Microassay of 5′-nucleotidase and adenosine deaminase in microdissected nephron segments of rats

F. Wu, Osamu Ito, P. L. Li, R. J. Roman, A. P. Zou, A. W. Cowley

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1 Citation (Scopus)

Abstract

Adenosine plays an important role in the regulation of renal vascular tone and tubular function. It has been reported that 5′-nucleotidase (5′-ND) and adenosine deaminase (ADA) contribute to the formation and deamination of adenosine in the kidney, respectively. However, localization of the adenosine production and deamination in the kidney remains unknown. In the present study, we have developed new techniques to determine the activities of 5′-ND and ADA in microdissected nephron segments in rats. Using isoelectric focusing and high resolution gel electrophoresis, we separated and concentrated the enzyme proteins from non-denatured lysate of nephon segments and then measured the activity of 5′-ND and ADA with substrate-specific staining. The activities of 5′-ND and ADA in different nephron segments are summarized as follows: Glm PCT PST CCD MCD CTAL MTAL 5′-ND (mU/mg prot.) 33.3 13.4 7.6 36.7 4.4 17.8 3.8 ADA (mU/mg prot.) 203.0 39.0 27.9 90.5 53.4 71.0 13.9 The preliminary results have shown that glomeruli (Glm) and cortical collecting ducts (CCD) had greater activity of 5′-ND when compared with other nephron segments. Similarly, the greatest ADA activity was also detected in the glomeruli and cortical collecting ducts. These results suggest that isoelectric focusing and electrophoresis-based microassay is a specific and sensitive method for the segmental analysis of adenosine metabolism.

Original languageEnglish
JournalFASEB Journal
Volume11
Issue number3
Publication statusPublished - 1997 Dec 1

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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