Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle

Thomas A. White, Sonja Johnson, Timothy F. Walseth, Hon Cheung Lee, Richard M. Graeff, Cyrus B. Munshi, Y. S. Prakash, Gary C. Sieck, Mathur S. Kannan

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Abstract

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of β-NAD+ to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4 ± 1.5 and 695.3 ± 171.2 μM and V(max) values of 330.4 ± 90 and 102.8 ± 17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca2+](i) in porcine TSM cells. (C) 2000 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)64-71
Number of pages8
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1498
Issue number1
DOIs
StatePublished - Oct 20 2000

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Keywords

  • ADP-ribosyl cyclase
  • Airway smooth muscle
  • Cyclic ADP-ribose
  • Cyclic ADP-ribose hydrolase

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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