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- Brodelius, Peter, et al.
(författare)
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Determination of Dissociation Constants for Binary Dehydrogenase-Coenzyme Complexes by (Bio)Affinity Chromatography on an Immobilized AMP-Analogue
- 1976
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Ingår i: Analytical biochemistry. - : Elsevier BV. - 0003-2697. ; 72:1-2, s. 629-636
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Tidskriftsartikel (refereegranskat)abstract
- Various alcohol and lactate dehydrogenases were adsorbed to an affinity column of immobilized N6-(6-aminohexyl)-AMP and subsequently eluted with gradients of coenzymes or coenzyme fragments. A linear relation was observed between the eluting concentration of nucleotide and the reported dissociation constants for the corresponding binary enzyme-nucleotide complexes. This relation has been utilized to determine unknown dissociation constants by affinity chromatography. The method presented can also be utilized for the estimation of dissociation constants betweendehydrogenases and coenzyme analogues.
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| 6. |
- Brodelius, Peter, et al.
(författare)
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Studies of Bovine Liver Glutamate Dehydrogenase by Analytical Affinity Chromatography on Immobilized AMP Analogs
- 1979
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 194:2, s. 449-456
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Tidskriftsartikel (refereegranskat)abstract
- Bovine liver glutamate dehydrogenase has been studied by analytical affinity chromatography on two immobilized AMP analogs, i.e., N6-(6-aminohexyl)-AMP and 8-(6-aminohexyl)-amino-AMP. The existence of various enzyme-coenzyme and enzyme-effector complexes has been verified. Also the cooperative formation of two ternary complexes, i.e., glutamic dehydrogenase (GHD)-NADP-glutamate and GDH-ADP-leucine, has been shown. The results of this study have been rationalized by the “ligand exclusion theory.” which has been proposed for the regulation of the glutamic dehydrogenase. It has been shown that the active site and the ADP-binding effector site are oriented close to each other on the enzyme. Furthermore, the data suggest that the adenylic site is not identical to the nonactive coenzyme binding site. A mechanism based on electrostatic interactions is suggested for the cooperative binding of oxidized coenzyme and substrate. Dissociation constants for complexes between the enzyme and two coenzyme fragments (P-ADPR and 2′,5′-ADP) have been estimated.
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| 7. |
- Brodelius, Peter, et al.
(författare)
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The Synthesis of 8-(6-Aminohexyl)-Amino-GMP and Its Applications as a General Ligand in Affinity Chromatography
- 1978
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 188:1, s. 228-231
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Tidskriftsartikel (refereegranskat)abstract
- The synthesis of a new general ligand, i.e., 8-(6-aminohexyl)-amino-GMP, has been achieved by bromination of GMP and subsequent substitution of the bromine for hexamethylene diamine. The overall yield of the synthesis has been 60 to 70%. This new general ligand was immobilized on BrN-activated Sepharose and used as an affinity adsorbent for inosinic acid dehydrogenase (E.C. 1.2.1.14) from Aerobacter aerogenes. Various elution methods were investigated in order to increase the specific activity of the purified enzyme.
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| 8. |
- 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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- Höjeberg, B, et al.
(författare)
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Affinity Chromatography and Binding Studies on Immobilized Adenosine 5'-Monophosphate and Adenosine 2'.5'-Bisphosphate of Nicotinamide Nucleotide Transhydrogenase from Pseudomonas aeruginosa
- 1976
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 66:3, s. 467-475
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Tidskriftsartikel (refereegranskat)abstract
- Nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa was purified to apparent homogeneity with an improved method employing affinity chromatography on N6-(6-aminoyl)-adenosine-2′,5′-bisphosphate-Sepharose 4B. Polyacrylamide gel electrophoresis of the purified transhydrogenase carried out in the presence of sodium dodecyl sulphate, indicated a minimal molecular weight of 55000 ± 2000. The kinetic and regulatory properties of the purified transhydrogenase resembled those of the crude enzyme, i.e., NADPH, adenosine 2′-monophosphate and Ca2+were activators whereas NADP+was inhibitory. Nicotinamide nucleotide-specific release of binding of the transhydrogenase to N6-(6-aminohexyl)-adenosine-2′,5′-bisphosphate-Sepharose and N6-(6-aminohexyl)-adenosine-5′-monophospliate-Sepharose suggests the presence of at least two separate binding sites for nicotinamide nucleotides, one that is specific for NADP(H) and one that binds both NAD(H) and NADP(H). Binding of transhydrogenase to N6-(6-aminohexyl)-adenosine-2′,5′-bisphosphate-Sepharose and activation of the enzyme by adenosine-2′,5′-bisphosphate showed a marked pH dependence. In contrast, inhibition of the Ca2+-activated enzyme by adenosine 2′,5′-bisphosphate was virtually constant at various pH values. This discrepancy was interpreted to indicate the existence of separate nucleotide-binding effector and active sites.
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