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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39 Scopus citations

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

Bibliographical note

Funding Information:
This work was supported by Grants from National Institutes of Health (HL057498 to M.S.K., DA11806 to T.F.W. and GM56686 to G.C.S.).

Keywords

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

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