Determination of D-3-hydroxybutyrate dehydrogenase in mouse pancreatic islets with a photokinetic technique using bacterial luciferase

Abstract

A sensitive assay for d-3hydroxybutrate dehydrogenase (EC 1.1.1.30) was developed for use with the minute amounts of material obtained from islets of Langerhans microdissected from freeze-dried pancreatic sections. NADH formed in the enzyme reaction was determined by photokinetic analysis of the luminescence obtained with bacterial luciferase from Achromobacter fishcherii. In this way, accurate determination was obtained with less than 0.1 mug dry weight of islet material. In obese hyperglycemic mice, the islet enzyme had an activity of 4.7 mumoles/min and g dry weight. Optimal enzyme activity was found at pH 8 for the islet enzyme. The enzyme activity was similar in pancreatic islets and acini, while considerably higher activity was found in cardiac muscle, liver and renal cortex. Normal mouse islets showed about equal enzyme activity as the islets from obese hyperglycemic mice.