TY - JOUR
T1 - Ion-exchange column chromatographic method for assaying purine metabolic pathway enzymes
AU - Ward, Herbert
AU - Baldwin, Douglas
AU - Wang, Tingchung
AU - Warner, Huber
AU - Seymour, Katherine
AU - Marquardt, Cathleen
AU - McFalls, Edward
AU - Foker, John E.
PY - 1998/4/10
Y1 - 1998/4/10
N2 - High energy phosphate levels fall rapidly during cardiac ischemia and recover slowly (more than one week) during reperfusion. The slow recovery of ATP may reflect a lack of purine metabolic precursors and/or increased activity of purine catabolic enzymes such as 5'-nucleotidase (5'-NT, EC 3.1.3.5) and adenosine deaminase (ADA, EC 3.5.4.4). The activity of enzymes involved in both the catabolism of ATP precursors (5-NT and ADA) and the restoration of ATP from slow synthetic pathways [adenosine kinase (AK, EC 2.7.1.20), adenine phosphoribosyl transferase (APRT, EC 2.4.2.7) and hypoxanthine phosphoribosyl transferase (HPRT, EC 2.4.2.8)] may directly affect the rate of ATP recovery. Strategies to enhance recovery will depend on the relative activity of these enzymes following ischemia. Their activity in different species and their response to ischemia are not well characterized. Hence, rapid assay methods for these enzymes would facilitate detailed time course studies of their activities in postischemic myocardium. We modified a single ion-exchange column chromatographic method using DEAE- Sephadex to determine the products of incubation of 5'-NT, AK, APRT and HPRT with their respective substrates. The uniformity of the final product measurement procedure for all assays permits the activities of the four enzymes to be rapidly determined in a single tissue sample and facilitates the study of a large number of samples. This technique should also be useful for enzymes of the pyrimidine metabolic pathway.
AB - High energy phosphate levels fall rapidly during cardiac ischemia and recover slowly (more than one week) during reperfusion. The slow recovery of ATP may reflect a lack of purine metabolic precursors and/or increased activity of purine catabolic enzymes such as 5'-nucleotidase (5'-NT, EC 3.1.3.5) and adenosine deaminase (ADA, EC 3.5.4.4). The activity of enzymes involved in both the catabolism of ATP precursors (5-NT and ADA) and the restoration of ATP from slow synthetic pathways [adenosine kinase (AK, EC 2.7.1.20), adenine phosphoribosyl transferase (APRT, EC 2.4.2.7) and hypoxanthine phosphoribosyl transferase (HPRT, EC 2.4.2.8)] may directly affect the rate of ATP recovery. Strategies to enhance recovery will depend on the relative activity of these enzymes following ischemia. Their activity in different species and their response to ischemia are not well characterized. Hence, rapid assay methods for these enzymes would facilitate detailed time course studies of their activities in postischemic myocardium. We modified a single ion-exchange column chromatographic method using DEAE- Sephadex to determine the products of incubation of 5'-NT, AK, APRT and HPRT with their respective substrates. The uniformity of the final product measurement procedure for all assays permits the activities of the four enzymes to be rapidly determined in a single tissue sample and facilitates the study of a large number of samples. This technique should also be useful for enzymes of the pyrimidine metabolic pathway.
KW - 5'-Nucleotidase
KW - Adenine phosphoribosyl transferase
KW - Adenosine kinase
KW - Enzymes
KW - Hypoxanthine phosphoribosyl transferase
KW - Purine
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U2 - 10.1016/S0378-4347(97)00577-X
DO - 10.1016/S0378-4347(97)00577-X
M3 - Article
C2 - 9613962
AN - SCOPUS:0032502946
VL - 707
SP - 295
EP - 300
JO - Journal of Chromatography B: Biomedical Sciences and Applications
JF - Journal of Chromatography B: Biomedical Sciences and Applications
SN - 0378-4347
IS - 1-2
ER -