Open AccessPreparation of Analogues of ATP, ADP and AMP Suitable for Binding to Matrices and the Enzymic Interconversion of ATP and ADP in Solid Phase
Author(s) -
LINDBERC Margareta,
MOSBACH Klaus
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb04089.x
Subject(s) - hexokinase , iodoacetic acid , biochemistry , chemistry , sepharose , atp synthase , atp–adp translocase , carbodiimide , adenine nucleotide , affinity label , enzyme , nucleotide , glycolysis , mitochondrion , inner mitochondrial membrane , gene
Alkylation of ATP with iodoacetic acid at pH 6.5 yielded 1‐carboxymethyl‐ATP which, after alkaline rearrangement, gave N 6 ‐carboxymethyl‐ATP. Condensation of this analogue with 1, 6‐di‐aminohexane in the presence of a water‐soluble carbodiimide generated N 6 ‐[(6‐aminohexyl)carbomoylmethyl]‐ATP in an overall yield of 40% based on the parent nucleotide ATP. The coenzymic activities of both N 6 ‐adenine‐substituted derivatives of ATP were tested with three kinases. Both derivatives showed coenzymic function against hexokinase with the “long” derivative having highest activity (95%) relative to unsubstituted ATP. Their activities towards the other two kinases tested was negligible except with the “long” analogue against glycerokinase (20%). The latter ATP analogue, when bound to Sepharose through its terminal amino group, could be dephosphorylated to the corresponding ADP analogue with soluble hexokinase yielding glucose 6‐phosphate in an enzymic “solid‐phase” fashion. The Sepharose‐bound ADP formed could subsequently be phosphorylated back to ATP using soluble acetate kinase. Sepharose‐ATP preparations were also used in preliminary affinity chromatography studies using citrate synthase. Alkylation of ADP following the above procedure yielded the corresponding ADP analogue, N 6 ‐[(6‐aminohexyl)carbamoylmethyl]‐ADP in an overall yield of 40%. Alkylation of AMP yielded the corresponding N 6 ‐[(6‐aminohexyl)carbamoylmethyl]‐AMP in an overall yield of 45%.