Abstract
Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate resulted in oxidation of the ribose linked to 3-aminopyridine ring and cleavage of the dinucleotide into 3-aminopyridine and adenosine moieties. These two moieties were separated by thin layer chromatography and were synergistically bound to pigeon liver malic enzyme (EC 1.1.1.40), causing inactivation of the enzyme. The inactivation showed saturation kinetics. The apparent binding constant for the reversible enzyme-reagent binary complex (KI) and the maximum inactivation rate constant at saturating reagent concentration (kmax) were found to be 1.1 +/- 0.02 mM and 0.068 +/- 0.001 min-1, respectively. L-Malate at low concentration enhanced the inactivation rate by lowering the KI value whereas high malate concentration increased the kmax. Mn2+ or NADP+ partially protected the enzyme from the inactivation and gave additive protection when used together. L-Malate eliminated the protective effect of NADP+ or Mn2+. Maximum and synergistic protection was afforded by NADP+, Mn2+ plus L-malate (or tartronate). Oxidized and cleaved 3-aminopyridine adenine dinucleotide phosphate was also found to be a competitive inhibitor versus NADP+ in the oxidative decarboxylation reaction catalyzed by malic enzyme with a Ki value of 4.1 +/- 0.1 microM. 3-Aminopyridine adenine dinucleotide phosphate or its periodate-oxidized cleaved products bound to the enzyme anticooperatively. Oxidized 3-aminopyridine adenine dinucleotide phosphate labeled the nucleotide binding site of the enzyme with a fluorescent probe which may be readily traced or quantified. The completely inactivated enzyme incorporated 2 mol of reagent/mol of enzyme tetramer. The inactivation was partially reversible by dilution and could be made irreversible by treating the modified enzyme with sodium borohydride. This fluorescent compound and its counterpart-oxidized 3-aminopyridine adenine dinucleotide may be a potential affinity label for all other NAD(P)+-dependent dehydrogenases.
Highlights
Treatment of 3-aminopyridine adenine dinucleotide Malic enzyme (malate dehydrogenase (NADP+),EC 1.1.1.40) from pigeon liver catalyzes of the ribose linked to 3-aminopyridine ring and cltehaev-oxidative decarboxylation of L-malate to give COz and age of the dinucleotide into 3-aminopyridine and adepny-ruvate with concomitant reduction of NADP+ to NADPH osine moieties
We introduce another reagent, the periodateoxidized 3-aminopyridine adenine dinucleotide phosphate
The fluorescence of AADP+was retained after oxidation. We found that this reagent is a potentially more convenient affinity label for malic enzyme.We believe that it might be useful for other NADP'-dependent dehydrogenases
Summary
Oxidation and Cleavage of oAADpC by Periodate-13C NMR spectrum of AADP+ showedthe high purity of the commercial product. In the'H NMR spectrum, the hydrogen signals of 3-aminopyridine and adenine were located at 6. Oxidation of AADP' with periodate was expected to result in the cleavage of the bond between C-2' and C-3' of the ribose ring bound to the 3-aminopyridine moiety and formation of a dialdehyde derivative (II in Scheme I). 'H and 13C NMR of oAADP+ did not show any free aldehyde groups at 6 9-11 and 6 180-210, respectively. NMR of oATP-ribose indicated no free aldehyde groups, but the hydrated aldehyde was clearly seen at 6 87.64 and 6. Together with the observations that many other periodate-oxidized nucleotides showed hydrated aldehyde by NMR spectroscopy (Lowe and Beechey, 1982;Borchardt et al, 1978),we suggested that the aldehyde groups of oAADP+existed as hydratedforms in the. 3-aminopyridine moiety; V, the adenosine moiety; VI and VII,dihydroxymorpholino and Schiff base derivatives of the dialdehyde 3-aminopyridine moiety and the enzyme
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