Fluorometric microassays of adenylate kinase, an enzyme important in energy metabolism.
The adenylate kinase system offers a mechanism for the rapid provision of energy by catalysing the production of ATP from ADP. Fluormetric micromethods were developed for determination of the activity of this enzyme using either formation of ADP or ATP, in each case measured by coupling to suitable dehydrogenase reactions. Both procedures yielded results in good agreement, but when ADP formation was measured an interfering phosphatase splitting of ATP had to be corrected for. Therefore, ADP was preferred as the substrate and its conversion to ATP was determined in a coupled hexokinase-glucose-6-phosphate dehydrogenase reaction yielding stoichiometric amounts of NADPH which were measured by the native fluorescence of this form of the nucleotide. The sensitivity and reproducibility of our micro-method permitted assay of small samples (50-500 ng) such as a layer of cerebellar cortical nerve cells and of insulin producing cells from the islets of Langerhans. Although not reaching the high values in muscle, these cells showed significantly higher activities than parenchymatous cells from the liver and the exocrine pancreas. The sensitivity attained is more than required for assay of clinical fine needle biopsies and is quite satisfactory for detection and estimation of adenylate kinase contaminants in enzyme preparations.
D000244:Adenosine Diphosphate / Q000096:biosynthesis; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000263:Adenylate Kinase / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D005069:Evaluation Studies as Topic; D005470:Fluorometry / Q000379:methods*; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D010770:Phosphotransferases / Q000032:analysis*
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000263:Adenylate Kinase