAD+. Condensation of this analogue with 1,6-diaminohexane gave the analogue NAD+- N6-[N-(6-aminohexyl)-acetamide]. The coenzymic activity of the two derivatives was tested with malate dehydrogenase, alcohol dehydrogenase and lactate dehydrogenase. The efficiency relative to unsubstituted NAD+ was in the range of 50–100%. NAD+-N6-[N-(6-aminohexyl)-acetamide] was attached to Sepharose 4B by the cyanogen bromide method. The immobilized NAD+ analogue thus obtained, exhibited cofactor activity when tested in a recycling three-enzyme system (malate dehydrogenease-citrate synthase-lactate dehydrogenase). The immobilized NAD+ analogue proved to be and effective ligand in affinity chromatography. Thus a mixture of albumin, alcohol dehydrogenase and lactate dehydrogenase was resolved with good recovery. The enzymes were eluted with NAD+ and NADH, respectively.
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