Eckhard Schlimme

Yeast hexokinase reaction with adenosine 5'-O-triphosphate and adenosine 5'-O-(1-thio-triphosphate) monitored by liquid chromatography.

Yeast hexokinase has been extensively studied but the mechanism of the enzymatic reaction is still a subject of lively discussion [ 1, 2 and cited ref.] . Systematically modified ANP* seem to be useful probes for better understanding of the hexokinase nucleotide interactions in binding and catalysis processes. In previous studies of this kind coupled indicator reactions were used [2, 31. However, direct monitoring of the nucleotide turnover is the best guarantee of true measurements and exact interpretations of results. Therefore, we should like to introduce high efficiency LC into enzymology [4,5] and to demonstrate the application of this direct analytical method in studies with ANP-thiophosphate analogues [6, 71 and hexokinase. Substitution of oxygen by sulfur in the aand @position of the phosphate moiety of the nucleotide may lead to diaisostereomerit forms [6, 81. Using an arbitrary nomenclature, peak 4 in the chromatogram in fig. 1 corresponds to

High-pressure liquid chromatography of modified purine mononucleotides and nucleosides

Abstract The useful application of high-pressure liquid chromatography in biochemical research with adenine nucleotide analogues is demonstrated by using a glass-walled separating column at inlet pressures between 100 and 200 atm. Three different aspects are highlighted: (1) purity control of the adenine nucleotide analogues; (2) direct reaction monitoring by high-pressure liquid chromatography; (3) high-pressure liquid chromatography data and mononucleotide conformation.

Properties of ADP- and ATP-1-N-oxide in the adenine nucleotide translocation in rat liver mitochondria

It has been shown that the adenine nucleotide exchange across the inner mitochondrial membrane is catalyzed by a highly specific carrier [l-3] . In addition to that the role of specific translocation inhibitors like atractyloside [3-51 and bongkrekic acid [6,7] has been investigated. Up to now, only some experimental results have become known about the properties of adenine nucleotide analogues. Adenosine 5’-tetraphosphate, ATPOP; adenosine 5’-O-(1,2methylenediphosphonate), AMPCHz P and other phosphonic acid analogues, adenosine S’-O-(diphosphoimido phosphate) ADPNHP, and dADP have been found to be active in adenine nucleotide exchange to some extent [3,7,8] . A change of the base, though, leads to a total inactivation in the translocation process as reported for guanine-hypoxanthine-, cytosineand uracil-nucleotides [3] . From this point of view it is interesting to investigate the contribution of the adenine base to the carrier binding, with respect to what extent chemical modifications will be tolerated. As far as we are aware no modification of the adenine base with regard to the translocation activity has been reported. This paper describes the role of adenine nucleotideI-IV-oxides (ox’ ADP, ox’ ATP, see formula) in the mitochondrial adenine nucleotide translocation. NH,