Bioluminescence Procedures for the Measurement of NAD(P) Dependent Enzyme Catalytic Activities in Submicrogram Quantities of Rabbit and Human Nephron Structures

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Abstract
Reduced flavin mononucleotide dependent luciferase (EC 1.14.14.3) from Photobacterium fischeri was used to measure NAD(P) dependent enzymes in submicrogram quantities of tissue homogenates and isolated structures of rabbit and human kidney. The procedure for measuring NAD(P)H was optimized, with internal standardization, to give a linear constant signal between 1 and 100 pmol. This method was applied to the measurement of G-6-P dehydrogenase (EC 1.1.1.49) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) along the various structures of the rabbit nephron, microdissected from fresh tissue slices. Blank and recovery measurements were performed on each structure, and enzyme catalytic activities were calculated on the basis of tubular length and protein. G-6-P dehydrogenase was found to be present in all nephron structures with highest catalytic activities in glomeruli, thin limbs of Henle''s loop and medullary collecting tubules. Lowest catalytic activities were detected in the pars recta of proximal tubules, the distal convoluted and the connecting tubule. The mitochondrial 3-hydroxybutyrate dehydrogenase exhibited a different distribution pattern: highest catalytic activities were found in the cortical ascending limbs of Henle''s loop, the proximal and distal convoluted tubule. An unusual internephron heterogeneity for this enzyme was found in the distal convoluted tubule. Catalytic activities in thin limbs of Henle''s loop, the medullary ascending limb, and the cortical and medullary collecting tubule were not significantly different from blank activities. The results obtained in isolated nephron segments agreed with those calculated from cortex and medullary homogenates. In a prelininary experiment on human kidney it could be demonstrated that the procedures can be applied to fresh human biopsy samples. The bioluminescence method offers several advantages (simplicity, rapidity) in comparison with the classical techniques used for ultramicro analysis of tissue enzymes (enzymatic cycling, radiochemical tests). The results confirm the biochemical heterogeneity of nephron structures and allow conclusions about species dependent functional differences of the pentose-phosphate cycle and renal ketone body metabolism along the nephron.