Microassay of 5 '-nucleotidase and adenosine deaminase activity in microdissected nephron segments

The present study describes a new method for microassay of the activity of 5'-nucleotidase (5'-ND) and adenosine deaminase (ADA) in the microdissected nephron segments. The nephron segments including glomeruli, proximal convo luted and straight tubules (PCT and PST), cortical and medullary thick asce nding limbs, and cortical and medullary collecting ducts were microdissecte d 5'-ND and ADA in the nondenatured lysate of 20-mm microdissected tubules and 20 glomeruli were separated by agarose gel electrophoresis and by isoel ectric focusing, respectively. The gels were incubated with specific substr ates and staining dyes to exhibit the dephosphorylation by 5'-ND or deamina tion by ADA. The enzyme activity was estimated by measuring the intensity o f the reaction bands on the gels, The 5'-ND activity was detected in all mi crodissected tubular segments and glomeruli. Among these nephron segments, PCT and PST exhibited the greatest enzyme activity, averaging 1142 and 939 mU/mg tissue protein, respectively. The activity of ADA was also detected i n all tubular segments and glomeruli. However, the greatest activity of thi s enzyme was found in the glomeruli (649.8 mU/mg protein). Using reverse tr anscriptase-polymerase chain reaction technique, we verified the presence o f mRNA of 5'-ND and ADA in all microdissected tubular segments and glomerul i. Based on these results, we conclude that 5'-ND and ADA are present in al l nephron segments studied, but the activity of these enzymes is nonuniform ly expressed along the nephron, This microassay is a highly specific, sensi tive, and reliable method for the segmental analysis of adenosine metabolis m in the kidney. (C) 1999 Academic Press.